Name: an inexpensive, scalable behavioral assay for measuring ethanol sedation sensitivity and rapid tolerance in drosophila
Uploaded: 2015-04-15T14:00:00.000+00:00
Duration: 7 min 28 s
Description: Watch this Scientific Journal Video about An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila at JoVE.com

These studies were supported by grants from the National Institutes of Health, National Institute for Alcoholism and Alcohol Abuse to M.G. (P20AA017828, R01AA020634, P50 AA022537). The authors thank Jill Bettinger for helpful discussions and Jacqueline DeLoyht for technical assistance.

분석 전에 1 일 <ol><li> 짧은 (1-5 분) CO 2에서 11 (단일 섹스)의 그룹 신선한 식품 유리 병에 파리를 수집합니다. </li><li> 파리는 환경 제어 공간에서 음식을 유리 병에 (일반적으로 25 ° C, 상대 습도 60 %, 12 시간 빛 / 어둠 사이클) O / N을 복구 할 수 있습니다. </li><li> 계획된 실험에 적합한 최종 농도 (들)에 정제 (≥18 MΩ) 물에 순수 (100 %) 에탄올을 희석하여 에탄올 용액 (들)을 준비합니다. 솔루션 (들) RT O / N로 복귀 할 수 있습니다. 참고 : 에탄올의 희석은 발열이다. </li></ol> 분석 2. 일 <ol><li> 파리의 각 바이알을 테스트하려면, 깨끗하고 빈 식품 유리 병을 (i)를 준비; 즉, 검사 바이알 (ⅱ) 새로운 셀룰로오스 아세테이트 플러그, (ⅲ) 실리콘 마개 및 (IV)의 에탄올 용액 (표 3 참조)를 1 ㎖. </li><li> 55-65%에 20 ~ 25 ° C의 온도와 상대 습도를 조절하여 테스트 룸을 준비합니다. </li><li> 다른 노동자 되세요튜브의 각 그룹에 고유 코드를 할당하고 이후의 코드를 기록합니다. 몇 분 동안 적응 시험 방에 파리 코드화 된 튜브를 놓습니다. </li><li> 2.3에서 비행 병에 코드를 일치하도록 빈 테스트 튜브 레이블. </li><li> 표 1과 유사한 스프레드 시트의 열로 코드 (하나의 열 / 병)을 입력하여 하드 카피 테스트 로그를 구축합니다. </li><li> 가이드로 테스트 로그를 사용하여 테스트 룸에서 배열을 일치로 파리와 빈 테스트 튜브 코드화 된 식품 유리 병을 준비. 참고 : 테스트하는 튜브의 합리적인 최대 수 (24) (즉, 4 병 각 6 세트). </li><li> 전송 일치 / 표시 빈 테스트 튜브로 음식 바이알에서 날아 셀룰로오스 아세테이트 플러그 유리 병 상판 아래 2cm가 될 때까지 즉시 테스트 튜브에 셀룰로오스 아세테이트 플러그를 삽입합니다. </li><li> 이하, 엇갈린 1 분 간격으로 집합으로 네 병의 각 행을 처리합니다. </li><li> 시간 0 평가의 경우, 개별적으로 w 각각의 유리 병을 파악i 번째 엄지 손가락과 집게 손가락은 유리 병의 바닥에 파리를 노크 30 초를 기다린 후 죽은 / 움직입니다 파리의 수를 계산하기 위해 테이블​​에 부드럽게 세 번 누릅니다. 시간에 하드 카피 테스트 로그에 0 분 각각의 병에 대한 부동 / 죽은 파리의 수를 기록한다. </li><li> 타이머는 시간 0에서 연속적으로 카운트 업을 시작하고, 즉시 4 튜브의 첫 번째 행 / 세트 바이알 셀룰로스 아세테이트 플러그에 에탄올 1 ㎖을 첨가 시작한다. 그들은 테스트 할 순서에 5 초 간격으로 바이알에 셀룰로오스 아세테이트 플러그에 에탄올 1 ㎖을 추가한다. 에탄올은 셀룰로오스 아세테이트 플러그 골고루 흡수되도록 원을 그리 셀룰로오스 아세테이트 플러그에 에탄올을 추가합니다. 에탄올은 세트의 모든 4 테스트 튜브에 첨가되었을 때, 그것을 밀봉하는 바이 얼 실리콘 플러그를 삽입한다. </li><li> 회 1, 2, 3, 4, 5 분에서, 각각의 튜브 (4) 두 번째, 세 번째, 네 번째, 다섯 번째 및 여섯 번째 세트에 에탄올 1 ㎖을 추가한다. 후 실리콘 마개를 삽입 계속4 튜브의 각 세트에 에탄올을 추가. </li><li> 시간 6 분에서, 엄지와 집게 손가락으로 각 유리 병을 잡고 유리 병의 바닥에 파리를 노크 테이블에 부드럽게 세 번을 눌러, 30 초 대기 후 계산과의 총 수를 기록하여 4 병의 첫 번째 세트를 테스트 진정시켰다 파리. 점수는 (i)는 유리 병의 바닥에 서 있지만, 또는 그들의 날개를 펄럭없이 자신의 허리에 도보 또는 (ⅱ) 거짓말하지 않는 경우 진정으로 날아갑니다. </li><li> 표 2의 스케줄을 사용하여 5 초 간격으로 셋 내의 각각의 바이알 핸들. </li><li> 제 세트에 대해 수행 배 7, 8, 9, 10 및 11 분에서 각각 튜브의, 제 3, 4, 5, 6 세트를 테스트한다. </li><li> 시간이 12 분으로 2.12에 기재된 바와 같이, 튜브 (4)를 다시 상기 제 1 세트의 테스트를 각각 13, 14, 15, 16 및 17 분에서의 바이알 제, 3, 4, 5, 6 세트의 연속 시험. </li><li> 모든 파리가들 때까지 2.12에 설명 된대로 테스트 파리 계속edated. </li><li> 하드 카피 테스트 로그의 각 유리 병에 파리의 총 수를 입력합니다. 파리의 총 수에서 시간 0에서 움직이지 / 죽은 파리를 검열. </li><li> 시간 (x 축) 대 % 활성 파리 (y 축)와 같은 각각의 평가 지점에서의 시간 퍼센트 활성 파리와 플롯 데이터를 계산한다. 다항식 또는 S 자형 곡선이 맞는 3 차에서 진정 작용 시간 (50) 값 (ST50, 50 %의 진정 작용에 시간) 보간 또는 곡선 아래의 면적을 계산하여 에탄올 진정 작용을 정량. </li><li> 디코딩 유리 병에서 데이터를 컴파일하고 통계 분석을 수행 (예를 들면, 일원 분산 분석 페로 니 여러 비교 테스트와) 실험 설계에 대한 적절한. </li></ol>

<ol>
	<li>Rehm, 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=Global+burden+of+disease+and+injury+and+economic+cost+attributable+to+alcohol+use+and+alcohol-use+disorders.">Global burden of disease and injury and economic cost attributable to alcohol use and alcohol-use disorders.</a> Lancet. 373, 2223-2233 (2009).</li><li>Prescott, C. A., Kendler, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+environmental+contributions+to+alcohol+abuse+and+dependence+in+a+population-based+sample+of+male+twins.">Genetic and environmental contributions to alcohol abuse and dependence in a population-based sample of male twins.</a> The American journal of psychiatry. 156, 34-40 (1999).</li><li>Devineni, A. V., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+Drosophila+melanogaster+as+a+model+for+alcohol+research.">The evolution of Drosophila melanogaster as a model for alcohol research.</a> Annual review of neuroscience. 36, 121-138 (2013).</li><li>Scholz, H., Mustard, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invertebrate+Models+of+Alcoholism.">Invertebrate Models of Alcoholism.</a> Current topics in behavioral neurosciences. 13, 433-457 (2011).</li><li>Rodan, A. R., Rothenfluh, A., Reilly, M. T., Lovinger, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Functional Plasticity and Genetic Variation: Insights into the Neurobiology of Alcoholism. 91, (2010).</li><li>Schumann, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+association+and+genetic+functional+studies+identify+autism+susceptibility+candidate+2+gene+(AUTS2)+in+the+regulation+of+alcohol+consumption.">Genome-wide association and genetic functional studies identify autism susceptibility candidate 2 gene (AUTS2) in the regulation of alcohol consumption.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 7119-7124 (2011).</li><li>Corl, A. B., 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=Happyhour,+a+Ste20+family+kinase,+implicates+EGFR+signaling+in+ethanol-induced+behaviors.">Happyhour, a Ste20 family kinase, implicates EGFR signaling in ethanol-induced behaviors.</a> Cell. 137, 949-960 (2009).</li><li>Moore, M. 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=Ethanol+intoxication+in+Drosophila:+Genetic+and+pharmacological+evidence+for+regulation+by+the+cAMP+signaling+pathway.">Ethanol intoxication in Drosophila: Genetic and pharmacological evidence for regulation by the cAMP signaling pathway.</a> Cell. 93, 997-1007 (1998).</li><li>Scholz, H., Franz, M., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hangover+gene+defines+a+stress+pathway+required+for+ethanol+tolerance+development.">The hangover gene defines a stress pathway required for ethanol tolerance development.</a> Nature. 436, 845-847 (2005).</li><li>Riley, B. P., 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=Alcohol+dependence+is+associated+with+the+ZNF699+gene,+a+human+locus+related+to+Drosophila+hangover,+in+the+Irish+affected+sib+pair+study+of+alcohol+dependence+(IASPSAD)+sample.">Alcohol dependence is associated with the ZNF699 gene, a human locus related to Drosophila hangover, in the Irish affected sib pair study of alcohol dependence (IASPSAD) sample.</a> Molecular psychiatry. 11, 1025-1031 (2006).</li><li>Morozova, T. V., 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=Alcohol+sensitivity+in+Drosophila:+translational+potential+of+systems+genetics.">Alcohol sensitivity in Drosophila: translational potential of systems genetics.</a> Genetics. 183, 733-745 (2009).</li><li>Ogueta, M., Cibik, O., Eltrop, R., Schneider, A., Scholz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+Adh+function+on+ethanol+preference+and+tolerance+in+adult+Drosophila+melanogaster.">The influence of Adh function on ethanol preference and tolerance in adult Drosophila melanogaster.</a> Chemical senses. 35, 813-822 (2010).</li><li>Han, 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=Integrating+GWASs+and+human+protein+interaction+networks+identifies+a+gene+subnetwork+underlying+alcohol+dependence.">Integrating GWASs and human protein interaction networks identifies a gene subnetwork underlying alcohol dependence.</a> American journal of human genetics. 93, 1027-1034 (2013).</li><li>Lind, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomewide+association+study+of+nicotine+and+alcohol+dependence+in+Australian+and+Dutch+populations.">A genomewide association study of nicotine and alcohol dependence in Australian and Dutch populations.</a> Twin Res Hum Genet. 13, 10-29 (2010).</li><li>Schuckit, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+level+of+response+to+alcohol+as+a+predictor+of+future+alcoholism.">Low level of response to alcohol as a predictor of future alcoholism.</a> The American journal of psychiatry. 151, 184-189 (1994).</li><li>Schuckit, M. A., Smith, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+8-year+follow-up+of+450+sons+of+alcoholic+and+control+subjects.">An 8-year follow-up of 450 sons of alcoholic and control subjects.</a> Archives of general psychiatry. 53, 202-210 (1996).</li><li>Tabakoff, B., Cornell, N., Hoffman, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol+tolerance.">Alcohol tolerance.</a> Ann Emerg Med. 15, 1005-1012 (1986).</li><li>Chan, R. 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=Contrasting+Influences+of+Drosophila+white/mini-white+on+Ethanol+Sensitivity+in+Two+Different+Behavioral+Assays.">Contrasting Influences of Drosophila white/mini-white on Ethanol Sensitivity in Two Different Behavioral Assays.</a> Alcohol Clin Exp Res. 38, 1582-1593 (2014).</li><li>Eddison, M., 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=arouser+reveals+a+role+for+synapse+number+in+the+regulation+of+ethanol+sensitivity.">arouser reveals a role for synapse number in the regulation of ethanol sensitivity.</a> Neuron. 70, 979-990 (2011).</li><li>Wen, T., Parrish, C. A., Xu, D., Wu, Q., Shen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+neuropeptide+F+and+its+receptor,+NPFR1,+define+a+signaling+pathway+that+acutely+modulates+alcohol+sensitivity.">Drosophila neuropeptide F and its receptor, NPFR1, define a signaling pathway that acutely modulates alcohol sensitivity.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 2141-2146 (2005).</li><li>Bhandari, P., Kendler, K. S., Bettinger, J. C., Davies, A. G., Grotewiel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+for+evoked+locomotor+behavior+in+Drosophila+reveals+a+role+for+integrins+in+ethanol+sensitivity+and+rapid+ethanol+tolerance.">An assay for evoked locomotor behavior in Drosophila reveals a role for integrins in ethanol sensitivity and rapid ethanol tolerance.</a> Alcohol Clin Exp Res. 33, 1794-1805 (2009).</li><li>Bhandari, P., 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=Chloride+intracellular+channels+modulate+acute+ethanol+behaviors+in+Drosophila,+Caenorhabditis+elegans+and+mice.">Chloride intracellular channels modulate acute ethanol behaviors in Drosophila, Caenorhabditis elegans and mice.</a> Genes, brain, and behavior. 11, 387-397 (2012).</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> Experimental gerontology. 40, 386-395 (2005).</li><li>Chen, J., Wang, Y., Zhang, Y., Shen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+Bacchus+reveal+a+tyramine-dependent+nuclear+regulator+for+acute+ethanol+sensitivity+in+Drosophila.">Mutations in Bacchus reveal a tyramine-dependent nuclear regulator for acute ethanol sensitivity in Drosophila.</a> Neuropharmacology. 67, 25-31 (2013).</li><li>Lasek, A. W., Giorgetti, F., Berger, K. H., Tayor, S., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lmo+genes+regulate+behavioral+responses+to+ethanol+in+Drosophila+melanogaster+and+the+mouse.">Lmo genes regulate behavioral responses to ethanol in Drosophila melanogaster and the mouse.</a> Alcohol Clin Exp Res. 35, 1600-1606 (2011).</li><li>Maples, T., Rothenfluh, A. <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+way+to+measure+ethanol+sensitivity+in+flies.">A simple way to measure ethanol sensitivity in flies.</a> J Vis Exp. , e2541 (2011).</li><li>Rothenfluh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+behavioral+responses+to+ethanol+are+regulated+by+alternate+RhoGAP18B+isoforms.">Distinct behavioral responses to ethanol are regulated by alternate RhoGAP18B isoforms.</a> Cell. 127, 199-211 (2006).</li><li>Rothenfluh, A., Troutwine, B. R., Ghezzi, A., Atkinson, N. S., Nohronha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+23.">Ch. 23.</a> Neurobiology of Alcohol Dependence. 23, 467-495 (2014).</li><li>Jones, M. A., Grotewiel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+age-related+impairment+in+locomotor+and+other+behaviors.">Drosophila as a model for age-related impairment in locomotor and other behaviors.</a> Experimental gerontology. 46, 320-325 (2010).</li><li>Martin, I., 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=Oxidative+damage+and+age-related+functional+declines.">Oxidative damage and age-related functional declines.</a> Mechanisms of ageing and development. 127, 411-413 (2006).</li><li>Devineni, A. V., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+ethanol+responses+in+Drosophila+are+sexually+dimorphic.">Acute ethanol responses in Drosophila are sexually dimorphic.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 21087-21092 (2012).</li></ol>

The authors have nothing to disclose.

재현성 의미있는 표현형을 정량 간단 분석은 행동의 분석을위한 큰 가치입니다. 여기에 설명 된 작품은 초파리에서 에탄올 진정 감도와 빠른 내성을 측정하기위한 분석의 몇 가지 실용적인 측면을 해결합니다. 이 작품이 아니 긴하지만, 행동 분석 연구에서 피험자에 대한 환경 및 유전 적 배경을 일정하게 유지에 의해 촉진된다. 또한, 비교는 일반적으로 사육과 나란히 테스트 파리의 그룹...

알코올 사용 장애 (AUD)는 전 세계적으로 (1 검토) 거대한 건강 문제입니다. AUD의 개발 구동 메커니즘 복잡하지만, 이러한 장애는 유전 주요 구성 요소를 가지고 (예를 들어, 2). AUD 및 보존 행동 반응의 대형 유전은 더 나은의 분자 기초를 이해하는 방향으로 에탄올과 관련된 행동의 특정 유전자의 참여를 조사하기 위해 유전자 모델 생물을 사용하여 강한 관심을 생성 한 (3,4 검토) 많은 종에서 에탄올 AUD. 초파리, 초파리 melanogaster의는 (3,4 검토) 에탄올과 관련된 행동의 분자 유전 학적 메커니즘을 탐험을위한 선도적 인 모델 생물로 떠오르고있다. 파리의 연구 (5 검토) 에탄올 행동 반응의 여러 신호 전달 경로에 대한 역할을 강조했다. 흥미롭게도, 유전자 및 경로의 일부 행동 응 답에 영향을 미치는 그파리에서 에탄올 ES는 또한 설치류 에탄올과 관련된 행동 및 / 또는 인간 AUD (예 : 6-14)에 연루되어있다. 초파리 모델 시스템에서 사용할 수있는 유전 적 도구의 스위트 룸과 함께 종에서 에탄올과 관련된 행동을, 구동 메커니즘의 보존, 에탄올 행동 반응의 유전학을 조사를 위해 초파리 모델의 유틸리티를 강조한다. 감도 (15, 16)과 인간에 에탄올 (17에서 검토) 허용 오차는 AUD의 발전에 연결되어 있습니다. 에탄올 이러한 행동 반응은 모두 (3,4 검토) 실험실 분석의 다양한 통해 파리에서 모델링 할 수있다. 저자에게 공지 플라이 분석 모두에서 진정 에탄올 중 시간에 따른 에탄올 - 유도 진정 / 운동 실조 또는 경시에 기초하여 복구된다. 에탄올 감도 및 R의 유전학에 우리 그룹에서 이전 기사에서초파리 APID 공차는, 파리의 에탄올 증기 유도 진정 작용에 기초하여 행동 분석은 (18)을 사용 하였다. 이 분석에서 시험이 라이브 성인 전송함으로써 개시 하였다는 셀룰로오스 아세테이트 플러그의 상부 (즉, 비 - 플라이 측)에 에탄올을 첨가, 셀룰로오스 아세테이트 플러그 바이알에서 파리 트래핑 음식 바이알 비우기 마취없이 날아간 및 실리콘 스토퍼 파리, 셀룰로오스 아세테이트 플러그 및 에탄올을 함유하는 바이알을 밀봉 (18 참조,도에서 개략적 S3 참조). 파리의 상이한 그룹을 나타내는 여러 바이알이 분석의 처리량을 증가 병렬 평가 하였다. 유리 병은 익명의 코드를 부여하고 실험자은 진정 작용의 평가에 의도하지 않은 편견을 방지하기 위해 치료 그룹에 눈을 멀게했다. 표준 실험에서, 바이알에 30 초 복구 후 각 바이알에 진정 파리의 수를 계수하고 converte시키고, 6 분 간격으로 부드럽게 탭핑 하였다 파리%의 활성 파리에 D. 파리 내부 에탄올 18 진정제 (이 보고서의 참조 (18) 및 CF도 1a 및도 1b)에서 점진적인 증가를 일으키는 시간 의존적 방식으로 셀룰로스 아세테이트 플러그에서 에탄올 증기를 흡수. 이 분석에서 진정 운영 체제는 산책을하거나 (II) 또는 날개를 펄럭없이 뒤에 거짓말의 부재에서 파리 (I) 서로 정의 하였다. 여기서, 에탄올이 진정 작용 분석법 상세히 설명에는, 사용과 관련된 추가의 최적화 동작이 제공되며, 분석은 비행 진정 감도 음식 보충 옵션 기여를 해결하기 위해 사용된다.

<table><tbody><tr><td>food vials</td><td>VWR</td><td>89092-772</td><td>narrow</td></tr><tr><td>Flugs</td><td>Genesee/flystuff.com</td><td>49-102</td><td>narrow</td></tr><tr><td>silicone stopper</td><td>Fisher Scientific</td><td>09-704-1l</td><td>#4</td></tr><tr><td>ethanol</td><td>Pharmaco-Aaper</td><td>111000200</td><td>200 proof</td></tr></tbody></table>

an inexpensive, scalable behavioral assay for measuring ethanol sedation sensitivity and rapid tolerance in drosophila

알코올 사용 장애 (AUD)는 건강에 심각한 도전이다. AUD로 큰 유전 요소에도 불구하고, 몇 가지 유전자는 분명하게 자신의 원인에 연루되어있다. 초파리, 초파리 melanogaster의는 알코올과 관련된 행동의 기초 분자 유전 메커니즘을 탐구하기위한 강력한 모델이므로 확인하고 AUD에 영향을 미치는 유전자의 기능을 이해하는 데 큰 약속을 보유하고있다. 이러한 유형의 연구를위한 모델 초파리의 사용은 안정적 에탄올 행동 반응을 측정하는 분석법의 가용성에 의존한다. 이 보고서는 파리에서 에탄올 감도와 빠른 내성을 평가하기위한 적절한 분석을 설명합니다. 이 분석에서 측정 에탄올 감도가 초파리 시험 직전 음식없이 수납되어 사용되는 부피의 에탄올 농도, 이전에보고 된 유전자 조작의 다양하고, 또한 시간의 길이에 의해 영향을 받는다. 한편, 에탄올 S이 분석에서 측정 ensitivity 플라이 처리, 파리의 성별, 항생제 또는 살아있는 효모와 성장 매체의 보충의 활력에 영향을받지 않습니다. 정량 에탄올 감도 세 가지 방법은 모두 본질적으로 구별 에탄올 감도 결과에 선도적 인 기술되어있다. 전반적인 단순 결합이 분석의 확장 성 성격, 그리고 상대적으로 낮은 비용으로 업을 설정 초파리 에탄올 감도와 빠른 관용의 크고 작은 규모의 유전 적 분석에 적합하게합니다.

이 에탄올 진정 작용 분석에서 원시 데이터는 에탄올 증기 노출 시간의 함수로서 진정시켰다 파리의 수이다. 원시 데이터는 시간의 함수로서 파리 퍼센트 활성으로 전환된다 (primary 데이터를도 1A, B, D - F). 곡선 적합로부터 기본 데이터에서 에탄올에 진정 감도 보간을 통해, 진정 작용 시간 50 (ST50)로 곡선 (AUC) 아래 진정제 또는 AEA가 날아간의 50 %에 필요한 시간을 정량화 ...

Watch this Scientific Journal Video about An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila at JoVE.com

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

에탄올 진정 감도 및 신속한 공차 측정을위한 저렴하고 확장 가능한 행동 분석<em&gt; 초파리</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

an-inexpensive-scalable-behavioral-assay-for-measuring-ethanol

Research

JoVE Journal

Neuroscience

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

10.2K 조회수.  Virginia Commonwealth University. Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

비디오: An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

행동 내분비학 및 실험 경제학을 결합 : 테스토 스 테론과 사회 의사 결정

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

연구

신경과학

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

파리에서 에탄올 민감도를 측정하는 간단한 방법

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

쥐에서 자당의 갈망의 보육을 평가하기위한 일반적인 방법

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

쥐에 활동 륜 실행에 녹음 및 Circadian 리듬 분석

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

The FlyBar: Administering Alcohol to Flies

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian clock modulates alcohol sensitivity, but not the formation of tolerance. Here, we describe our protocol in detail. Alcohol is administered to the flies using the FlyBar. In this setup, saturated alcohol vapor is mixed with humidified air in set proportions, and administered to the flies in four tubes simultaneously. Flies are reared under standardized conditions in order to minimize variation between the replicates. Three-day old flies of different genotypes or treatments are used for the experiments, preferably by matching flies of two different time points (e.g., CT 5 and CT 17) making direct comparisons possible. During the experiment, flies are exposed for 1 hr&#160;to the pre-determined percentage of alcohol vapor and the number of flies that exhibit the Loss of Righting reflex (LoRR) or sedation are counted every 5 min. The data can be analyzed using three different statistical approaches. The first is to determine the time at which 50% of the flies have lost their righting reflex and use an Analysis of the Variance (ANOVA) to determine whether significant differences exist between time points. The second is to determine the percentage flies that show LoRR after a specified number of minutes, followed by an ANOVA analysis. The last method is to analyze the whole times series using multivariate statistics. The protocol can also be used for non-circadian experiments or comparisons between genotypes.

Drosophila has emerged as a significant model system for dissecting the cellular and molecular underpinnings of behavioral responses to alcohol. Here we present a protocol for the collection of alcohol sensitivity data in a circadian context that can be easily applied to other experiments and is well-suited for undergraduate research.

의 flybar : 파리에 주류 관리

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian ...

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

행동분석학

Flypub To Study Ethanol Induced Behavioral Disinhibition and Sensitization

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the underlying neurobiological mechanisms, for which diverse experimental approaches and model systems are needed. The main ingredient of alcoholic beverages is ethanol, which causes adaptive changes in the central nervous system and behavior upon chronic intake. Behavioral sensitization (i.e., escalated responses) in particular represents a key adaptive change underlying addiction. Most ethanol-induced behavioral sensitization studies in animal models have been conducted on the locomotor activating effect of ethanol. A prominent effect of ethanol is behavioral disinhibition. Behavioral sensitization to the disinhibition effect of ethanol, however, is underrepresented. To address this issue, we developed the Flypub assay that allows measuring the escalated increase in disinhibited courtship activities upon recurring ethanol exposure in Drosophila melanogaster.&#160;Here, we report the step-by-step Flypub assay including assembly of ethanol exposure chambers, setup of the assay station, criteria for fly care and collection, ethanol delivery, quantification of disinhibited courtship activities, data processing and statistical analysis. Also provided are how to troubleshoot critical steps, overcome limitations and expand its utility to assess additional ethanol-induced behaviors. The Flypub assay in combination with powerful genetic tools in Drosophila melanogaster will facilitate the task of discovering the mechanism underlying ethanol-induced behavioral sensitization.

The Flypub assay measures the behaviors that the fruit fly Drosophila melanogaster displays under the influence of ethanol. The assay can be readily mastered by experimenters at all levels and applied to various vaporized stimuli, facilitating substance abuse and addiction studies.

플라이펍, 에탄올 유발 행동 억제 및 과민성 연구

Alcohol use disorder (AUD) remains a serious problem in our society. To develop effective interventions for addiction, it is important to understand the ...

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.

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 ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

의 로코 속도에 에탄올의 효과를 측정하는 분석<em&gt; 예쁜 꼬마 선충</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

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 ...

에탄올 진정 감도 및 신속한 공차 측정을위한 저렴하고 확장 가능한 행동 분석

요약

더 많은 비디오 탐색

이 비디오의 챕터

행동 내분비학 및 실험 경제학을 결합 : 테스토 스 테론과 사회 의사 결정

파리에서 에탄올 민감도를 측정하는 간단한 방법

쥐에서 자당의 갈망의 보육을 평가하기위한 일반적인 방법

쥐에 활동 륜 실행에 녹음 및 Circadian 리듬 분석

의 flybar : 파리에 주류 관리

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

플라이펍, 에탄올 유발 행동 억제 및 과민성 연구

사회 회피의 정량화에 대한 간단한 분석

의 로코 속도에 에탄올의 효과를 측정하는 분석

사용 보상 추구 행동의 변화를 측정하는 간단한 방법