이 작품은 NSF 기금 IOS 1,121,692에 의해, 워싱턴의 연구 재단의 대학에 의해 지원되었다.

1. 휘발성 Follection <ol><li> 이 예제에서, 우리는 M.에서 휘발성 샘플을 사용하여 lewisii 꽃 - 캘리포니아에 고산 야생화의 기본. 휘발성 물질이 Riffell 외에 따라 동적 흡착 방법을 사용하여 수집하고 있습니다. 14. 간단히,이 방법은 꽃이 테플론 백에 동봉되는 폐 루프 트래핑 시스템을 사용합니다. 불활성 진공 펌프 사용하여 꽃 주변의 공기는 Porapak Q 행렬로 가득 파스퇴르 피펫으로 구성 &quot;덫&quot;을 통해 형편입니다. 펌프의 반환 공기는 활성탄에 의해 필터링됩니다. 소정의 기간 후에, 우리의 경우 24 H에, Porapak Q 행렬은 집중 추출물를 수집하기 위해, 비 극지 용매, 일반적으로 헥산으로 용출되어 있습니다. 추출물 그런 다음 -80 ° C에서 분석 될 때까지 저장됩니다. 필요한 경우, 샘플은 질소 가스의 흐름에 따라 사전 분석에 집중 할 수 있습니다. 샘플은 이미 잘 - 특징 않는 한,를 통해 그것을 나누어지는을 실행기체 크로마토 그래프 - 질량 분석기 (GC-MS)는 샘플을 사용하기 전에 휘발성 구성 요소를 식별합니다. </li></ol> 2. Electrophysiological 준비 <ol><li> 1000 μl 피펫 팁의 끝에서 약 1cm를 자른다. 피펫 팁의 기본에 범블비 (Bombus impatiens)를 놓고는 머리가 노출 될 때까지 부드럽게 끝의 반대쪽으로 밀어. </li><li> 치과 왁스를 녹여 및 보안에 대한 화합물 눈 위에와 왁스 준수는 꿀벌의 머리가 완전히 고정되어 만들 수 있는지 확인하기, 노출 된 머리 주위를 뜨는거야. 벌의 안테나에 어떤 왁스를하지 않도록주의하십시오. </li><li> 머리가 안전하면, 면도기 블레이드 - 차단기 나 큐티클을 잘라 할 메스 적절한 크기를 사용하여 헤드 캡슐로 광장, 윈도우 같은 절개를합니다. 블레이드 - 차단기를 사용하여 즉시 화합물 눈에 안테나와 인접 하나 뒤에, 머리 캡슐의 등 측면에서 시작합니다. 절단한 화합물 눈에서 contralateral 화합물 눈에 직선. 반대 화합물 눈에 직선을 절단 한 후, 그 가슴 근처의 헤드 캡슐 곡선과 끝날 때까지 dorsally 절개을 시작합니다. 이 시점에서, 머리 캡슐의 반대 끝 부분 절단하기 시작합니다. 마지막으로, 한 번 줄이 반대쪽으로 절단되었습니다 초기 절개의 시작 위치로 다시 줄을 잘라. 이 전극의 삽입을 방해하므로 그것은 안테나에 인접 해 있습니다 큐티클을 제거하는 것이 중요합니다. </li><li> 큐티클을 잘라되면 다음 벌의 뇌를 노출하고, 더 중요한 것은 antennal 엽해야 벌의 큐티클을 제거 포셉 한 쌍의를 사용합니다. 바로 뇌 dessicated이되지 않도록, 곤충 식염수 뇌를 superfusing 시작합니다. 뇌가 노출되면 조심스럽게 즉시 antennal 엽 위의 perineural 피복을 제거하는 아주 좋은 포셉 한 쌍의를 사용합니다. 정말 조심해야하지에포셉과 구멍 벌의 뇌를. </li></ol> 3. 멀티 채널 녹음과 가스 크로마토 그래피 <ol><li> 벌 &quot;준비&quot;- 뇌의 노출이있는 튜브에서 수정이 - 지금 electrophysiological 레코딩을위한 준비가되었습니다. 공기 테이블에 자리 잡고 있습니다 자기베이스에 고정 클램프의 벌을 배치합니다. </li><li> IV 가방, 흐름 컨트롤러, 그리고 생리가 계속 뇌를 superfuses 있도록 튜브를 (곤충 생리가 가득) 준비합니다. </li><li> 벌의 눈에, 텅스텐 와이어로 만든 micromanipulator, insertareference 전극을 사용합니다. </li><li> 별도의 micromanipulator를 사용, 이러한 코일 와이어 tetrode, 또는 실리콘 멀티 채널 전극 (Neuronexus 기술)로, 멀티 채널 전극을 삽입 벌의 antennal 엽에. 이 전극 등의 가열 치사 시스템의 Z-버스 bioamp 프로세서에 가열 치사 시스템의 S-3 Z 시리즈와 같은 사전 증폭기에 연결되어 있습니다. 가스 Chro의 출력차폐 된 BNC 케이블을 통해 matogram의 감지기가 모두 신경 및 GC 신호가 동기화되는 등의 증폭기 및 데이터 수집 시스템과 인터페이스 할 수 있습니다. </li><li> 안정화 할 수있는 신경 레코딩을위한 약 30~60분 기다립니다. 자연 활동 및 녹화 채널에 단위의 파형 모양이 일관성이되었습니다되면, 벌을 자극하고 냄새에 기록 채널의 반응을 관찰하는 악취 주사기를 사용합니다. </li><li> 에 의해 뉴런의 세포 스파이크를 기록 자동 임계 값 각 기록 채널에서 신호의 3.5-5 시그마하여 녹화 채널을. 수동 임계 값은 전기 노이즈의 오염을 방지하기 위해 일부 채널 필요할 수 있습니다. 뉴런의 활동 전위가 기록 채널에 전압 스파이크로 표시됩니다. 채널의 전압이 임계 값을 초과하면 시스템은이를 실려, 임계의 교차로 전후 몇 밀리 초 버퍼 및 저장GA는 파형의 스냅 샷, 또는 스파이크. </li><li> 바로이 공기 테이블 옆에있는 GC입니다. GC에 꽃 추출물을 주입하기 전에 GC 실행의 온도 램프를위한 방법이 올바른지 확인하십시오. 우리 예제에서, 우리는 10 ° C / 분의 속도로 온도의 증가에 이어 4 분 50에서 시작 온도 방법 ° C를 사용합니다. 220 ° 우리가 추가 6 분의 GC을 보유하고있는 시간에 C,. 우리는 캐리어 가스로 헬륨이있는 DB-5 GC 컬럼 (J &amp; W 과학, Folsom, CA, USA)를 사용합니다. 입구는 200 ° C.로 설정 온도, splitless입니다 불꽃 이온화 검출기 온도는 230 ° C.로 설정되어 있습니다 </li><li> GC 칼럼으로 adsorbed 휘발성 물질을 해제 할 수있는 GC의 온수 주입 포트에 꽃 천정 공간의 추출물 샘플을 주입. 열에서 유출 물은 불꽃 이온화 검출기 (FID) 및 유리 &quot;Y&quot;커넥터 (J &amp; W 과학)를 사용하여 벌 안테나 사이에 1:1 분할되어 있습니다. REC를 시작당신은 GC에 샘플을 투입으로 전극에서 ording. </li><li> GC 실행 완료 후, 5 15 분을 위해 준비 나머지를 보자. 그런 다음 GC에 다른 샘플을 주입 또는 단일 휘발성 화합물 또는 화합물의 혼합물을 사용하여 준비를 자극한다. 준비를 자극이 후자의 방법에서는 일정한 공기 흐름에서 공기의 펄스는 화합물이 증착 된되는 필터 종이를 포함하는 유리 주사기를 통해 우회합니다. 냄새 자극은 소프트웨어에 의해 제어 솔레노이드가 활성화 된 밸브에 의해 펄스되었습니다. </li><li> 단위 활동이 갑자기 중단하거나 변경할 경우, 생리 똑을 확인하고 15 분 동안 준비 나머지를 보자. 자연 활동이 이전 수준을 회복하지 않으면 사용 가능한 경우 다음 준비는 폐기하고 다른 꿀벌이 사용되어야합니다. </li><li> 실험 후, 여전히 조직의 프로브와 20 분에 포르말린 5퍼센트으로 뇌를 해결할 수 있습니다. 다음, 소비세 뇌그리고 4 시간에 2 % 글 루타 알데히드에 배치하고, 이후 등급 에탄올 탈수 시리즈를 수행하고 살리실산 메틸과 뇌를 취소합니다. 조직, 전극의 조직은 공 촛점 현미경에 의해 명확하게 알아볼 수 있어야합니다 구멍을 AL에있는 위치의 고정과 개간을 기반으로합니다. </li></ol> 4. 데이터 분석 <ol><li> 분석은 기록 된 신경 단위를 분리하고 식별 할 수있는 실험 후 데이터를 수집했습니다. 이러한 피크 또는 계곡 진폭, 최대 반폭 등의 스파이크 모양, 또는 감소 조치에 따라 별도의 파형, 또는 &quot;스파이크&quot;(주요 구성 요소) 17 일반적인 소프트웨어 프로그램을 (오프라인 정렬 기, MClust 및 SClust)를 사용 18 (그림 2). 3 차원 공간에서 분리 (PC1-PC3)과 서로의 통계적 다른 것을 스파이크의 만 클러스터를 사용합니다 (변수 ANOVA, P &lt;0.05) (그림 2) 추가 분석을 위해. 를 참조하십시오tetrode 기록과 스파이크 - 정렬 방법에 대한 자세한 설명을위한 인용 # S 17-19. </li><li> 시간 스탬프 각 클러스터에 스파이크하고, 래스터 플롯과 발사 속도 응답 (도 2, 3A)를 만들 MATLAB 또는 Neuroexplorer을 (Nex 기술, 윈스턴 세일럼, NC)를 사용하여 분석을 위해 이러한 데이터를 내보낼 수도 있습니다. </li><li> 동시에 기록 GC 데이터를 사용 휘발성 물질의 유지 시간을 확인합니다. 그 시간 지점에서 단위 반응을 검사 할 크로마토 그램의 피크의 꼭대기에 의해 결정 휘발성 물질의 보유 시간을 사용합니다. </li><li> GC 실행을 통해 각 단위 반응을 확인하려면, 100 밀리 초 간격으로 스파이크의 개수 및 bin 및 휘발성 물질을 용출의 보존 기간을 참조하여 속도 응답을 해고의 시간 과정을 검토합니다. 100 밀리 초 간격의 스파이크의 binning는 GC에서 용출 odorant에 neuronal 응답의 시간 코스에 대해, 충분히 상세하게, 또는 신호를 제공합니다. </li><li> EXA에다른 용출의 휘발성 물질에 인구 응답을 채굴, 전에 3 초 샘플링 창을 통해 1.5 초를 개별 단위의 발사 속도 응답을 통합하고, 1.5 초 후의 보존 기간 휘발성 (그림 3). 이 기간은 GC에서 휘발성 용출 기간의 전형적인 있습니다. 및 (열) GC 유출에 앙상블 응답을 나타내는 각 행과 활동 매트릭스로를 준비하고 (; 우리는이 색상 코딩을 (파란색은 낮은 응답입니다 빨간색이 높은 발사 속도 응답이다) 단위의 속도 응답을 발사 보여 그림 3). </li></ol>

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A., Pellmyr, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+headspace+analysis+of+floral+volatiles:+a+comparison+of+methods.">Dynamic headspace analysis of floral volatiles: a comparison of methods.</a> Oikos. 81, 238-254 (1998).</li><li>Rodriguez-Saona, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbivore-induced+blueberry+volatiles+and+intra-plant+signaling.">Herbivore-induced blueberry volatiles and intra-plant signaling.</a> J Vis Exp. , e3440 (2011).</li><li>Nguyen, D. 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=Micro-drive+array+for+chronic+in+vivo+recording:+tetrode+assembly.">Micro-drive array for chronic in vivo recording: tetrode assembly.</a> J Vis Exp. , e1098 (2009).</li><li>Schjetnan, A. G. P., Luczak, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+large-scale+neuronal+ensembles+with+silicon+probes+in+the+anesthetized+rat.">Recording large-scale neuronal ensembles with silicon probes in the anesthetized rat.</a> J Vis Exp. , e3282 (2011).</li><li>Deisig, N., Giurfa, M., Lachnit, H., Sandoz, 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=Neural+representation+of+olfactory+mixtures+in+the+honeybee+antennal+lobe.">Neural representation of olfactory mixtures in the honeybee antennal lobe.</a> European Journal of Neuroscience. 24, 1161-1174 (2006).</li><li>St&#246;kl, 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=A+deceptive+pollination+system+targeting+drosophilids+through+olfactory+mimicry+of+yeast.">A deceptive pollination system targeting drosophilids through olfactory mimicry of yeast.</a> Current Biology. 20, 1846-1852 (2010).</li><li>Schneider, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elektrophysiologische+untersuchungen+von+chemo-+und+mechanorezeptoren+der+antenne+des+seidenspinners+Bombyx+mori+L.">Elektrophysiologische untersuchungen von chemo- und mechanorezeptoren der antenne des seidenspinners Bombyx mori L.</a> Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology. 40, 8-41 (1957).</li><li>Arn, H., St&#228;dler, E., Rauscher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electroantennographic+detector:+a+selective+and+senstitive+tool+in+the+gas+chromatographic+analysis+of+insect+pheromones.">The electroantennographic detector: a selective and senstitive tool in the gas chromatographic analysis of insect pheromones.</a> Zeitschrift f&#252;r Naturforschung. 30c, 722-725 (1975).</li><li>Schneider, D., Boeckh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rezeptorpotential+und+nervenimpulse+einzelner+olfaktorischer+sensillen+der+insektenantenne.">Rezeptorpotential und nervenimpulse einzelner olfaktorischer sensillen der insektenantenne.</a> Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology. 45, 405-412 (1962).</li><li>Blight, M. M., Pickett, J. A., Wadhams, L. J., Woodcock, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+perception+of+oilseed+rape+Brassica+napus+(Brassicaceae)+volatiles+by+the+cabbage+seed+weevil+Ceutorhynchus+assimilis+(Coleoptera,+Curculionidae).">Antennal perception of oilseed rape Brassica napus (Brassicaceae) volatiles by the cabbage seed weevil Ceutorhynchus assimilis (Coleoptera, Curculionidae).</a> Journal of Chemical Ecology. 21, 1649-1664 (1995).</li><li>Lin, D. Y., Shea, S. D., Katz, 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=Representation+of+natural+stimuli+in+the+rodent+main+olfactory+bulb.">Representation of natural stimuli in the rodent main olfactory bulb.</a> Neuron. 50, 937-949 (2006).</li><li>Lei, H., Reisenman, C. E., Wilson, C. H., Gabbur, P., Hildebrand, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiking+patterns+and+their+functional+implications+in+the+antennal+lobe+of+the+tobacco+hornworm+Manduca+sexta.">Spiking patterns and their functional implications in the antennal lobe of the tobacco hornworm Manduca sexta.</a> PLoS ONE. 6, e23382 (2011).</li><li>Syed, Z., Leal, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+olfactory+response+of+Culex+mosquitoes+to+a+human-+and+bird-derived+attractant.">Acute olfactory response of Culex mosquitoes to a human- and bird-derived attractant.</a> Proceedings of the National Academy of Sciences. 106, 18803-18808 (2009).</li></ol>

관심 없음 충돌이 선언 없습니다.

곤충 후각로 인한 행동이 복제, 호스트 사이트 선택, 적절한 음식 자원의 식별 등을 포함하여 다양한 프로세스를 운전합니다. 이러한 프로세스의 연구는 소스뿐만 아니라 행동을 중재 아르 이러한 화합물을 식별 할 수있는 능력에서 방출 된 휘발성 물질을 식별 할 수있는 능력이 필요합니다. 문제를 복잡하게하는 것은 냄새가 함께 각각의 성분 6,7,13,19,20 다르게 인식되고 독특한 향을 만?...

<table border="1"><tbody><tr><td> 항목의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> Porapak 타입 Q 80-100 메쉬 </td><td> 워터스 </td><td> WAT027060 </td><td></td></tr><tr><td> 레이놀즈 오븐 가방 </td><td> 레이놀즈 </td><td></td><td></td></tr><tr><td> GC </td><td> 애질런트 </td><td> 7820A </td><td></td></tr><tr><td> GC 컬럼 </td><td> J &amp; W 과학, Folsom, CA, USA </td><td> DB-5 (30m, 0.25 mm, 0.25 μm) </td><td></td></tr><tr><td> 분석 헬륨 캐리어 가스 </td><td> Praxair </td><td> HE K </td><td> 한 참조 / 분 </td></tr><tr><td> 16 채널 실리콘 전극 </td><td> Neuronexus Technologies </td><td> a4x4-3mm50-177 </td><td></td></tr><tr><td> 정밀 와이어 NiCr, 0.012 mm 직경) </td><td> Sandvik Kanthal HP 리드 </td><td> PX000004 </td><td> 사용자 정의 tetrodes 및 stereotrodes을 만들기위한 </td></tr><tr><td> 사전 증폭기 </td><td> 터커 - 데이비스 시스템 </td><td> PZ-2 </td><td></td></tr><tr><td> 증폭기 </td><td> 터커 - 데이비스 시스템 </td><td> RZ-2 </td><td></td></tr><tr><td> 데이터 수집 시스템 - OpenEx 스위트 </td><td> 터커 - 데이비스 시스템 </td><td></td><td></td></tr><tr><td> 온라인 스파이크 - 정렬 소프트웨어 - SpikePac </td><td> 터커 - 데이비스 시스템 </td><td></td><td></td></tr><tr><td> 오프라인 스파이크 - 정렬 소프트웨어 - Mclust 스파이크 - 정렬 도구 상자 </td><td> 데이비드 Redish, 신경 과학, 대학학과미네소타 </td><td> 에서 무료로 다운로드 <a href="http://redishlab.neuroscience.umn.edu/MClust/MClust.html" target="_blank">http://redishlab.neuroscience.umn.edu/MClust/MClust.html</a> </td><td> MATLAB 도구 상자 </td></tr></tbody></table>

identification of olfactory volatiles using gas chromatography-multi-unit recordings (gcmr) in the insect antennal lobe

모든 생물은 환경에 자신의 행동 및 생리적 반응을 결정하는 감각 자극으로 가득 찬 세상을 살고 있습니다. 후각은에 응답하고, 사이, 복잡한 냄새 자극을 차별하기 위해 후각 시스템을 사용 곤충에 특히 중요합니다. 이 냄새는 재생산 및 서식지 선택 1-3로 프로세스를 중재하는 행동을 이끌어내는. 또한, 수분 4-6 식품 작물 7 herbivory, 질병 8,9의 전송을 포함하여 농업과 인간의 건강을 위해 매우 중요 곤충의 mediates 동작에 의한 화학 감지. 후각 신호와 곤충 행동에서의 역할 확인은 생태 학적 프로세스와 인간의 음식 자원과 웰빙을 모두 이해하는 따라서 중요합니다. 현재까지, 곤충의 행동을 유도 휘발성 물질의 식별이 어렵고 종종 지루한했다. 현재 기술은 다음과 같습니다가스 크로마토 그래피 결합 electroantennogram 기록 (GC-EAG) 및 가스 단일 sensillum 레코딩 (GC-SSR) 10-12를 크로마토 그래피 - 커플 링. 이 기술은 bioactive 화합물의 식별에 중요한 것으로 판명. 13,14, 우리는 antennal 엽에있는 뉴런 (곤충의 기본 후각 센터 AL)에서 멀티 채널 electrophysiological 레코딩 ( &#39;GCMR&#39;이라고한다)에 커플 링 된 가스 크로마토 그래피를 사용하는 방법을 개발했습니다. 이 최신 기술은 우리가 냄새 정보가 곤충 뇌에 표시하는 방법을 조사 할 수 있습니다. 후각 처리의 수준 냄새에 신경 응답이 AL의 뉴런에 안테나의 수용체 뉴런의 수렴의 정도에 매우 민감 인하여 때문에 또한, AL 녹음은 자연 냄새 효율적이고 고감도로의 활성 성분의 검출을 할 수 있습니다. 여기 GCMR을 설명하고 사용 예를 제공합니다. 몇몇 일반적인 단계는 invol 아르bioactive 휘발성 물질과 곤충 응답의 검출에 ved. 휘발성 물질 먼저 관심 소스에서 수집 (이 예에서 우리는 속 Mimulus (Phyrmaceae)에서 꽃을 사용) 및 표준 GC-MS 기술에게 14-16를 사용하여 필요에 따라 특징해야합니다. 곤충은 기록 전극이 신경 녹음이 시작 antennal 엽 및 다중 채널에 삽입 된 후, 최소한의 절개를 사용하여 학습을위한 준비가되어 있습니다. 신경 데이터의 후 처리는 특정 odorants는 곤충 신경계에서 중요한 신경 반응의 원인이되는 드러내고있다. 우리가 여기에 제시 예는 수분 연구에 특정한이지만, GCMR는 연구 생물 및 휘발성 소스의 광범위한 확장 할 수 있습니다. 예를 들어,이 방법은 odorants 벡터 곤충, 농작물 해충을 끌거나 repelling의 식별에 사용할 수 있습니다. 또한, GCMR는 또한 PO 등의 유익 곤충에 대한 attractants를 식별하는 데 사용할 수 있습니다llinators. 기술은뿐만 아니라 비 곤충 분야로 확대 할 수 있습니다.

M.를 사용하여 GCMR 분석에서 lewisii 꽃 향기가, 우리는 GC에 추출물의 3 μl를 삽입. GC를 통해 용출 휘발성 물질의 총 수는 일반적으로 60-70 휘발성 물질입니다. M.의 향기 lewisii는 주로 여섯 탄소 등 2 헥산 올과 같은 휘발성 물질, 그리고 천정 공간의 &lt;1 %를 구성하는 sesquiterpenoids로 구성된 향기의 나머지 부분과 함께, β-myrcene (acyclic)와 α-피넨 포함 monoterpenoids?...

Watch this Scientific Journal Video about Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe at JoVE.com

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

후각 신호는 곤충의 여러 가지 행동을 중재하고, 휘발성 화합물의 수백 수십 구성 종종 복합 혼합물이다. 곤충 antennal 엽의 멀티 채널 녹음과 가스 크로마토 그래피를 사용하여, 우리는 bioactive 화합물의 식별하는 방법을 설명합니다.

곤충 Antennal 엽에 가스 크로마토 그래피 - 멀티 유닛 레코딩 (GCMR)를 사용하여 강한 휘발성 물질의 식별

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

identification-olfactory-volatiles-using-gas-chromatography-multi

Research

JoVE Journal

Neuroscience

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

14.2K 조회수.  University of Washington. 후각 신호는 곤충의 여러 가지 행동을 중재하고, 휘발성 화합물의 수백 수십 구성 종종 복합 혼합물이다. 곤충 antennal 엽의 멀티 채널 녹음과 가스 크로마토 그래피를 사용하여, 우리는 bioactive 화합물의 식별하는 방법을 설명합니다.

비디오: Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

에어 - 상 Electroolfactogram 기록을 사용하여 마우스 강한 감각 뉴런의 분석 응답

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

연구

신경과학

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

의 냄새-evoked 응답의 칼슘 이미징 Drosophila Antennal 엽

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

흡입 피펫 기법을 사용하여 강한 수용체 뉴런에서 기록 Odorant 유발 응답

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

급성 Hippocampal 슬라이스에 Synaptically-evoked Astroglial와 Neuronal 응답의 듀얼 Electrophysiological 레코딩

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

메뚜기에 신경 활동을 특성화하기 위해 다중 단위 기록 방법 (<em&gt; Schistocerca 아메리카나</em&gt;) 강한 회로

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects foraging for food or searching for mates3: the olfactory search is constrained by the physics of turbulent transport4. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7 but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells8 or toxic and illicit substances9-11. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14 or using nanostructured gas sensors that mimic insect antennae15.

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

후각 검색을위한 자율 로봇에 곤충 Electroantennogram 센서를 사용하여

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

본래 Vomeronasal 기관 및 액세서리 후각 망울의 생체 준비

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

The electrophysiological technique of intracellular recording is demonstrated and used to determine spectral sensitivities of single photoreceptor cells in the compound eye of a butterfly.

에서 곤충 모델에서 시각 세포 스펙트럼 감도의 결정<em&gt; 생체</em&gt; 세포 내 녹음

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect

Robotic odor source localization has been a challenging area and one to which biological knowledge has been expected to contribute, as finding odor sources is an essential task for organism survival. Insects are well-studied organisms with regard to odor tracking, and their behavioral strategies have been applied to mobile robots for evaluation. This "bottom-up" approach is a fundamental way to develop biomimetic robots; however, the biological analyses and the modeling of behavioral mechanisms are still ongoing. Therefore, it is still unknown how such a biological system actually works as the controller of a robotic platform. To answer this question, we have developed an insect-controlled robot in which a male adult silkmoth (Bombyx mori) drives a robot car in response to odor stimuli; this can be regarded as a prototype of a future insect-mimetic robot. In the cockpit of the robot, a tethered silkmoth walked on an air-supported ball and an optical sensor measured the ball rotations. These rotations were translated into the movement of the two-wheeled robot. The advantage of this "hybrid" approach is that experimenters can manipulate any parameter of the robot, which enables the evaluation of the odor-tracking capability of insects and provides useful suggestions for robotic odor-tracking. Furthermore, these manipulations are non-invasive ways to alter the sensory-motor relationship of a pilot insect and will be a useful technique for understanding adaptive behaviors.

The capability to localize an odor source is necessary for insect survival and is expected to be applicable to artificial odor-tracking. The insect-controlled robot is driven by an actual silkmoth and enables us to evaluate the odor-tracking capability of insects through a robotic platform.

곤충 제어 로봇 : 이동 로봇 플랫폼은 곤충의 냄새 추적 능력을 평가하는

Robotic odor source localization has been a challenging area and one to which biological knowledge has been expected to contribute, as finding odor ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

후각 감각 축색 돌연변이 분자 식별 코드의 시각화를위한 뒷 전구의 4 배 면역 면역 측정법

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Methods Article

곤충 Antennal 엽에 가스 크로마토 그래피 - 멀티 유닛 레코딩 (GCMR)를 사용하여 강한 휘발성 물질의 식별