This work was supported by the Singapore National Research Foundation (grant NRF-RF2010-06 to J.Y.Y.).

1. 샘플 준비 <ol><li> (; P {20XUAS-IVS-GCaMP5G} attP40 1,118w) UAS-GCaMP5G (19) 파리에 GAL4 3,22 드라이버 라인을 교차. 십자가가 25 ° C에서 성장 할 수 있습니다. 참고 : 생성은 형광 신호 강도를 향상하는 데 도움이 할 수있는 GAL4 또는 UAS-GCaMP 유전자의 여러 복사본으로 날아갑니다. Gr68a-GAL4 드라이버의 제어하에 발현 될 때 UAS-GCaMP 트랜스 (UAS-GCaMP3)의 이전 버전들은 매우 약한 형광 강도를 나타내었다. <ol><li> 수집 섹스 새로 eclosed 성인 파리로 구분하고 25 ° C에서 올립니다. 참고 : GCaMP5G의 개화의 기준 레벨이 14~28일 세 사이 파리에 최적입니다. 섹스에 의해 파리를 분리하면 신경 반응에 영향을 미칠 수있는 사회적 상호 작용의 가능성을 배제. </li></ol></li><li> CO 2의 온화한 기류 하에서 비행을 마취. </li><li> f를를 부착LY 먼저 0.17 mm의 커버 슬립의 중심 매니큐어의 작은 방울을 배치하여 유리 커버 슬립에 관한 것이다. 부드럽게 명확한 매니큐어의 드롭 옆에 비행을 배치하고 전체 드롭이 즉시 적용되어 있는지 확인합니다. </li><li> 8 배의 배율로 사용되는 해부 실체 현미경 아래 단계 1.5 및 1.6를 수행합니다. </li><li> 파리의 앞다리를 확장 젖은 붓을 사용합니다. 첫 번째와 다섯 번째 눈꺼풀 세그먼트 (그림 2A)를 통해 테이프의 두 개의 얇은 스트립을 배치하여 위치에 앞다리를 고정합니다. 이 영상 (그림 2B)에 대한 눈꺼풀 부분을 T2-T4를 노출합니다. </li><li> 프로보 시스에 이미지 뉴런 위해, labellum (그림 2C, D)를 노출하는 코의 연단을 통해 테이프의 얇은 스트립을 배치합니다. </li><li> 커버 슬립 표면 위로 흐르는에서 솔루션을 방지하기 위해 PAP 펜으로 비행 주위에 소수성 장벽을 그립니다. 설치 후 30 분 이내에 측정을합니다. </li></ol> 자극 2. 준비해결책 <ol><li> 친 유성 리간드를 희석 PBST 용액 (예 : 본 연구에서 사용 CH503 등) : 137 mM의 염화나트륨, 2.7 밀리미터의 KCl, 10 mM의 나 2 HPO 4, 1.8 mM의 KH 2 PO 4, 0.1 % 트리톤 X-100 (V / V ); pH가 7.4. <ol><li> PBST를 사용하여 리간드를 1 ㎎ / ㎖의 스톡 용액을 만든다. PBST를 사용하여 모액의 모든 연속 희석액을 준비한다. 주 : 관심있는 리간드의 용해도에 따라 다양한 용매는 리간드를 용해 필요할 수있다. 다양한 용매 CH503의 용해도는하기 표 1에 기재되어있다. 다른 용매는 달리 PBST 형광의 증가를 생성하지 않았다. </li></ol></li></ol> 미각 신경 및 이미지 획득 3. 자극 <ol><li> 10 μL의 피펫을 사용하여 눈꺼풀 세그먼트에 PBST 10 μl를 배치합니다. 참고 :이 단계는 다리를 습하게하고 표류에서 눈꺼풀 세그먼트를 방지 할 수 있습니다. </li><li> SU의 카메라를 지닌 광학 시스템을 사용fficient 속도와 감도가 높은 시간 해상도에서 좋은 형광 신호를 달성했다. 참고 :이 연구에서 우리는 회전 디스크 공 초점 현미경과 sCMOS 카메라 (자료 목록 참조)를 사용했다. </li><li> 특정 눈꺼풀 세그먼트와 신경 세포를 선택하는 이미징합니다. 세 가지 사전 자극 공 촛점 Z - 스택을 획득. <ol><li> 이상적으로, 사용 취득 설정이 여전히 셀 전체 볼륨을 캡처하는 동안 최대 시간 해상도를 허용하기에 충분히 빠르다. 예를 획득 설정은 다음과 같습니다 매 2 초를 캡처 × 2 2, 6 × 0.5 μm의 2 광학 부분을, 비닝 200 밀리 초 노출. 주 : 고정화 다리는 GCaMP 신호를 식별 할 수있는 표백으로 30 초 간격으로 최대 10 분 동안 성공적으로 묘화하고있다. 긴 녹화 시간의 경우, 다리가 커버 슬립의 위치에 아주 단단히 고정되어 있는지 확인합니다. </li><li> 높은 신호대 전체 IMA 동안 최소 광표백로 달성을위한 레이저 출력을 최적화정권 카. 여기에 설명 된 실험을 위해, 영상 30 %의 전송에 다리와 코에 Gr68a 및 ppk23의 미각 신경 세포를 모두 491 nm의 다이오드 레이저 (100 mW의)를 사용합니다. 여전히 충분한 공간 해상도를 달성하면서 짧은 노출 시간을 허용하는 2 × 2 비닝 (binning)을 사용합니다. 주 : 레이저 출력 설정은 1.2 내지 4.5 배 증가의 범위에서 형광 변화의 검출을 허용하도록 최적화되었다. </li></ol></li><li> 피펫 눈꺼풀 세그먼트 상에 자극 용액 10 μL. 제어 실험을 위해, PBST의 또 다른 10 μl를 추가합니다. 즉시 117 이후 자극 이미지를 수집. 참고 취득한 후 자극 매수의 형광의 최대 변화에 도달 한 시간의 양 (약 2 분)에 기초한다. <ol><li> 중요한 단계 :이 앞다리 위치에서 이동하게하기 때문에 준비에 솔루션을 피펫 팅하는 동안, 피펫 팁으로 비행을 터치하지 않습니다. 참고 : 또는 사용정확하게 눈꺼풀 부분에 자극 가까이를 포함하는 유리 모세관을 배치하는 미세 조작기는 이미지화된다. 제조자의 지시에 따라 세포 마이크로 인젝션 디스펜서를 이용하여 자극의 볼륨 조절을 제공한다. </li></ol></li></ol> 4. 이미지 처리 및 분석 : 응답의 정량화 <ol><li> 피지 (사용 공 촛점 Z - 스택의 일련의 열기 <a href="http://fiji.sc/Fiji" target="_blank">http://fiji.sc/Fiji</a> ) 23 또는 ImageJ에 ( <a href="http://imagej.nih.gov/ij/" target="_blank">http://imagej.nih.gov/ij/</a> ) (24) 이미지 분석 및 처리 소프트웨어를. </li><li> 120의 시점마다 여섯 Z 슬라이스의 합계 강도 투영을 만든다. <ol><li> &quot;이미지&quot;에서 &quot;스택&quot;옵션을 선택합니다. 또한, &quot;Z 투사&quot;옵션을 선택하고 정지 슬라이스로 시작 슬라이스 및 &quot;4&quot;로 &quot;1&quot;을 입력합니다. 프로젝션 타입의 경우, R을 선택20; 합 조각 모든 시간 프레임을 &quot;풀다운 메뉴에서, 그리고 선택&quot; &quot;. </li></ol></li><li> 셀 몸이나 신경 돌기 주위에 경계를 그립니다 자유 선택 도구를 사용하여 관심 (ROI)의 영역을 정의합니다. 은 &quot;분석&quot;레이블을 클릭하고 &quot;도구&quot;에서 &quot;투자 수익 (ROI) 관리자&quot;옵션을 선택합니다. </li><li> 은 &quot;분석&quot;메뉴 아래의 &quot;설정 측정&quot;옵션을 선택하고 통합 밀도, 지역의 상자를 선택하여 투자 수익 (ROI)의 형광을 정량화 평균과 최대 회색 값과 레이블입니다. 그 후, 투자 수익 (ROI) 관리자 메뉴에서 &quot;추가&quot;옵션을 선택하고, 또한 &quot;멀티 측정&quot;옵션을 선택합니다. 참고 :이 선택한 각 매개 변수에 대한 값을 팝업 상자를 생성합니다. </li><li> 화소의 중에 포화 도달 했으므로되도록하여 최대 계조 값을 확인 (예를 들어, 16 비트의 카메라 65535). 참고 : 실험을 위해 t에 표시그의 종이, 응답은 ΔF / F의 최대 증가가 관찰되었다 위치에 따라 세포 기관 또는 신경 돌기에서 어느 측정 하였다. </li><li> 다음과 같은 시점 t에서 형광 (ΔF / F)의 최대 상대적인 변화를 계산이다 : <img alt="식 (1)" src="/files/ftp_upload/53392/53392eq1.jpg" /><ol><li> 각 F (셀) 값에서 빼기 배경 형광. 배경 형광을 정량화하기 위해, 감지 뉴런없는 앞다리 (또는 코)의 영역에서 동등한 크기와 모양의 ROI의 통합 밀도를 측정한다. </li><li> 포스트 자극 형광 F (셀) t 정량화 자극 후의 시각 &quot;t&quot;에서 전지 본체 또는 프로세스의 ROI의 집적 밀도를 측정한다. </li><li> 예비 자극 형광을 정량하기 위해, F (셀) t = 0, 동일한 방법으로 상기 전지 본체 또는 프로세스의 사전 자극 이미지를 분석포스트 자극 신호. F (셀) t = 0을 결정하기 위해 3 이미지의 평균 집적 밀도를 사용합니다. </li></ol></li></ol>

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Biol. 19, 1245-1254 (2009).</li><li>Bray, S., Amrein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+putative+Drosophila+pheromone+receptor+expressed+in+male-specific+taste+neurons+is+required+for+efficient+courtship.">A putative Drosophila pheromone receptor expressed in male-specific taste neurons is required for efficient courtship.</a> Neuron. 39, 1019-1029 (2003).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat. Methods. 9, 676-682 (2012).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9, 671-675 (2012).</li><li>Kaissling, K. E., Zack Strausfeld, C., Rumbo, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+processes+in+insect+olfactory+receptors.+Mechanisms+and+behavioral+significance.">Adaptation processes in insect olfactory receptors. Mechanisms and behavioral significance.</a> Ann. N. Y. Acad. Sci. 510, 104-112 (1987).</li><li>Marder, E., Bucher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+pattern+generators+and+the+control+of+rhythmic+movements.">Central pattern generators and the control of rhythmic movements.</a> Curr. Biol. 11, 986-996 (2001).</li><li>Koepsell, K., Wang, X., Hirsch, J. A., Sommer, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+function+of+neural+oscillations+in+early+sensory+systems.">Exploring the function of neural oscillations in early sensory systems.</a> Front Neurosci. 4, 53 (2010).</li><li>Berridge, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+trisphosphate+and+calcium+signalling.">Inositol trisphosphate and calcium signalling.</a> Nature. 361, 315-325 (1993).</li><li>Busch, K. E., 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=Tonic+signaling+from+O(2)+sensors+sets+neural+circuit+activity+and+behavioral+state.">Tonic signaling from O(2) sensors sets neural circuit activity and behavioral state.</a> Nat Neurosci. 15, 581-591 (2012).</li></ol>

The authors declare that there are no competing financial interests.

우리는 여기에 2 개의 다른 감각 기관에 초파리 주변 신경 세포의 라이브 칼슘 이미징을 수행하는 방법을 설명합니다. 칼슘 2 +는 CH503은 용량 의존적 양적했다 페로몬 리간드에 의해 유도 Gr68a - 뉴런에서 GCaMP 형광 반응을 -evoked. 이 같은 phasic와 토닉 응답 등의 다른 신경 응답 패턴을 식별하는 것이 가능했다. phasic 반응을 나타내는 신경 연속 자극에 빠른 적응?...

동물들은 생존과 재생에 필수적인 의사 결정을 중재하는 후각과 미각 정보에 의존합니다. 감지 신경계에 의해 처리하는 방법을 화학 감각 단서 이해하는 감각 수용체 (들) 및 해당 화학 리간드의 확인이 필요합니다. 초파리는 휘발성 및 비 휘발성 화합물의 엄청난 다양성을 감지하고 생리를 연구하기에 좋은 모델입니다 메커니즘은 chemosensation을 기본. 후각 기관은 휘발성 분자를 감​​지하는 동안, 미각 기관은 낮은 휘발성 화합물을 검출하는 전문으로하고 있습니다. 여기서는 직접 저 휘발성 친 리간드 초파리 melanogaster의 맛 기관에서 신경 반응을 측정하는 방법을 제시한다. 파리의 미각 기관은 앞다리, 코, 날개 등이 있습니다. S 응답 sensilla 알려진 모발 형 구조는 미각 기관의 표면에 분포ugars, 쓴 맛, 소금, 물, 페로몬 8. sensilla 맛 강모으로 형태 학적으로 분류 된 맛은 9 쐐기. 긴 (L 형)으로 분류 된 labellum에 약 31 맛 강모, 짧은 (S 형) 및 중간 (i 형) 모폴로지가 있습니다. 설탕에 생리 학적 반응하는 &#39;L&#39;과 &#39;의&#39;sensilla 집 4 감각 뉴런 (S 셀), 저염합니다 (L1 세포), 높은 소금과 쓴맛 화합물 (2 계층 셀) 및 물 (승 전지) (10) 11. 다른 응답 동안 높은 소금 (12)에 낮은 소금과 설탕에 모두 응답 하나의 &#39;난&#39;sensilla 집이 감각 뉴런,. 남성의 약 41 맛 sensilla와 앞다리에 각각 분산 여성 26 sensilla이 있습니다. 둘 다 남성과 여성의 경우, midleg 21 sensilla 및 뒷다리 (13)에 대해 22 sensilla있다. 다리에 맛 sensilla로 둘러싸인 미각 신경 세포도 다 있습니다L1, L2, W 및 S 타입으로 lassified. 단일 뉴런에서 전기적 활성도를 측정하기위한 하나의 표준 방법은 이온 흐름을 기록하는 세포 또는 세포의 전극을 사용한다. 전극 측정은 신경 기능은 초파리 종, 나방, 신경 라벨링에 대한 광범위한 유전 적 도구가 부족 꿀벌로 비 모델 생물에서 공부 할 수 있습니다. 전기 생리 학적 방법을 일상적으로이 방법을 적용 4,13,14 sensilla 초파리 맛의 활동을 측정하는 데 사용되었지만 그러나, 여러 기술적 과제를 제시한다. 먼저, 칫솔모가 형태학 및 공간적 위치에 기초하여 식별되어야 맛. 식별되면, 전기 생리 학적 측정은 sensilla의 작은 크기에 의해 방해 될 수 의한 위치로 액세스 가능성을 제한 및 맛 화학적 자극 제어 된 양을 적용하는데 어려움이 모. 또한 sensilla의 자극은 하나 이상의 신호를 생성 할 수있다신경 세포 유형 15. 둘째, 전기 신호의 검출은 전자 기기로부터의 기계적인 진동과 노이즈로 인한 배경 잡음에 의해 혼동 될 수있다. 잘못 사용되는 경우 14 번째, 날카롭게 전극의 사용은 플라이 제조를 손상 할 수있다. 마지막으로, 전기 생리학 장비를 조립하는 자극 전달, 신호 기록 및 데이터 분석을위한 특수한 전자 부품을 필요로하고 비용이 많이들 수있다. D.에서 melanogaster의 유전자 인코딩 툴의 가용성은 뉴런의 작은 집단의 반응을 조사 할 수 있도록 영상 기술의 발달을 촉진 하였다. 하나의 이러한 접근은 CaLexA 기자 (16)의 사용이다. 이 방법에서는 LEXA-VP16 전사 인자 및 칼슘 민감성 NFAT 단백질을 코딩하는 유전자 서열은 융합된다. 단백질 포스파타제 칼시 뉴린의 칼슘 활성화 차례, facilita에서 NFAT의 탈 인산화를 촉진하고핵으로의 수입을 TES. 핵 내부에서 LEXA 도메인 따라서 기능적으로 활성화 된 뉴런의 지속적인 식별을 허용, 녹색 형광 단백질 (GFP) 기자의 발현을 지시하는 LexAop-DNA 결합 모티프에 결합한다. 이 접근법은 취기 (16)에 라이브 파리 노광 다음 더듬이 로브 특정 후각 사구체의 응답을 측정하기 위해 성공적으로 사용되어왔다. 최근 IR52c 미각 수용체 신경의 생리적 반응은 CaLexA 7을 사용하여 실시간 파리에서 측정 하였다. 최대 6 주 GFP의 전사를 향상시키기위한 그러나,이 연구를 위해,이 나이에 파리에 필요했다. 항 GFP 항체로 면역 염색하면 CaLexA 신호를 강화하는데 사용될 수 있지만,이 방법에 따라서, 라이브 세포 이미징을위한 가능성을 배제 조직의 고정을 필요로한다. 유 전적으로 인코딩 된 칼슘 표시기 단백질 GCaMP는 광범위의 숫자에 신경 반응을 연구하는 데 사용되었습니다종 17. 단백질 GCaMP은 신경 자극하기 전에 낮은 강도와​​ 형광. 자극의 적용은 칼슘의 유입의 결과로 신경 세포의 활동 전위를 트리거합니다. 칼슘 결합 GCaMP, 그것은 밝게 강도 (그림 1)과 형광 원인, 구조적 변화를 겪는다. 최근에 개발 된 단일 뉴런 칼슘 촬상 방법은 앞다리 특정 Gr61a의 미각 신경 (18)에 대한 리간드로서 포도당을 식별하는 데 사용 하였다. 본 연구에서는 Gr61a의 미각 뉴런 GCaMP 발현 형질 전환 초파리 해부 앞다리는 촬상 전에 아가의 층으로 덮었다. 그러나, 해부 조직의 사용에 따라서 측정 시간 및 검출 감도를 제한 GCaMP 발현의 최종 런을 일으킬 수있다. 또한, 아가로 인해 높은 형광 배경 레벨 및 광 산란 특성에 감도를 제한 할 수 있습니다. 티ㅇ 이러한 단점들을 해결, 우리는 앞다리와 코 그대로​​ 동​​물의 미각 신경 세포의 생리적 반응을 기록하는 GCaMP 매개 칼슘 이미징의 사용을 설명합니다. 우리는 초파리 지질 페로몬 유 전적으로 인코딩 GCaMP5G 19 발현 생리 Gr68a의 반응과 ppk23 뉴런 (3 R, (11) Z 19 Z) -3- 아세 톡시 11,19-octacosadien -1- 올 (CH503) 20 표시 21. 신경 반응은 페로몬 자극 동안 GCaMP5G 형광 신호의 증가를 정량함으로써 측정된다. 이 프로토콜에서, 신경 세포에서 신경 개별 활성화 패턴을 구별하기에 충분한 120 초 총 지속 기간 동안 영상화된다.

<table>
	<tbody>
		<tr>
			<td>Gr68a-Gal4</td>
			<td></td>
			<td></td>
			<td>Gift from H. Amrein (Texas A&#38;M Health Science Center, TX, USA) and J. Carlson (Yale University, CT, USA)</td>
		</tr>
		<tr>
			<td>ppk23-Gal4</td>
			<td></td>
			<td></td>
			<td>Gift from K. Scott (Univ. of California, Berkeley, CA, USA)</td>
		</tr>
		<tr>
			<td>UAS-GCaMP5&#160;</td>
			<td></td>
			<td>42037</td>
			<td>Bloomington Drosophila&#160; Stock Center</td>
		</tr>
		<tr>
			<td>0.17 mm coverslip (Gold-Seal coverslip)</td>
			<td>Electron Microscopy Services</td>
			<td>63790-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish, &#34;Hard as Nails Clear&#34;&#160;</td>
			<td>Sally Hansen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Z377821</td>
			<td></td>
		</tr>
		<tr>
			<td>Paint brush</td>
			<td></td>
			<td></td>
			<td>fine-tipped brush</td>
		</tr>
		<tr>
			<td>Tape&#160;</td>
			<td>Scotch brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>13021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, lab grade</td>
			<td>Merck</td>
			<td>10094</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane, HPLC grade</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>H303SK-4</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>PBST</td>
			<td></td>
			<td></td>
			<td>Recipe described in the protocol section</td>
		</tr>
		<tr>
			<td>CH503</td>
			<td></td>
			<td></td>
			<td>Synthesis described in Mori et al., 2010</td>
		</tr>
		<tr>
			<td>sCMOS Camera (ORCA Flash4.0)</td>
			<td>Hamamatsu&#160;</td>
			<td>C11578-22U</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (Ti-Eclipse)</td>
			<td>Nikon</td>
			<td>Ni-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning Disk Scan head&#160;</td>
			<td>Yokogawa</td>
			<td>CSU-X1-A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquistion Software (MetaMorph Premier)</td>
			<td>Molecular Devices</td>
			<td>40002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td>open source</td>
			<td></td>
			<td><a href="http://fiji.sc/Fiji">http://fiji.sc/Fiji</a></td>
		</tr>
	</tbody>
</table>

measuring physiological responses of drosophila sensory neurons to lipid pheromones using live calcium imaging

포유 동물과는 달리, 이러한 초파리와 같은 곤충은 여러 맛 기관이있다. 다리, 코, 날개 초파리의 산란관의 화학 감각 뉴런 미각 수용체 1,2, 이온 채널 3-6, 휘발성 및 비 휘발성 감각 큐의 검출에 참여하는 이온 성 수용체 (7)를 표현한다. 이 신경 세포는 직접 식품, 유해 물질 및 페로몬 등 tastants 문의 따라서 이러한 공급, 달걀 누워와 결합 많은 복잡한 행동에 영향을 미친다. 전극 기록 칼슘 촬상 널리 이러한 tastants 의해 유발 된 신경 세포 반응을 정량화하는 곤충에 사용되어왔다. 그러나, 전기 생리학은 세포 유형, 크기 및 위치에 따라 기술적으로 어려울 수있는 하나의 맛 sensillum 특화된 장비 및 구하는 측정을 필요로한다. 또한, 초파리에서 단일 신경 세포 해상도는 맛 sensilla 허우 때문에 달성하기가 어려울 수 있습니다화학 감각 신경 세포의 자체 이상의 유형입니다. 여기에 설명 된 실시간 칼슘 이미징 법 라이브 파리 단일 미각 뉴런의 반응을 측정 할 수있다. 이 방법은 수성 용매에 대한 낮은 용해도를 지질 페로몬 및 기타 리간드 유형의 연결 반응을 영상화에 특히 적합하다.

GCaMP 칼슘 표시기는 유 전적으로 Gr68a-GAL4 또는 ppk23-GAL4 드라이버를 사용하여 표현 하였다. 앞다리 신경 세포와 비 신경 지원 세포의 고유 한 인구는 각 드라이버 (그림 3A-C)에 의해 표시된다. 친 유성 페로몬 CH503 리간드에 특이 적 반응 Gr68a-GAL4 관찰하고 ppk23-GAL4 세포 GCaMP (도 3D)을 표현. 상대적인 GCaMP5G 신호 (ΔF / F)의 형광 강도의 변화는 CH...

Watch this Scientific Journal Video about Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging at JoVE.com

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

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

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

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

measuring-physiological-responses-drosophila-sensory-neurons-to-lipid

Research

JoVE Journal

Immunology and Infection

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

8.5K 조회수.  Temasek Life Sciences Laboratory. The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone. 

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

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

전체 리프 이미징을 사용하여 박테리아 세포를 라이브 Stomatal 응답을 평가

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

연구

면역학 및 감염병학

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

감염된 마우스에 세균 하중과 면역 응답 측정 리스테리아 monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

라이브 셀 이미징 바실러스 subtilis과 연쇄상 구균 pneumoniae 자동 시간 저속 현미경을 사용하여

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

라이브 세포 형광 현미경을 사용하여 박테리아 독소 유도 응답의 시각화

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

Alphaherpes 바이러스 선행 성 전송 및 확산의 라이브 셀 이미징

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

파지 Phenomics : 생리 학적 접근 방법은 소설 바이러스 성 단백질의 특성을

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

<em&gt; 체외</em&gt; HIV 공동 감염의 맥락에서 말라리아에 면역 반응을 측정하기위한 모델

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

방법은 지질 호중구의 변경 및 호중구 세포 외 트랩의 후속 형성을 연구하기 위해

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

사용 하 여 MDCK SIAT1 세포 분석 실험 측정 인플루엔자 항 체 응답 Microneutralization에 의해 인간의 세라에 A(H3N2) 바이러스를 중화

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.