우리는이 원고의주의 읽기에 대한 스콧 해밀턴과 O&#39;Halloran 연구실의 구성원을 감사드립니다. 우리는 또한 우수한 제안과 통찰력 의견을 익명 리뷰어를 감사합니다.

1. GFP의 건설 EGL-4 표현 동물 태그 <ol><li> odr-3 유전자 (직접 상류 시작 코돈의 사용 2,678 BP)를위한 발기인 아래 병진 융합 GFP :: EGL-4를 복제 (P) odr-3 :: GFP :: EGL-4. odr-3 프로모터 드라이브 amphid 뉴런 쌍 표현 : AWA, AWB, AWC, 그리고 약하게 ASH 인치 </li><li> (P) ofm-1 : 플라스미드 (P) odr-3 :: GFP :: 50 wildtype (N2) 동물에 EGL-4 표준 세균 라인 변환 기술 8을 사용 공동 분사 마커와 μl / NG를 삽입 : : GFP 9 일 (μl / 25 NG) 및 (P) odr-1 :: dsRed 10 (25 NG / μl). AWC 및 AWB amphid 뉴런 쌍의 odr-1 프로모터 드라이브 표현하고 coelomocytes의 ofm-1 프로모터 드라이브 표현. </li><li> 울트라 바이올렛 (UV) / trimethylpsoralen (TMP) 통합 프로토콜 단계 1.2에 설명 된 extrachromosomal 배열을 표현 형질 전환 동물을 대상11. </li><li> 블리치는 동물 12 동기화하고 OP50 E.를 포함하는 선충류 성장 미디어 (NGM) 접시에 올려 배양 L4 단계에 대장균. OP50 E.를 제거하는 M9 버퍼로 NGM 플레이트에서 L4 동물을 씻어 대장균 박테리아. </li><li> M9 버퍼를 제거하고 30 μg / 1.5 ML의 microcentrifuge 관 웜 펠렛로 ML의 TMP 작업 솔루션의 50-100 μl를 추가합니다. 주변 빛을 차단하고, 호일로 싼 1.5 ML의 microcentrifuge 관에서 15 분 동안 회전하는 것에 부드럽게 활발히 논의하기 위해 호일에 1.5 ML의 microcentrifuge 관을 넣어. 큰 unseeded NGM 판에 TMP / 웜 솔루션에 웜을 전송합니다. </li><li> Stratalinker 2400을 사용하여 UV 350 μJ (X100)에 NGM 접시에 웜을 쉽게받을 수 있습니다. 그런 다음 OP50 포함 NGM 플레이트에 웜을 추가로 5 시간 동안 짙은 어둠 속 품다. </li><li> 약 10 시드 플레이트 (2-5 웜 각 판) 50 유전자 변형 웜을 선택합니다. 전송 P0 새로운 판에 3 일 동안은 24 시간입니다. </li><li> 250 clonal F1을 선택nimals (판 당 1 동물). 각 F1 판 2-4 F2 동물을 복제. ofm-1 (P)에서 :: 해부 형광 현미경으로 GFP 표현 패턴을 보면 100 % 전송 라인을 찾아 F3 동물을 확인합니다. </li><li> Outcross wildtype의 N2 동물과의 최종 통합 라인 (들) 5 회. 우리는 pyIs500으로 최종 통합 라인을 참조하고, GFP 등 GFP 태그 EGL-4 분자 :: EGL-4입니다. </li></ol> 2. 원자력 Translocation의 Assays을위한 재배와 동물의 유지 보수 <ol><li> 25 pyIs500 동물을 육성 ° OP50 E. 스탠다드 10cm NGM 플레이트에 C (제조법은 아래를 참조) 시드 대장균 박테리아 13 일 (그림 1). </li><li> 1 일에서 5 개의 10cm OP50 E.에 25 재배 플레이트 4-5 L4 pyIs500 동물 ° C를 선택 대장균은 25 접시 배양을 놓는 ° C (그 판 당 4-5 L4 동물입니다.) </li><li> 4 일에서 성인 P를 씻어S-자연적으로 흐르는 것과 버퍼를 사용하여 대형 NGM 플레이트에서 pyIs500 동물의 opulations (제조법은 아래를 참조). 그들은 플라스틱 피펫 팁의 측면에 집중하므로, 동물을 전송하는 일회용 유리 피펫을 사용합니다. </li></ol> 3. 장기 냄새 유발 원자력 Translocation의 Assays <ol><li> pyIs500 동물에게 세균을 제거 할 S-자연적으로 흐르는 것과 3 번 씻으십시오. 세차 사이에 동물을 원심 분리기하지 마십시오 아니라 동물 고정 1.5 ML의 microcentrifuge 튜브에 중력에 의해 정착 할 수 있습니다. 다 박테리아가 제거되어 있는지 확인하는 것이 중요합니다, 그 NGM의 pates는 잔여 박테리아가 부정적인 translocation 분석의 결과에 영향을 미칠 것이기 때문에, 오염에서 무료입니다. 또한, 우리는 1.5 ML의 microcentrifuge 튜브를 autoclaving하는 측면에 집중하기 위해 동물을 일으키는 등 비 autoclaved 1.5 ML의 microcentrifuge 튜브를 사용하는 관의 내부 벽에 &quot;끈적&quot;재산을 생산하는 것으로 확인되었습니다. </li><li> 동물이 세차 사이에 정착하고 있지만, t를 구성하는그는 적응 솔루션을. 100 ML의 졸업생 실린더에 S-자연적으로 흐르는 것과 버퍼의 100 ML을 추가합니다. S-자연적으로 흐르는 것과에게 냄새를 적응 추가 Parafilm의 스트립을 사용하여 실린더를 밀봉. butanone 적응이 S-자연적으로 흐르는 것과 100 ML에 2 butaone 11 μl를 추가에 대한 benzaldehyde 적응은, S-자연적으로 흐르는 것과 100 ML에 benzaldehyde의 7.5 μl를 추가합니다. 수행하는 경우 부드럽게 (흔들하지 않음)이 포함 된 졸업 실린더가 S-자연적으로 흐르는 것과과 냄새 30 회 균일 한 에멀젼을 만드는 반전. </li><li> S-자연적으로 흐르는 것과의 마지막 세척 후, 동물이 모든 액체를 해결하고 제거 할 수 있습니다. 라벨 한 튜브 &quot;+ VE&quot;또는 &quot;적응&quot;과 다른 튜브 &quot;VE&quot;또는 &quot;unadapt&quot;. 그런 다음 적응 microcentrifuge 튜브 (+ VE)에 적응 믹스 1 ML을 추가하고 unadapted 제어 microcentrifuge 관 (-VE)에 S-자연적으로 흐르는 것과 1 ML를 추가합니다. 각 튜브에 100와 200 사이의 동물의 숫자를 유지하려고합니다. 연습으로, 그것은 눈으로 펠릿에 동물의 대략적인 수를 추정 할 수 있습니다. </li><li> 1.5 ML microcentrifuge 튜브 캡 보호를 삽입각 튜브 (터지는 오픈에서 뚜껑을 방지)와 80 분의 회선 근에 튜브를 삽입합니다. 우리는 60 분 및 80 분 사이의 냄새에 노출 비슷한 결과를 (장기 적응 응답에서 두 결과) 산출 것으로 나타났습니다. 그러나, 80 분 노출 우리 손에 더 일관된 결과를 제공합니다. 따라서 우리의 모든 장기 적응 assays는 80 분 냄새에 노출 (그림 2)를 사용하여 표준화합니다. </li><li> 80 분 후, S-자연적으로 흐르는 것과의 동물에게 3 번 씻는다. 동물이 각 빨래 사이에 중력에 의해 해결 할 수 있습니다. </li></ol> 4. 단기 냄새 유발 원자력 Translocation의 Assays <ol><li> 단기 냄새 적응의 경우, 그러나 대신 80 분에 냄새를 적응 pyIs500 동물 잠복기 때문에, 30 분 노출을 사용하여 당 장기 냄새 유발 translocation의 assays (단계 3.1-3.4)와 같은 프로토콜을 사용 . 아래에 설명 된 악취 유발 핵 translocation의 assays의 정량화은 (5 단계) 장기 및 단기 적이에 대해 동일합니다m 노출. </li></ol> 5. 냄새 유발 원자력 Translocation 이벤트 점수를 <ol><li> DH 2 0에서 젤 전기 영동 아가로 오스를 용해하여 5 MM 나트륨 azide (할머니 3) 포함 2% 아가로 오스 패드를 확인합니다. 유리 현미경 슬라이드에 낸 세를 포함하는 용융 아가로 오스의 한 방울을 넣으십시오. 즉시 용융 아가로 오스의 드롭을 평평하게하는 최초의 슬라이드에 다른 유리 현미경 슬라이드를 놓습니다. 30 초에두고 부드럽게 현미경은 ~ 1 mm의 평면 아가로 오스 패드와 하나 현미경 슬라이드를두고 슬라이드 떨어져 애타게. </li><li> S-자연적으로 흐르는 것과의 마지막 세척 후, 웜은 1.5 ML의 microcentrifuge 관에 정착하고 모든 액체를 제거 할 수 있습니다. 그런 다음 몇 가지 현미경 슬라이드의 아가로 오스 패드로 드롭 현명한 동물을 추가합니다. 이 50과 100 사이에 슬라이드 당 동물의 번호를 유지하는 것이 중요합니다. 슬라이드 당 너무 많은 동물이 골을 복잡하고 종종 푸른 빛이 여기 뒤에 산란 빛이 생성됩니다. 초과 액체가 수도김 부 작은을 사용하여 아가로 오스 패드에서 악한합니다. 패드에 0.5 mm의 유리 커버 슬립을 놓고 동물을 고정하는 할머니 3 수 있도록 2 분에 서 보자. 참고 동물 채점 후 슬라이드에서 복구 할 수 있습니다. M9 버퍼 한 방울로 시드 NGM 플레이트로 전송하면 동물 ~ 1 시간 후 할머니 3 마취에서 회복됩니다. </li><li> 이 시점에서는 각 실험은 슬라이드 실험 (+ VE) 슬라이드하고있는 슬라이드 (-VE) 슬라이드 컨트롤입니다 추적 할 키를 고안하고, 맹목적으로 GFP를 점수를 다른 사람에게 슬라이드를 통과 필수적입니다 : EGL-4 현지화 패턴. 이 모든 실험 바이어스에 대해 적용됩니다. </li><li> , 10X 40X 63X 배율과 목표 및 GFP / RFP 필터를 수직 형광 현미경을 사용하여 슬라이드 당 50과 100 동물 사이의 점수. 첫째, 밝은 분야 조명을 사용하는 10X 배율에 따라 동물을 찾습니다. 둘째, brigh를 해제하여 AWC 뉴런을 찾습니다t-필드 조명 및 RFP 필터를 사용합니다. AWB 및 AWC 뉴런의 (P) odr-1 :: dsRed 드라이브는 표현. 모양의 작고 원형 아르 AWB 셀 기관 (그림 2)와 비교 AWC 뉴런은 독특한 타원형 모양의 세포 기관을 갖추고 있습니다. </li><li> AWC 전지 본체가 위치한되면, GFP 필터로 전환 GFP의 지역화 기록 : 핵이나 세포질로 EGL-4. 카운터를 사용하면 실험이 슬라이드를 골면서 접안 렌즈로 계속 찾고 있습니다. 참고 : 단계 5.1-5.5는 통계적으로 의미가있는 데이터 (그림 1-2) 생산 별도의 일에 최소 3 번 반복됩니다. </li></ol> 6. 장기 냄새 적응에서 복구하는 동안 EGL-4 GFP 태그 모니터링 <ol><li> 단계 3.5 이후 시간 포인트 제로에서 AWC의 여러 aliquots과 핵 대 세포질 GFP에 대한 점수 :: EGL-4에 pyIs500 동물의 인구를 분할 (즉 80 분 노출 후) 및 multiple 이후의 시간 점 (그림 3). </li><li> NGM 접시에 일회용 유리 피펫을 사용하여 컨트롤 (-VE) 및 실험 (+ VE) 동물을 모두 전송합니다. </li><li> 동물이 시간의 다양한 길이에 복구 할 수 있도록 각 시점에서 GFP :: EGL-4in AWC의 재검토 현지화 5.1 5.5 단계에 있습니다. </li></ol> 7. 자료 NGM 번호판 :; 3g의 나트륨 염화물 (NaCl) 2.5 g Bacto-펩톤 21g Bacto-한천 : 1 L에 대해 1 L ddH 2 0에 추가 할 수 있습니다. 압력솥 미디어. 온도는 54 ° C.를 읽고 autoclaving, 멋진 한천이 끝날 때까지 , 1 ML 1 M의 황산 마그네슘 (MgSO 4), 1 ML 1 M의 염화칼슘 (CaCl 2), 에탄올 1 ML의 콜레스테롤 (5 MG / ML 주식) 25 ML 1M 칼륨 인산 버퍼 다음 솔루션을 추가합니다. 1 M 이염 기성 K 2 HPO 4 174.2 g / 몰 : 500 ML를 들어 : 400 ML ddH 2 O에 용해 87.1 g DibasiC K 2 HPO 4. ddH 2 O. 500 L에 볼륨 조절 500 ML 무균 필터 유닛을 사용하여 소독 필터링 할 수 있습니다. 1 M 일 염기의 KH 2 PO 4 136.1 g / 몰 : 1 L의 경우 : 800 ML ddH 2 O에서 136.1 g 일 염기의 KH 2 PO 4 분해. ddH 2 O. 1 개 L로 볼륨을 조정 500 ML 무균 필터 유닛을 사용하여 소독 필터링 할 수 있습니다. 1 M의 칼륨 인산 (K 3 PO 4) 버퍼 산도 6.0 : 1 L의 경우 : 1 L 무균 필터 유닛에서 1 M 일 염기의 KH 2 PO 4 868 ML를 추가합니다. 단위를 통해 필터링 할 수 있습니다 다음, 1 M 이염 기성 K 2 HPO 4 132 ML를 추가합니다. 장치가 모든 액체를 필터링 할 수 있습니다. 필터 장치를 제거하고 6.0 산도를 조정합니다. 실온에서 보관하십시오. S-자연적으로 흐르는 것과 : 1 L의 경우 : 50 ML 100 K 3 PO 4 버퍼를 추가 산도 ~ 6.0; 20 ML 5M NaCl AQ. 1 개 L에 기입ddH 2 O. 솔루션을 압력솥. M9 : 3g KH 2 PO 4, 6g 오세영 2 HPO 4, 5g NaCl, 1 ML 1 M MgSO 4, H 2 O. : 1 L의 경우 ddH 2 0 1 개 L로 입력합니다. autoclaving하여 소독.

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C., 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=Efficient+gene+transfer+in+C.elegans:+extrachromosomal+maintenance+and+integration+of+transforming+sequences.">Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.</a> EMBO Journal. 10, 3959-39 (1991).</li><li>Miyabayashi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+function+of+members+of+a+divergent+nuclear+receptor+family+in+Caenorhabditis+elegans.">Expression and function of members of a divergent nuclear receptor family in Caenorhabditis elegans.</a> Dev. Biol. 215, 314 (1999).</li><li>L'Etoile, N. D., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfaction+and+odor+discrimination+are+mediated+by+the+C.+elegans+guanylyl+cyclase+ODR-1.">Olfaction and odor discrimination are mediated by the C. elegans guanylyl cyclase ODR-1.</a> Neuron. 25 (3), 575 (2000).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods Cell Biol. 48, 451 (1995).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> Wormbook. , (2006).</li><li>Brenner, 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+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71 (1974).</li><li>Kauffman, A., Parsons, L., Stein, G., Wills, A., Kaletsky, R., Murphy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+Positive+Butanone+Learning,+Short-term,+and+Long-term+Associative+Memory+Assays.">C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays.</a> J. Vis. Exp. (49), e2490 (2011).</li><li>Berkowitz, L. A., Hamamichi, S., Knight, A. L., Harrington, A. J., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+C.+elegans+Dopamine+Neuron+Degeneration+Assay+for+the+Validation+of+Potential+Parkinson's+Disease+Genes.">Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes.</a> J. Vis. Exp. (17), e835 (2008).</li><li>Pittenger, C., Kandel, 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=In+search+of+general+mechanisms+for+long-lasting+plasticity:+Aplysia+and+the+hippocampus.">In search of general mechanisms for long-lasting plasticity: Aplysia and the hippocampus.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 358 (1432), 757 (2003).</li></ol>

관심 없음 충돌이 선언 없습니다.

GFP 태그 EGL-4 분자의 냄새 유발 핵 항목은 여기에 설명하는 것은 C.에서 후각 적응의 강력한 분자 기록을 제공합니다 elegans. 냄새 유발 핵 translocation의 assays은 간단하고 준비 시간을 몇 일을해야합니다. 우리가이 assays에 건설 한 pyIs500 동물은 AWC 신경 세포뿐만 아니라 GFP 태그 EGL-4 단백질을 표현을 비춰 마커를 표현합니다. 따라서, 우리는 뉴런의 클래스로 작업없이 경험이 거의...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> Bacto 한천 </td><td> Difco </td><td> DF0140 - 07-4 </td><td> NGM 플레이트 </td></tr><tr><td> 나트륨 염화물 </td><td> 피셔 화학 </td><td> S671-10 </td><td> NGM 플레이트 </td></tr><tr><td> Bacto 펩톤 </td><td> Difco </td><td> DF0118-07-2 </td><td> NGM 플레이트 </td></tr><tr><td> 이염 기성 인산 칼륨 </td><td> 피셔 화학 </td><td> S375-500 </td><td> S-자연적으로 흐르는 것과 버퍼와 NGM 플레이트 </td></tr><tr><td> 인산 칼륨 일 염기의 </td><td> 피셔 화학 </td><td> P285-500 </td><td> S-자연적으로 흐르는 것과 버퍼와 NGM 플레이트 </td></tr><tr><td> Kimwipes - 작은 </td><td> 킴벌리 - 클락 </td><td> LS2770 </td><td></td></tr><tr><td> 에탄올 백퍼센트 </td><td> 골드 방패 화학(주) </td><td> 43196-115 </td><td> chemotaxis assays를위한 diluting 냄새 </td></tr><tr><td> 칼슘 염화물 </td><td> 시그마 - 알드리치 </td><td> C8106-500G </td><td> NGM 플레이트 </td></tr><tr><td> 마그네슘 황산염 </td><td> MP의 Biomedicals </td><td> 150136-500G </td><td> NGM 플레이트 </td></tr><tr><td> 나트륨 Azide 99% </td><td> 피셔 과학 </td><td> ICN10289180 </td><td> 마취제 </td></tr><tr><td> 아가로 오스 - UltraPure </td><td> Invitrogen </td><td> 16500-500 </td><td> 아가로 오스 패드 </td></tr><tr><td> Benzaldehyde </td><td> 시그마 - 알드리치 </td><td> B1334-100G </td><td> AWC 냄새 </td></tr><tr><td> Butanone, ACS 학년 </td><td> 시그마 - 알드리치 </td><td> 360473-500ML </td><td> AWC 냄새 </td></tr><tr><td> Microcentrifuge 튜브 - 1.5 ML 컬러 </td><td> Denville </td><td> LS8147 </td><td></td></tr><tr><td> 파스퇴르 Pipet 일회용 유리 5-3/4 &quot; </td><td> 피셔 과학 </tD&gt; <td> 13-678 - 20B </td><td></td></tr><tr><td> Stratalinker </td><td> Stratagene </td><td> Stratalinker 2400 </td><td> UV 통합 </td></tr><tr><td> 필터 진공 병 - 500 ML </td><td> Nalgene </td><td> 09-740 - 25B </td><td></td></tr></tbody></table>

a molecular readout of long-term olfactory adaptation in c. elegans

지속적인 자극하는 동안 대부분의 감각 뉴런은 신호의 감도를 감소하여 반응을 적응합니다. 적응 반응은 모양의주의를 할 수 있으며, 오버 자극 세포를 보호합니다. C.의 후각 회로에 적응 elegans 먼저 콜버트와 Bargmann 1,2에 의해 설명되었다. 여기에서 저자들은 Amphid 윙 셀 타입 C (AWC) 감각 뉴런에 의해 감지 휘발성 냄새에 적응하는 능력에 돌연변이가 결함 분리하는 유전자 화면을 설계하는 데 사용되는 후각 적응 패러다임의 매개 변수를 정의. 때 wildtype C. elegans 동물들은 냄새로 자신의 응답을 수정합니다 30 분에 매력적인 AWC - 감지 냄새 셋에 노출 된 후 ~ 1 시간의 chemotaxis 행동 분석에 적응 냄새를 무시합니다. 때 wildtype C. elegans 동물들은 후 AC에 적응 냄새를 무시합니다 ~ 1 시간을위한 매력적인 AWC - 감지 냄새에 노출되어~ 3 시간에 hemotaxis 행동 분석. C.의 후각 적응의 두 단계 elegans는 단기 후각 적응 (30 분 냄새에 노출 후 유도), 그리고 장기 후각 적응 (60 분 냄새에 노출 후 유도)로 설명했다. 나중에 L&#39; Etoile의 외.에서 일, 4 AWC 뉴런의 단기 및 장기 후각 적응 모두 필요합니다 EGL-4라는 단백질 키나제 G (PKG)을 발견. EGL-4 단백질은 장기 후각 적응 반응하지만 AWC 4 단기 후각 적응 응답을위한 소모품에 필요한 핵 현지화 과정이 포함되어 있습니다. 녹색 형광 단백질과 태그 EGL-4함으로써, 장시간 냄새에 노출시 AWC의 EGL-4의 현지화를 시각화 할 수했습니다. 이 모든 기능을 사용하여 우리가 AWC 5 장기 후각 적응의 분자 표시 장치를 개발 할 수 있었던 EGL-4 (GFP :: EGL-4) 분자 GFP를 태그. 이 m을 사용하여후각 적응의 olecular 판독 우리는 AWC 6,7에서 GFP의 결함이 subcellular 지방화 패턴 :: EGL-4을 전시 돌연변이 동물을 식별하는 모두 순방향 및 역방향 유전 화면을 수행 할 수있었습니다. 여기 설명 : GFP 1) 건설 :: EGL-4 표현 동물, 2) 장기 냄새 유발 핵 translocation의 assays를위한 동물의 재배를위한 프로토콜 및 장기적인 악취 유발 3) 채점 핵 translocation 이벤트와 핵 GFP :: EGL-4 상태에서 복구 (재 sensitization).

AWC 전, 연장 냄새에 노출 후 GFP :: EGL-4in의 지역화 패턴의 예는 그림 2에 표시됩니다. 장시간 악취 노출, GFP :: EGL-4 이전은 AWC (그림 2B)의 세포 기질에 지역화하고 있으며, 80 분 냄새 노출 GFP 후 :: EGL-4는 AWC (그림 2D)의 핵에 번역되어 있습니다. 행동 수준에서 cytosolic GFP :: EGL-4 AWC에있는 동물은 냄새의 포인트 소스 (- chemotaxis 지수 3 가까운 1 되오니, 이용?...

Watch this Scientific Journal Video about A Molecular Readout of Long-term Olfactory Adaptation in C. elegans at JoVE.com

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

여기에 장기 후각 적응의 분자 기록을 설명<em&gt; Caenorhabditis elegans</em&gt;. 단백질은 키나제 G, EGL-4는, AWC라는 기본 감각 뉴런 쌍의 안정적인 적응 응답이 필요합니다. 세포 기질의 AWC의 핵에 장시간 냄새에 노출 EGL-4 translocates 동안.

의 장기 강한 적응의 분자 판독<em&gt; C. elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

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JoVE Journal

Neuroscience

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

10.7K 조회수.  George Washington University. 여기에 장기 후각 적응의 분자 기록을 설명 Caenorhabditis elegans. 단백질은 키나제 G, EGL-4는, AWC라는 기본 감각 뉴런 쌍의 안정적인 적응 응답이 필요합니다. 세포 기질의 AWC의 핵에 장시간 냄새에 노출 EGL-4 translocates 동안.

비디오: A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

C. 엘레간스 긍정적인 Butanone 학습, 단기 및 장기 연관 메모리 Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

연구

신경과학

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

C로 생체내 레이저 Axotomy에서 엘레간스

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

에서 시냅틱 형광의 자동 부량<em&gt; C. elegans</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

C. elegans 추적 및 행동 측정

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.의 생체 Neuronal 칼슘 이미징에서 elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

자유롭게 수영 약하게 전기 생선의 장기 행동 추적

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

C.에서 영속 특정 신경 세포 리모델링의 생체 내 이미징에서 엘레

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

의 장기 칼슘 이미징을위한 아가로 오스 Microchambers<em&gt; 예쁜 꼬마 선충</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

의 자동 분석<em&gt; C. 엘레</emCeleST 소프트웨어를 사용&gt; 수영 동작

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

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 ...