I thank Dr. Maureen Barr, under whose supervision the dauer IL2 remodeling phenotype was initially characterized. Thanks to Dr. C. Britt Carlson for critical reading of the manuscript. Funding in the Barr lab was from the NIH (5R01DK59418) and postdoctoral fellowships from the USDA (2010-65106-20587) and the New Jersey Commission on Spinal Cord Research (CSCR12FEL004). Current funding for this work is from a University of Illinois Urbana-Champaign College of ACES FIRE grant.

1 원유 영속 페로몬 생산 <ol><li> OP50 E. 성장 하나의 식민지 O에서 / N 37 ° C에서 대장균은 200 rpm에서 LB 배지 100 ㎖에 떨고있다. 참고 :이 O / N 문화가 사전에 4 ° C에 저장할 수 있습니다. </li><li> N2 C. 성장 엘레 15 - 20 OP50 E.의 50 μL 시드 NGM 한천 (표 1의 레시피 참조) 6cm 배양 접시 대장균 박테리아까지 거의 19 고갈된다. </li><li> 1 L 삼각에 S 미디어의 5 배 250 ㎖에 (표 1의 레시피를 참조하십시오) 확인은 19 플라스크. 참고 :이 솔루션은 사전에 할 수있다. </li><li> OP50 E.의 문화를 성장 E.에서 OP50의 1 ㎖로 접종하여 LB 국물 O의 4 L / N에서 대장균 대장균 및 16 시간 동안 37 ° C에서 떨고. </li><li> (S 기저 용액 (표 1의 제조법 참조) 및 단계 1.3에서 제조 된 S 미디어 4 일 L 삼각 플라스크 각각에 4 ~ 5 판에서 전송 선충 1 ㎖와 함께 접시 (단계 1.2)에서 4를 선충을 씻으 5 판각 실린더 용) 19. </li><li> 10 분 동안 650 XG에서 단계 1.4에서 OP50을 원심 분리기와 뜨는을 제거합니다. S 기저 용액 10 ㎖ 중의 각 세균 펠렛을 재현 탁. 선충의 각 플라스크에 재 부유 세균의 같은 양을 전송합니다. </li><li> 이 솔루션은 음식의 고갈을 나타내는 취소 시작할 때까지 5 일 - (- 25 ° C에서 20) 100 - 4 150 rpm으로 실온에서 진탕에 플라스크를 배치합니다. </li><li> 음식이 고갈되면, OP50 E.의 또 다른 O / N 문화를 성장 LB 국물의 4 배 1 L에 대장균. 10 분 동안 650 XG에서 박테리아를 원심 분리기 및 선충류와 S 미디어 플라스크에 각각 한 플라스크에서 펠렛을 추가합니다. 음식이 다시 소진 될 때까지 추가 3~4일를 들어, 단계 1.7에서와 같이 진탕에 S 미디어 플라스크를 돌려줍니다. </li><li> 각 플라스크 용액의 ~ 1 ㎖의 분취 량을 가지고 각 1 ㎖의 분취에서 1 μl를 서브가 샘플링 및 선충의 수를 계산하여 선충의 농도를 추정한다. 참고 :75,000 선충 / ㎖ - 선충 인구는 50,000 있어야합니다. 이 농도에서 선충 대부분 dauers이다. </li><li> 오염 된 플라스크를 폐기하십시오. 참고 : 오염 자주 단계 1.9 동안 본 곰팡이 균사로 나타난다. </li><li> 4 ° C에서 10 분 동안 4,000 XG에 원심 분리기 선충. 선충 펠렛을 버리고 상층 액을 유지합니다. 대안 적으로, 낮은 - 역가 페로몬을 만들기 위해 동물을 사용하여 이들에 대한 설명을 참조. </li><li> # 1 와트 만 여과지를 통해 뜨는을 필터링합니다. 주 : 진공 플라스크에이 과정을 촉진 할 것이다. </li><li> 또한 0.45 μm의 멸균 진공 여과 장치를 통해 뜨는을 필터링 할 수 있습니다. 참고 : 여과 액은 4 ° C에서 O / N을 유지 또는 1.14로 진행 할 수 있습니다. </li><li> 교반 막대와 2 L 비이커에 여과 액을 추가합니다. 가열 볶음 접시에 흄 후드에 배치합니다. 교반하면서 끓기 약 200 ㎖의 증발. 500 ㎖의 비이커에 용액을 전송. 약 50 ㎖를 제작 때까지 끓는 계속피트 NOTE : 용액 위에 비등하는 것이 가능한 한 용품은이 단계에서 수행되어야한다. 50 ml의 최종의 색은 어두운 갈색이다. 조리 과정 중에 언제든지 열이 해제 될 수 있고, 여과 액을 RT 또는 4 ℃에서 O / N을 기억. </li><li> g 차가운하자 X 1000에서 5 분 동안 원심 분리. 100 ㎖의 비이커에 뜨는을 붓고 펠렛을 폐기합니다. </li><li> 갈색 빵 껍질에 뜨는을 졸이다. 열에서 제거하고 주걱 지각을 휴식. 지각을 커버하기에 충분한 200 증거 에탄올을 추가합니다. 파라 필름으로 비이커를 커버하고 RT에서 O / N 앉아 보자. 깨끗한 비커에 에탄올을 붓고 옆에 넣어. 지각을 커버하는 신선한 에탄올을 추가합니다. O / N의 추출을 3 회 반복합니다. 추출물을 풀입니다. </li><li> 에탄올 추출물을 건조 진공 펌프를 사용합니다. 진공 펌프를 사용할 수없는 경우 흄 후드에서 교반하면서, 50 ° C로 깨끗한 비커에 따뜻한로 에탄올 추출물을 추가합니다. 참고 : 에탄올 리터의 끈끈한 잔류 물을 증발되면광명 브라운 컬러는 유지되어야합니다. 가열이 사용되는 경우,주의가 너무 오랫동안 가열하여 용액을 구울주의해야한다. </li><li> 멸균 증류수 10ml에 잔류 물을 용해. 필터는 1 ㎖의 분액에서 -20 ° C에서 0.22 μm의 주사기 필터 및 저장이 솔루션을 소독. 참고 : 물 1 ㎖의 원액 선충 문화의 100 ML에 대한 잔류 물을 용해하는 데 사용됩니다. 액체 배양 플라스크 중 하나는 오염으로 인해 폐기되는 경우, 예를 들어, 다음, 잔사를 용해하는 데 사용되는 물의 양을 조절한다. </li></ol> 페로몬 복용량 응답의 2 결정 <ol><li> 펩톤없이 노블 한천로 NGM 미디어의 6 배 5 mL를하여 영속 페로몬을 준비하고, 50 / ㎖ 스트렙토 마이신, 황산 μg과 영속 페로몬의 범위와 보충. 4, 19 (표 1에서 제조법 참조) 13x100 mm의 구성 요소를 믹스 유리 문화 튜브, 분젠 버너를 통해 종기, 다음 3cm 페트리 접시에 붓는다. </li><li> 오전의 무게를icrocentrifuge 관. E.의 O / N 문화의 한 ML을 추가 microcentrifuge 관에서 ~ 17,000 XG에 튜브와 원심 분리기에 대장균 OP50. 상층 액을 모두 제거합니다. 다시 튜브를 달아 세균 펠릿의 중량을 결정한다. 4 %의 최종 농도를 위해 M9 완충액 (표 1의 레시피를 참조) 펠렛을 재현 탁 (/ w v). 나누어지는 각 페로몬 판 (4)의 중심 상에 세균 현탁액 10 μL. 저장 판 O / RT에서 N. </li><li> 페로몬 판 (4) 상에 성인으로 탈피 한 후 10 ~ 15 임신하는 N2 야생 형 자웅 동체에게 일일를 선택합니다. 4 시간 - 3 25 ° C에서 번호판을 저장합니다. </li><li> 성인 자웅 동체의 모든를 선택합니다. 각 접시에 누워 계란의 수를 기록한다. 파라 필름과 판을 밀봉하고 삼일 (17)에 대해 25 ° C로 돌아갑니다. </li><li> dauers의 비율을 계산한다 (그림 2). 참고 : 두 가지 방법의 경우는주의하는 것이 중요하다 dauers (그리고에 낮은 전십t 비 dauers)의 벽을 오르고, 페트리 접시의 뚜껑 위에 것이다. 이 선충에 대한 요리의 모든 표면을 검사하는 것이 중요하다. </li><li> 용량 - 반응 곡선의 데이터를 장착하고도 3에 도시 된 바와 같이 (dauers을 형성하기 위해 동물의 90 %를 유도하기에 충분한 페로몬) 페로몬의 EC (90)의 농도를 추정 참고. 우리는 보통 EC (90)을 추정하기 위해 로지스틱 회귀 모델을 사용 하지만 서로 다른 통계 소프트웨어 패키지는 유사한 추정치를 생산하는 다른 방법이있을 수 있습니다. </li></ol> 영속 IL2 뉴런의 3 라이브 영상 <ol><li> 단계 2.6에서 결정된 EC 90 값을 사용하여 프로토콜이 같이 영속 페로몬 판을 준비합니다. </li><li> 통합 IL2 리포터 유전자를 운반하는 균주 (myIs13 [P의 KLP-6 :: GFP] myIs14 [P의 KLP-6 :: GFP] myIs16 [P와 프로토콜이있는 방법을 사용하여 dauers을 유도 <em&gt; KLP-6 :: tdTomato] 또는 qIs56 [P 지연-2 :: GFP]) 12, 20, 21 참고 :. Q Is56를 사용하는 경우, GFP의 발현에 몇 시간까지 IL2Qs에 표시되지 않습니다 영속 탈피하고 초기 리모델링 이벤트 중 일부는 표시되지 않습니다. P 래그-2 : GFP 유전자에서 형광 신호가 P의 KLP-6 형광 리포터보다 영속 동안 IL2s 궁극적 밝게하면서 또한,이 P 래그-2 : GFP 유전자는 머리 근육에 약하게 발현되는 미세 IL2 분기에 마스크를 적용 할 수 있습니다. </li><li> 달걀 누워 다음, 성인 자웅 동체를 제거 파라 필름과 번호판을 봉인하고 25 ° C 17에서 34 시간 동안 배양한다. 참고 : 영속 탈피에 시간의 길이는 개인 사이에서 약간 다릅니다. 영속 탈피의 시작 5 시간 (39 - - 수상 돌기 arborization의 가장 빠른 기간은 4를 시작합니다 (44)시간 포스트 달걀 누워 25 ° C) 12시. </li><li> 촬영 전날, EC 90 농도의 영속 페로몬과 M9 버퍼에 10 %의 용액 (w / v)의 아가로 오스를 구성합니다. 나누어지는 아가로 오스의 약 100 μl의 현미경 슬라이드 상에 신속하게, 조심스럽게, 평면 아가로 오스 표면을 만들기 위해 상단에 다른 슬라이드를 배치합니다. 아가로 오스가 고형화 후, 습도 챔버 (젖은 종이 타월로 피펫 팁 상자)에 사란 랩 및 저장소에 슬라이드를 래핑합니다. </li><li> 34 시간의 잠복기 후, 인두 펌핑을 중지 선충에 대한 페로몬 판을 검사합니다. 개별 슬라이드 한 슬라이드 일면에만 그것에 아가 갖도록. 10 % 아가로 오스 패드와 0.1 미크론 폴리스티렌 비즈 22의 한 μ 리터에 하나 이상의 선충을 선택합니다. 참고 : 잘 동기화 인구, 접시에 선충의 대부분은 L2D에서 영속에 털갈이로 입력됩니다. 그러나, 나는 사이에서 변동이있을 수 있습니다ndividuals. 그것은 동물이 영속 탈피 내왔다 시간 (예를 들어 대표적인 결과 참조)의 양을 추정하기 위해 추가 기능 (인공 항문 폐쇄, 인두 리모델링)을주의하는 것이 중요합니다. </li><li> 선충의 방향을 (좌 / 우, 지느러미 / 복부)를 결정합니다. 가능한 최저 형광 강도와 100X 배율 매 15 분으로 IL2Q 덴 드라이트의 Z-스택 이미지를 캡처하거나 특정 실험에 필요한. (인두가 펌핑 시작) 선충은 L2D 표피에서 분리 될 때까지 캡처 이미지를 계속 동물이 영속을 복구하는 경우 고정이 불가능하거나. 적극적 묘화하지 않을 때, 습도 챔버에 슬라이드를 배치했다. 참고 : 우리는 전동 무대와 미분 간섭 대비 (DIC) 광학과 epifluorescent 현미경을 사용합니다. </li></ol>

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+chemosensory+control+of+dauer+formation+in+Caenorhabditis+elegans.">Genetic analysis of chemosensory control of dauer formation in Caenorhabditis elegans.</a> Genetics. 130, 105-123 (1992).</li><li>Vowels, J. J., Thomas, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+chemosensory+defects+in+daf-11+and+daf-21+mutants+of+Caenorhabditis+elegans.">Multiple chemosensory defects in daf-11 and daf-21 mutants of Caenorhabditis elegans.</a> Genetics. 138, 303-316 (1994).</li><li>Ailion, M., Thomas, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dauer+formation+induced+by+high+temperatures+in+Caenorhabditis+elegans.">Dauer formation induced by high temperatures in Caenorhabditis elegans.</a> Genetics. 156, 1047-1067 (2000).</li><li>Neal, S. J., Kim, K., Sengupta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pheromone Signaling. , 273-283 (2013).</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, 71-94 (1974).</li><li>Ouellet, J., Li, S., Roy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signalling+is+required+for+both+dauer+maintenance+and+recovery+in+C.+elegans.">Notch signalling is required for both dauer maintenance and recovery in C. elegans.</a> Development. 135, 2583-2592 (2008).</li><li>Blelloch, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gon-1+gene+is+required+for+gonadal+morphogenesis+in+Caenorhabditis+elegans.">The gon-1 gene is required for gonadal morphogenesis in Caenorhabditis elegans.</a> Dev. Biol. 216, 382-393 (1999).</li><li>Fang-Yen, C., Gabel, C. V., Samuel, A. D., Bargmann, C. I., Avery, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microsurgery+in+Caenorhabditis+elegans.">Laser microsurgery in Caenorhabditis elegans.</a> Methods Cell Biol. 107, (2012).</li><li>Nelson, F. K., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+study+of+the+Caenorhabditis+elegans+secretory-excretory+system+using+laser+microsurgery.">Functional study of the Caenorhabditis elegans secretory-excretory system using laser microsurgery.</a> J. Exp. Zool. 231, 45-56 (1984).</li><li>Sulston, J. E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-embryonic+cell+lineages+of+the+nematode,+Caenorhabditis+elegans.">Post-embryonic cell lineages of the nematode, Caenorhabditis elegans.</a> Dev. Biol. 56, 110-156 (1977).</li><li>Singh, R., Sulston, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+observations+on+moulting+in+Caenorhabditis+elegans.">Some observations on moulting in Caenorhabditis elegans.</a> Nematologica. 24, 63-71 (1978).</li></ol>

The authors have nothing to disclose

영속 별 신경 가소성 및 기타 영속 관련 형태 학적 변화를 검사 통제되고 하나 유전자 변이 (즉, DAF-C 돌연변이)를 통해 또는 페로몬 야생 형 동물의 노출에 의해 dauers의 안정적인 형성이 필요합니다. dauers를 유도하는 유전자 변이의 사용이 편리하지만, 그 결과를 혼란시킬 수있다. 따라서 DAF-C 돌연변이로 수집 된 데이터는 이후 영속 페로몬에 노출 영속 단계를 입력하도록 유도 야생?...

발달 및 행동 가소성을 포함 표현형 소성은 불리한 환경 조건에 적응하기위한 중요하고 진화 한 원동력으로 간주됩니다. C. 엘레 자주 개발 및 신경 생물학의 연구에 사용되는 모델 생물이다. 이상적인 조건에서, C. 엘레 성인 생식 단계에 들어가기 전에 사 애벌레 단계를 통해 개발하고 있습니다. 그러나, 낮은 음식 가용성과 높은 인구 밀도, C의 조건에서 엘레 발달 스위치를 받아야하고 수명이 긴 스트레스에 강한 영속 2 단계로 입력 할 수 있습니다. Dauers 가능성이 스트레스 저항을 중재 비 dauers에서 여러 형태 학적 및 행동의 차이를 표시합니다. 영속 항목을 규제하는 환경 단서 및 유전 적 경로가 광범위하고, 5, 4, 3 6, 7을 설명했다. 그러나, 각각의 표현형의 변화를 제어하는 분자 메커니즘을 거라고 발생하는uring의 영속 형성이 덜 잘 이해된다. 영속 특정 표현형 검사는 영속 동물의 생산을 필요로한다. 이 세 가지 방법으로 수행 할 수 있습니다 : 1) C의 결핍 문화 따기 엘레 간스, 2) 영속 형성 제정 (DAF-C) 돌연변이, 정제 페로몬을 통해 영속 형성 3) 유도의 사용. 그것은 C.있는 표준 NGM 플레이트에서 dauers을 선택하는 비교적 간단하다 그 박테리아의 식품 공급을 소진했다 elegans의 인구. 우리의 손으로, 표준 NGM 플레이트는 원래 삼일 성장이 허용 이후 3 접종 OP50 대장균의 50 μL, 시드 - 4 성인 자웅 동체는 일주 내에서 식량 공급을 배출하고 수천을 생산합니다 25 ° C로 유지 추가 3 일 이내 dauers (공복 수많은 비 dauers 이외에). 그러나,이 방법은 D로 탈피 중에 발생하는 검사 공정에 불만족AUER. 그것은 영속에 입력하는 일반적인 굶주린 접시에 수천의 동물을 결정하기가 어렵습니다. 또한, 고갈 플레이트의 사용은 동기화를위한 가능성을 제공하지 않는다. DAF-C 돌연변이 체의 사용은 동기화 및 dauers의 안정적인 생성을 허용한다. 그러나, 특정 DAF-C 돌연변이가 단독으로 또는 추가 돌연변이와 함께 다른 영속 특정 표현형에 영향을 미치지 않는다는 보장은 없습니다. 영속 페로몬의 존재는 첫째 Cassada 러셀에 의해 암시하고 나중에 황금과 수수께끼에 의해 입증되었다. 영속 페로몬은 이후 화학적, 열 9, 8,이 특징으로하고있다. 정제 된 페로몬은 다른 형태로 9, 원유 영속 페로몬 추출물 11. 사용이 가능 모두 행동 및 발달 과정을 조절 ascarosides의 복잡한 혼합으로 구성 야생형 백그라운드에서 동기화 dauers의 안정적인 제어 유도. </p&gt; 신경계 및 관련 아교 세포는 영속 유충 형성 12, 13, 14시 개장을받을. 이러한 변화 중 일부는 영속 13 일 14시 신경교 세포의 리모델링으로 되돌릴 수 있습니다. 그러나 다른 리모델링 이벤트가 유리한에 반환시 되돌릴 수 있습니다 환경 조건에 따른 것입니다. 수상 돌기 arborization, multidendritic 신경 세포와 축삭이 12 개조에 하나의 돌기에서 스위치 : 예를 들어, 네 IL2Q 뉴런의 집합을 포함 영속 형성하는 동안 신속하고 가역적 신경 리모델링을 거쳐. 방법은 여기에 모델 생물 스트레스에 의한 신경 가소성의 신뢰할 수있는 생체 내 이미징 할 수 있습니다. 원유 생산 및 영속 페로몬의 테스트를 위해 제시된 방법은 앞서 여러 소스 4, 8, 15, 16, 17, 18에서 설명한 컴파일된다.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">N2 C. elegans Bristol strain</td>
			<td style="width:152px;">Caenorhabditis Genetics Center</td>
			<td style="width:212px;"></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PT2660 myIs13[Pklp-6::gfp] III</td>
			<td style="width:152px;">Upon request from Schroeder lab</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PT2762 myIs14[Pklp-6::gfp] V</td>
			<td style="width:152px;">Upon request from Schroeder lab</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">JK2868 qIs56[Plag-2::gfp] V</td>
			<td style="width:152px;">Caenorhabditis Genetics Center</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">OP50 E. coli</td>
			<td style="width:152px;">Caenorhabditis Genetics Center</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes</td>
			<td style="width:152px;">Tritech Research</td>
			<td>60 mm T3308, 35 mm T3501</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="132">
			<td height="132" style="height:132px;">NGM agar</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">3 g NaCl, 17 g agar, 2.5 g peptone in 975 mL H2O autoclaved cooled to 55&#176;C followed by 1 mL of 5 mg/ml cholesterol in ethanol, 25 mL of 1 M KPO4 buffer (pH 6.0), 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4</td>
		</tr>
		<tr height="356">
			<td height="356" style="height:356px;">S Media</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">S media consists of S basal solution (1.46 g NaCl, 0.25g K2HPO4, 1.5 g KH2PO4, 0.25 ml of 5 mg/ml cholesterol in ethanol and H2O to 250 mL. Autoclaved.) To the S basal solution add 2.5 mL of potassium citrate pH 6.0 (2 g citric acid monohydrate, 29.4 g tri-potassium citrate monohydrate, H2O to 100 mL, autoclaved), 2.5 mL of Trace Metals Solution (1.86 g disodium EDTA, 0.69 g FeSO4&#160;&#8226;7 H2O, 0.2 g MnCl2&#8226;4 H2O, 0.29 g ZnSO4&#160;&#8226;7 H2O, 0.025 g CuSO4&#160;&#8226;5 H2O, H2O to 1 liter. Autoclaved and stored in the dark), 0.75 mL of 1 M CaCl2, and 0.75 mL of 1 M MgSO4.</td>
		</tr>
		<tr height="392">
			<td height="392" style="height:392px;width:259px;">Dauer pheromone plates for dose-response assay</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">Mix 0.085 g Noble agar, 0.5 mL of 0.513 M NaCl and 5 &#181;L of 5 mg/ml cholesterol dissolved in ethanol in each of 6x 15 mL culture tubes. Add sufficient crude pheromone to each tube to create a range of concentrations. Add H2O to bring the volume to 4.725 mL.&#160; Boil over a Bunsen burner until the agar is dissolved and then cool to 55 &#176;C. Add 125 &#181;L 1 M KPO4 buffer (pH 6.0), 50 &#181;l 0.1 M CaCl2, 50 &#181;L 0.1 M MgSO4 and 50 &#181;l of 5 mg/ml streptomycin sulfate to each tube.&#160; Pour the contents of each tube into a 35 mm diameter Petri dish. Let the agar solidify at room temperature overnight (if longer, store in enclosed container to prevent excessive drying).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsphere beads 0.1 micron</td>
			<td style="width:152px;">Polysciences Inc.</td>
			<td>00876-15</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;">M9 buffer</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">Mix 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 liter. Sterilize by autoclaving</td>
		</tr>
	</tbody>
</table>

in vivo imaging of dauer-specific neuronal remodeling in c. elegans

동물의 스트레스에 의한 표현형의 가소성을 조절 메커니즘은 생체 내에서 공부를 자주 복잡하고 어렵습니다. 스트레스에 의한 소성의 전형적인 예는 C의 영속 무대 엘레 간스. Dauers 박테리아 음식과 영속 페로몬의 높은 농도의 낮은 농도의 조건에서 형성된 대안 발달 애벌레 단계이다. Dauers 광범위한 발달 및 행동 가소성을 표시합니다. 예를 들어, 네 개의 내부 - 순측 사분면의 세트 (IL2Q) 뉴런 영속 형성 동안 광범위한 가역 리모델링을 겪는다. 대형 선충 액체 배양에서 원유 영속 페로몬의 생산을위한 잘 알려진 경로 환경을 미치는 영속 항목, 이전에 설정된 방법을 활용하는 것이 입증된다. 이 방법으로, 50000의 농도 - 배양액 75000 선충 / ㎖가 고도로 강력한 원유 영속 페로몬을 생산하기에 충분하다. 조 페로몬의 효능 dete입니다용량 반응 생물 검정법에 의해 rmined. 마지막으로, 영속 형성하는 동안 IL2Qs의 생체 시간 경과 영상에 사용되는 방법이 설명되어 있습니다.

더위와 추위 안정적인있는 노란색 액체의 원유 영속 페로몬 결과 (그림 1)의 생산. 원유 페로몬 일정량 활동의 명백한 손실 무기한 -20 ° C에 저장됩니다. 페로몬의 제조에 이어, 단일 용량 - 반응 생물 검정법의 효능은 대략적으로 충분하다. 3 회, 각 실험으로부터 평균을 취 - 그러나, 대부분의 실험에는 생물 검정 2를 반복 할 필요가있다. 이 페로몬 생물 검정에서 대표 결과는 그...

Watch this Scientific Journal Video about In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans at JoVE.com

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 ...

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

11.1K 조회수.  University of Illinois Urbana-Champaign. Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant ‘dauer’ juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer. 

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

마우스 망막 신경절 세포 Transfection로 생체내에 Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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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 ...

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 ...

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, ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

글루타민산 염과 GABA의 빠른 마이크로 이온 도입 : 시냅틱 통합을 조사하는 유용한 도구

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Super-resolution Imaging of Neuronal Dense-core Vesicles

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization.
Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

신경 밀도 코어 소체의 수퍼 - 해상도 이미징

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

결합 된 방법론을 활용하여 축삭 분기 및 신경 세포 형태 형성 동안 플라즈마 멤브레인 배달의 역할을 정의합니다

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

남성 선 충 C. 머리 신경에 있는 페로몬에 대 한 응답에서 신경 활동의 이미징에 대 한 적응된 미세 후 각 칩을 사용 하 여

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease.
C. elegans adult neurons that express aggregating proteins can extrude large (~4 &#181;m) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called &#8220;exophers&#8221; and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases.
While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons.
Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 &#181;m) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.