1. 추적 현미경에 대한 설명 <ol><li> 한천 플레이트는 섬유 광원에 의해 조명과 카메라와 이미징 있습니다. 이 시스템은 X, Y 번역 단계에 장착된다. </li><li> 무대는 스테퍼 모터 컨트롤러에 연결되어있는 표준 스테퍼 모터에 의해 이동된다. </li><li> 컨트롤러와 카메라가 컴퓨터에 연결되어 LabVIEW로 작성된 사용자 정의 프로그램에 의해 제어됩니다. </li><li> 카메라는 이미지 한천 플레이트의 표면을과 가벼운 배경에 어두운 물체를 식별 할 수 있습니다. </li><li> 컴퓨터 프로그램이 실시간으로 객체를 수량화 할 수 있도록 이미지 화질이 조정됩니다. 게인, 밝기, 카메라의 셔터 속도는 흰색 배경에 검은 물체를 제공하기 위해 조정할 수 있습니다. </li><li> 자동 보정 과정에 대한 보정 마크는 웜 곡괭이로 한천 표면을 농담으로 구성되어 있습니다. </li><li> 추적 프로그램에 의해 사용되는 필터링, 바이너리 이미지를 확인하실 수 있습니다. </li><li> 그래서ftware는 테스트 개체에게 고정 된 거리를 이동하여 보정 행렬을 계산하는 자동 보정 기능이 있습니다. </li><li> 픽셀 거리가 보정 매트릭스하여 스테퍼 모터에 의해 촬영 단계로 조정됩니다. </li><li> 보정 한 후, 시스템은 갈 준비하고 배율이 변경되지 않는 한 또는 카메라 위치를 변경하는 경우 recalibrated 할 필요가 없습니다. </li></ol> 2. 추적 접시와 C. 준비 추적에 대한 elegans <ol><li> 구리 반지가 벌레를 마차를 둥글게 둘러 친 진과 접시의 가장자리로 마이그레이션에서 그들을 유지하는 데 사용됩니다. 구리는 지역 화학 장벽을 제공하며 실험 (&lt;1 시간)의 기간 동안 달리 웜의 움직임에 영향을주지 않습니다. 열 블록 또는 이와 동등한에 배치하여 먼저 링을 가열. </li><li> 신선한 한천 플레이트로 장소 링 (1.7 % Bacto 한천, 0.25 % Bacto-펩톤, 0.3 % NaCl, 1 MM CaCl 2, 1 MM MgSO 4, 25 MM 인산 칼륨 버퍼, 5 μg / ML콜레스테롤)과 한천 표면에을 삽입 할 약간 아래로 누르십시오. </li><li> 식품 잔류 물을 닦아야 일부 NGM 버퍼 (0.3 % NaCl, 1 MM CaCl 2, 1 MM MgSO 4, 25 MM의 칼륨 인산 버퍼)로 가득 한천 플레이트에 L4 단계 또는 젊은 성인 웜를 선택합니다. 웜은 몇 분 동안 수영을 보자. </li><li> 조심스럽게 링의 중심 근처의 추적 플레이트에 하나의 웜을 배치합니다. 그리고 웜 추적기로 판을 놓습니다. </li></ol> 3. 웜 추적 <ol><li> LabVIEW 프로그램을 실행하고 필요한 경우 (이미지에 대한 위치, 이미지, 측정, 카메라 설정의 종류) 옵션을 선택합니다. </li><li> 조이스틱이 웜의 이미지까지 현미경을 이동 사용하면 시야 (컴퓨터 화면)에 있습니다. 추적 프로그램을 참여하려면 &quot;트랙&quot;을 누르십시오. </li><li> 컴퓨터 프로그램은 실제로 같이 바이너리 필터링 이미지의 움직임을 측정하고 실시간으로이 이미지에 대한 측정을 할 수 있습니다 </li><li> 고물에웜의 로컬 형상 변경 사항이 상세하게 볼 수있는 동안 글로벌 궤도의 재건을 추적 어,이 스테퍼 모터의 움직임에서 만들어 질 수있다. </li></ol> 4. 데이터 분석 <ol><li> 웜의 모양을 매개 변수화 할 수 skeletonizing 스크립트 (MATLAB)를 실행합니다. (그림 2) </li><li> skeletonized 데이터 eigenmodes을 계산합니다. (그림 3,4) </li><li> 그림 5를 표시합니다. 그림 범례 또는 대리인 결과를 읽어 보시기 바랍니다. </li></ol>

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관심 없음 충돌이 선언 없습니다.

운동과 자연 행동의 연구는 데이터 감소 기술과 파트너의 비 침습적 추적 기술이 필요합니다. 여기 C.에 대한 자세한 이미지를 기록 추적 시스템을 사용하기 쉬운을 증명 한 는 한천 플레이트의 표면에 크롤링으로 elegans 행동. 이러한 이미지에 포함 된 정보의 양이 광대 높은 차원이며, 그래서 우리는 네 개의 근본적인 조치로 데이터의 차원을 줄이기 위해 방법을 개발했습니다. 이러한 조치는 포괄적이며 웜의 행동에 대해 해석하기 쉽습니다. 이 작품의 경우는 순방향 및 역방향의 undulatory주기의 서로 다른 점을 눈으로 할 어려운 조치에 우선적으로 국가 사이의 그 웜 전환을 보여줍니다. 이 작품은 측정 할 수 있습니다 또는 neuronal acti을 조절 시스템도 낮은 배율 8,9,10에서 웜의 동작을 측정하기 위해 개발 된 시스템을 추적에 보완하며,유전자 인코딩 시약에게 11,12을 사용하여 vity. 하나의 유기체 추적 행동을 정량화하기위한 강력한 방법이지만 기술에 대한 몇 가지 제한 사항이 있습니다. 첫 번째는 시스템이 한 번에 하나의 생물을 추적 있다는 사실입니다. 여러 생물 9,10을 추적 할 수 있습니다 고정 카메라 추적에 비해 우리 추적기의 처리량이 낮습니다. 그러나, 우리는 기아, 계란 부설하고, foraging 등 시간이 오래 규모 동작을 수량화하는 것이 중요합니다 멀티 웜 추적,보다 시간이 훨씬 더 오래 기간 동안 하나의 유기체의 행동을 측정 할 수 있습니다. 또한 추적은 명확 배경에서 웜을 식별 이미지가 필요합니다. 이 웜을 필터링 할 수있는 영상 처리 시스템의 시각 복잡하거나 너무 복잡 환경에서 웜의 움직임을 공부에서 우리를 precludes. 이 시스템은 유연하고 behavi 다른 종류의 사용할 수 있습니다구두 추적. 대신 동질적인 환경의 다른 추적 시스템과 유사한 공간과 측두엽 감각 정보를 제출 반면, 웜은 추적 할 수 있습니다. 예를 들어, 열 자극은 레이저 자극 7,14를 통해 적용 할 수있는, 또는 화학 정보는 한천 3 일까지 공간 그라디언트를 통해 적용 할 수 있습니다. 전체 시스템은 설계에 유연하고 Drosophila의 애벌레 같은 다른 크롤링 시스템에서 사용할 수 있습니다.

<table border="1"><tbody><tr><td> 부분의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> CCD 카메라 </td><td> Basler </td><td> A601f </td><td></td></tr><tr><td> 렌즈 </td><td> 에드먼드 광학 </td><td> MMS 시리즈 </td><td></td></tr><tr><td> 섬유 조명 </td><td> Dolan 제너 </td><td> DC-950H </td><td></td></tr><tr><td> 번역 단계 </td><td> Deltron </td><td> LS3-4 </td><td></td></tr><tr><td> 스테퍼 모터 </td><td> 미국 디지털 </td><td> MS23C </td><td></td></tr><tr><td> 스테퍼 모터 드라이브 </td><td> 도마뱀 붙이 </td><td> G201 </td><td></td></tr><tr><td> 스테퍼 모터 제어 </td><td> SimpleStep </td><td> SSXYZ </td><td></td></tr><tr><td></td><td></td><td></td><td> 모든 프로그래밍 코드는 사용할 수 있습니다. 해당 작성자에게 요청 이메일을 보내 주시기 바랍니다. </td></tr></tbody></table>

c. elegans tracking and behavioral measurement

우리는 C.의 locomotory 동작을 수량화하기 위해 계측, 이미지 처리 및 데이터 분석 기술을 개발했습니다 elegans는 한천 플레이트의 표면에 크롤링 있습니다. 행동, C.의 유전자, 생화학, 그리고 neuronal 기초 연구에 대한 이 현미경 의무, 유전자 다루기 쉬운이며, 택시, 학습, 사회 상호 작용 1,2 등의 복잡한 행동의 수를 보여주기 때문에 elegans는 이상적인 유기체입니다. 그들은 한천 플레이트에 크롤링하는 웜의 움직임을 추적에 따라 행동 분석은 감각 동작 3, 운동 4, 및 일반 mutational phenotyping (5)의 연구에 특히 유용했습니다. 우리 시스템은 웜이 더 기계적 자극이 웜에 전송되지 않습니다 보장 고정 한천 플레이트에 크롤링으로 카메라와 조명 시스템을 이동하여 사용할 수 있습니다. 우리의 추적 시스템은 사용하기 쉬운이며, 반자동 보정 기능이 포함되어 있습니다. 차모든 동영상 추적 시스템의 llenge는 차원 본질적으로 높은 데이터의 엄청난 금액을 생성하는 것입니다. 우리 영상 처리 및 데이터 분석 프로그램은 종합적으로 만 3-4 치수 6,7의 기능으로 웜 동작을 재구성 독립적 인 구성 요소의 집합으로 웜 모양을 줄임으로써 이러한 문제를 처리합니다. 과정의 예를 들어 우리는 웜이 위상 특정 방식으로의 반전 상태를 입력하고 종료 보여준다.

예 : foraging, C. elegans 전환 앞으로의 자주 전진 상태로 돌아 가기 전에 reorientation을 (오메가 턴) 수행, 운동을 반대로합니다. 이 전환을 정량화하는 것은 운동의 foraging 패턴을 이해하고 또한 웜의 모터 제어에 중요합니다. 운동 행동의 미묘한 세부 사항을 공개 할 수있는 능력은 우리의 추적 장치를 사용하여 볼 수 있습니다. 예를 들어 우리는 반대로 ~ 30 분의 한천 플레이트에 자유롭게 크롤링 웜의 고해상도 이미지를 캡처하여 전환을 전달 반대로 앞으로 좀 봐. 실행됩니다. eigenmode 분석 (그림 3) 우리가 웜의 undulatory 운동의 위상을 측정 할 수 있습니다. 차례로 위상 측정은 우리가 웜과 전환 (그림 4) 바꿀 수있는 앞으로의 속도를 계산 할 수 있습니다. 웜의 속도와 단계의 공동 확률을하려함으로써, 우리는 그 트랜스 볼 수 있습니다순방향 및 역방향 우대 단계 (그림 5A)와 probabilistically 일 사이 ition. 우리가 반대로 전환을 전달하고 반전 이벤트를 입력하고 종료 웜의 위상의 조건부 확률 보는 역 앞으로 분리하는 경우, 우리는 사람들이 서로 다른 위상 분포를 (그림 5B, C)하신 것으로 알고 있습니다. <img src="/files/ftp_upload/4094/4094fig1.jpg" alt="그림 1" /> 그림 1. 현미경을 추적. 이미징 시스템보기의 중심에 웜을 유지하기 이동 중에 한천 플레이트에 크롤링 웜의 샘플은 고정 남아 있습니다. 이미징 시스템은 스테퍼 모터 (3)에 의해 구동되는 2D 번역 무대에서 내장되어 있습니다. CCD 카메라 (Basler A601f)와 렌즈 (25mm 초점 거리) 이미지 아래에서 웜 (1)과 섬유 등 (에드먼즈 광학, 모델) (2) 위에서 켜집니다. 간단한을 사용 수제 스테퍼 모터 컨트롤러EP 컨트롤러 보드 (SimpleStep, SSXYZ)는 이미징 시스템의 움직임을 제어합니다. 동시에 필드의 중앙에 웜을 유지 모터 컨트롤러와 통신하는 동안 사용자 정의 LabVIEW 프로그램 (내쇼날 인스트루먼트, 오스틴, TX, USA)는 웜의 이미지를 획득하고 처리합니다. <img src="/files/ftp_upload/4094/4094fig2.jpg" alt="그림 2" fo:content-width="5in" fo:src="/files/ftp_upload/4094/4094fig2highres.jpg" /> 그림 2. 이미지 처리. (A) 웜은 시간에서의 곡률을 변경하여 이동하고 그래서 우리는 센터 라인을 parameterizing하여 웜 이미지를 처리​​했습니다. 이미지는 원시 그레이 스케일 이미지 (B)를 thesholding하여 바이너리를 만든, 개인 웜은 크기 (C)에 의해 개체를 필터링하여 식별되었습니다. 웜의 질량의 중심과 이미지의 중심 사이의 거리가 다시 센터 앨리스의 적절한 거리를 계산 한 다음 단계는 이동했습니다RM. 이러한 보정 거리가 웜의 궤도를 계산하는 데 사용되었습니다. 웜 주변의 곡률은 꼬리 (최대 곡률)과 머리를 (보조 최대) 식별하는 데 사용되었다. (D) 이미지는 다음 중심선을 찾습니다 skeletonized했다. (E) 결과 곡선은 다음엔 101 세그먼트로 보정되었고 세그먼트 사이의 계산 된 각도는 곡선을 매개 변수화하는 데 사용되었다. <a href="http://www.jove.com/files/ftp_upload/4094/4094fig2large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> . <img src="/files/ftp_upload/4094/4094fig3.jpg" alt="그림 3" fo:content-width="4.75in" fo:src="/files/ftp_upload/4094/4094fig3highres.jpg"/> 그림 3. Eigenmode 계산. 웜 형태의 기본 곡선은 원리 구성 요소 분석 5 사용하여 계산되었습니다. 각 웜 곡선은 고유 벡터의 선형 합 (또는 eigenworms)로 표시되는<html> 각 구성 요소의 진폭은 eigenmode (모드)로 알려져 있습니다. 우리는 시간이 지남에 따라 웜 모양의 처음 3 eigenmodes을 측정하여 웜의 동작을 수량화 할 수 있습니다. <img src="/files/ftp_upload/4094/4094fig4.jpg" alt="그림 4" fo:content-width="5in" fo:src="/files/ftp_upload/4094/4094fig4highres.jpg"/> 그림 4. 위상 및 속도. 제한주기위한 첫 두 모드 (A) 공동 분포. (b) 본 제한 사이클의 단계 나 위치는 웜의 undulatory 위치의 위상과 연결합니다. (C) 웜 앞으로 위상 속도를 사용 및 역방향 움직임이 정량화 될 수 있습니다. (D)이 제한주기를 따라 속도와 방향은 웜의 속도를 나타냅니다. <a href="http://www.jove.com/files/ftp_upload/4094/4094fig4large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> . 4094fig5.jpg &quot;ALT =&quot;그림 5 &quot;구경 : 콘텐츠 폭 =&quot;4in &quot;강한 : SRC =&quot;/ files/ftp_upload/4094/4094fig5highres.jpg &quot;/&gt; 그림 5. 단계 따라 속도 스위칭. 순방향 및 역방향 상태 사이의 전환은 왕복주기를 따라 무작위로 발생하지 않습니다. 그들은 (하지만 여전히 stochastically) 특정 단계에서 발생합니다. (가) 위상 속도와 속도의 공동 배포 분명 웜은 특정 단계에서 우선적으로 앞으로 상태를두고 입력을 나타냅니다. (b)는 반전 이벤트의 입구에 에어컨이 단계의 음모를 보여줍니다. (C) 반전 이벤트의 출구에 에어컨이 단계를 보여줍니다. 검은 선은 출구 또는 입구에 에어컨이없는 위상 각도의 배포입니다.

Watch this Scientific Journal Video about C. elegans Tracking and Behavioral Measurement at JoVE.com

C. elegans Tracking and Behavioral Measurement

우리는 기록하고 계량화 할 수있는 비디오 속도 추적 현미경 시스템을 개발 한<em&gt; C. elegans</em높은 해상도와 높은 속도로&gt; 동작. 우리는 또한 완전히 웜의 모양을 설명하는 측정의 기본 집합에 웜 이미지의 차원을 줄이기 위해 계산 방법을 개발했습니다.

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 ...

c-elegans-tracking-and-behavioral-measurement

Nanyang Technological University

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JoVE Journal

Neuroscience

C. elegans Tracking and Behavioral Measurement

19.0K 조회수.  University of Toronto. 우리는 기록하고 계량화 할 수있는 비디오 속도 추적 현미경 시스템을 개발 한 C. elegans 동작. 우리는 또한 완전히 웜의 모양을 설명하는 측정의 기본 집합에 웜 이미지의 차원을 줄이기 위해 계산 방법을 개발했습니다.

비디오: C. elegans Tracking and Behavioral Measurement

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

유전자 변형을 사용하여 시금 β - 아밀로이드 독성 C. 엘레간스 모델

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

연구

신경과학

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 ...

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 ...

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

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

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

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

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 ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Hippocampal 인슐린 Microinjection 및<em&gt; 생체에</em공간 메모리 테스트 기간 동안&gt; Microdialysis

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

개별 피라미드 뉴런의 고 충실도 Optogenetic 제어하는​​ 방법<em&gt; 생체 내</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

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 ...

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

동안 Neuroblast 세포질 분열을 시각화<em&gt; C. 엘레</em&gt; 배아

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...