작품은 NIH 임상 과학자 개발 수상 K08NS002083, Shervert 프레이 연구소 그랜트, 에드거 A. Buttner에 NARSAD 젊은 탐정 수상에 의해 지원되었다.

1. 발달 지연 / 치사율 분석 : 야생형 (N2)와 두 개의 돌연변이 (Mut1 및 Mut2) 균주가 12 - 웰 플레이트에 세 클로자핀 농도 테스트 <ol><li> 1 일에 (2cm 직경 아니라 각) 12 - 웰 플레이트의 각 웰에 2 ㎖ NGM 매체 (10)을 부어 하룻밤 실온 (RT)에 벤치에 강화 할 수 있습니다. 같은 날, 하룻밤 220 rpm으로 회전에서 대장균 OP50 박테리아의 식민지를 선택 50 ㎖ LB 솔루션의 병을 감염, 그리고 37 ° C 통 문화를. </li><li> 2 일에, 각 웰 NGM의 중심에 20 μL OP50 박테리아 문화를 전송할 수 있습니다. 37 ° C 배양기에서 접시를 품어 또는 세균 잔디가 두껍게 성장 할 수 있도록 실온에서 밤새을 둡니다. </li><li> DMSO (디메틸 설폭 사이드) 용매에 클로자핀을 용해하여 80 mM의 클로자핀 주식 솔루션을 확인합니다. 그 후 1.7 mM의 아세트산 B에게 희석 원액과 80 ㎕의 작업 솔루션을표 1에 ased. 참고 : 관심의 특정 약물에 대한 작업 솔루션은 돌연변이 체에서 야생 유형을 구별하기위한 가장 적절한 농도를 찾기 위해 예비 농도 일련의 테스트를 수행하여 결정해야합니다. </li><li> 전송 80 μL 클로자핀 잘 시드 NGM 판에 솔루션을 작동 액이 표면에 균등하게 분배 할 수 있도록 판을 소용돌이 친다. 플레이트가 마를 때까지 기다립니다. 같은 날에 분석을 위해 플레이트를 사용하거나 다음 날 사용할 수 있도록 20 ° C의 배양기에 배치합니다. 참고 : 작업 솔루션은 특히 높은 농도로 인해 클로자핀의 낮은 용해도에 약 서스펜션입니다. 따라서, 약물 농도가 정확하다는 것을 보장하기 위해 약물 플레이트 용액을 전송하기 전에 튜브를 소용돌이. </li><li> M9 버퍼 많은 임신하는 성인을 포함하는 3.5 cm 플레이트 웜을 세척 한 다음 15 ㎖의 튜브를 스핀 다운1 분 동안 2,000 rpm에서. </li><li> 뜨는을 대기음. 5 ㎖ 표백제 용액 (: 차아 염소산 : NaOH를 4시 1분 5초에서 DDH 2 O)를 추가하고 부드럽게 튜브를 반전하여 선충을 방해. </li><li> 그 중 절반이 약 5 분에서 용해 될 때까지 동물을 관찰한다. 1 분 동안 2,000 rpm에서 계란을 스핀 다운. </li><li> 뜨는을 대기음 빠르게 14 ㎖의 M9 버퍼를 추가합니다. </li><li> 1 분 동안 2,000 rpm에서 계란을 회전 약 100 ㎕의 솔루션을 남겨 뜨는을 대기음. 계란을 일시 중단 소용돌이. </li><li> 전송 2 μL 달걀 전송 얼마나 많은 계란을 테스트 할 수있는 정기적 인 NGM 플레이트에 M9에서 일시 중단. 약 30 ~ 35 알을 각 전송에 있다는 것을 확인하기 위해 일시​​ 중단 된 계란의 볼륨을 조정합니다. 분석 플레이트의 각 웰에 30 ~ 35 알을 전송 한 후 24 시간 동안 20 ° C 배양기에서 배양한다. </li><li> 검정 첫날, 부화 계란의 개수 점수. picki에 의해 25/well에 부화 동물의 총 수를 조정필요한 경우 별도의 벌레를 겨. </li><li> 다음과 같은 일에, 동물을 관찰하고 그들에게 매 24 시간을 점수. 발달 단계는 동물의 크기 및 외음부 (11)의 형상에 기초하여 획득된다. 실험은 일반적으로 세 번째 또는 네 번째 날에 본 가장 강력한 효과 5-6 일을 지속. </li><li> Excel 파일에 입력 데이터와 실험의 매일 각 발달 단계에서 동물의 비율을 계산합니다. &#39;100 % 메뉴 &#39;&#39;2-D 열에서&#39;열 스택 기능을 그래프를 생성합니다. </li></ol> 2. 인두 양수 분석 : 야생형과 돌연변이 균주에 대한 젊은 성인 동물은 클로자핀 분석 플레이트에 채점 <ol><li> 분석 플레이트가 발달 지연 / 사멸 분석에 사용되는 동일한 프로토콜 뒤에 이루어진다. </li><li> 24 시간 분석하기 전에 시드 NGM 플레이트에 각 변형 50 L4 동물을 선택하고 20 ℃에서 벌레를 품어 </li><li> ASSA에 10 마리를 선택Y는 잘 접시. 그런 다음 잘 때마다 15 ~ 20 분에 10 마리를 선택합니다. </li><li> 첫 번째 잘 동물이 30 분 동안 약물에 노출 된 후, 해부 현미경으로 인두 펌프를 관찰하여 분석을 시작하고 20 초 9의 펌프를 점수. 인두 펌핑이 득점되면, 플레이트 떨어져 웜을 선택합니다. </li></ol>

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저자는 참여 관심 충돌이없는 것을 선언합니다.

여기, 우리는 C의 개발 및 행동에 APDS의 효과를 테스트하는 방법을 설명합니다 엘레. 약이 물에 상대적으로 불용성이기 때문에 DMSO 또는 에탄올, 클로자핀을 용해하는 데 사용됩니다. 용매는 C. 영향을 미치는 것으로보고되었다 있으므로 엘레 생물학 (12), DMSO 형 또는 에탄올 형 제어 그룹은 필수적이다. 우리의 분석에 사용 된 DMSO의 높은 농도는 C.에 명백?...

예쁜 꼬마 선충은 대규모의 순방향 및 역방향 유전자 화면과 화학 유전 화면에 순종 간단한 유전 유기체이다. C. elegans의 생리 활성 화합물의 광범위에 민감하고, 따라서 이러한 화합물의 여러 가지의 작용 메커니즘을 정의하는 데 성공적으로 사용되어왔다. 예를 들어, 생체 활성 화합물은 웜 약물 유전학은 아세틸 콜린 수용체 작용제 (예를 들어 레바 미솔, 니코틴, morantel 및 pyrantel), 마취제 (예 : 할로 탄), 카페인, 콜린 에스 테라 제 억제제 (예 알디 카르 브, lannate 및 trichlorfon), 플루오 라이드, GABA-관련을 포함하여 연구 화합물 (예를 들어, GABA 및 muscimol), 이버 멕틴, 파라콰트, 포르 볼 에스테르, 세로토닌 관련 약물 (예, 세로토닌과 imiprimine) 3. 또한, C. 엘레하므로 대규모 저분자 스크린에 사용 된 새로운 생리 활성 화합물의 발견s 및 새로운 유전자 표적 4의 식별. C. elegans의 게놈은 신경 전달 물질의 합성과 시냅스의 구조와 기능 5에 필요한 단백질을 코딩하는 유전자를 포함하여 잠재적 인 항 정신병 약물 (APD) 인간의 보존 목표를 포함한다. 따라서, C. 엘레 neurogenetics와 APDS의 행동의 새로운 분자 메커니즘을 발견하기위한 신경 생물학 제공 방법. 선충에서 APD 노출은 개발 초기 발달 지연, 그리고 더 높은 농도에서 치사 2,6를 생성합니다. 성인기 동안 APD 노출 행동 표현형을 생산하고 있습니다. 예를 들어, 클로자핀 노출은 운동과 인두 펌프를 억제하고 1,2,7 누워 계란을 향상시킵니다. APD 유도 발달 지연 및 치사율은 대규모 화학 유전 스크린에 대한 강력한 표현형이다. 그들은 아마도 더 이상의 세포 및 유전자 BASI이 이러한 표현형은 지금까지의 복잡의. 따라서, 이러한 유전 스크린은 간접 약물 표적의 다양성을 수득 할 것으로 예상된다. 그러나, 우리의 실험실은 후보 유전자 화면과 APD 유도 발달 지연과 치사율의 억제를위한 게놈 전체의 RNAi 화면을 수행하고 성공적으로 가능성이 도파민, 인슐린 및 니코틴 아세틸 콜린 수용체 2,8 등의 직접적인 목표를 인코딩 유전자를 회복했다. 성인의 APD-유도 행동에 따라 유전 스크린은 새로운 APD 목표의 식별을 이끌고있다, 우리는 지금 포유류 7 발달과 행동 두 화면에서 대상의 유효성을 검사합니다. 따라서, APDS의 행동의 새로운 분자 메커니즘을 발견하는 무척추 화학 물질의 유전 방식은 5,8 가능한 것으로 보인다. C. elegans의 인두 (20) 신경 (20) 근육 세포 및 기저막에 의해 래핑 (20) 액세서리 세포를 포함하는 기관이다. 포유 동물의 심장과 유사하게, 인두는 자율입니다ND 지속적으로 외부 환경 9에서 음식을 펌프. 인두 펌핑 속도의 억제는 음식의 흡수를 저해하고, 따라서 돌연변이 또는 약물 인두 펌핑 원인 발달 지연을 억제하거나 9를 체포하는. APDS 개발과 생존의 1,2에 미치는 영향에 대한 부분에서 회계, 인두 펌핑 속도를 억제한다. 여기, 우리는 선충 개발 및 인두 펌핑 약물 분석을 설명하기 위해 예를 들어 전형적인 APD의 클로자핀을 사용합니다.

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="22">
			<td>Clozapine</td>
			<td>Sigma</td>
			<td>C6305</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D8418</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Acetic acid (HAC)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP2401</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sodium hydroxide(NaOH)</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0590-13</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Hypochlorite</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">425044</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:236px;">Centrifuge 5810</td>
			<td style="width:188px;">Eppendorf</td>
			<td style="width:200px;">5810 000.017</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:236px;">Incubator shaker</td>
			<td style="width:188px;">New Brunswick Scientific</td>
			<td style="width:200px;">M1246-0006</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:236px;">Low temperature incubator 815</td>
			<td style="width:188px;">Precision Scientific</td>
			<td style="width:200px;">J1790-1B</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:236px;">Stereo microscope</td>
			<td style="width:188px;">Olympus</td>
			<td style="width:200px;">SZX12</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:236px;">Petri dish 35 x 10 mm</td>
			<td style="width:188px;">Fisher Scientific</td>
			<td style="width:200px;">NC9434271</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:236px;">12-well tissue culture plate</td>
			<td style="width:188px;">BD Falcon</td>
			<td style="width:200px;">REF 353043</td>
		</tr>
	</tbody>
</table>

methods for studying the mechanisms of action of antipsychotic drugs in caenorhabditis elegans

예쁜 꼬마 선충은 앞으로 대규모의 의무와 유전 화면과 화학 유전 화면을 반전 간단한 유전 유기체이다. C. elegans의 게놈은 신경 전달 물질의 합성과 시냅스의 구조와 기능에 필요한 단백질을 코딩하는 유전자를 포함하여 잠재적 인 항 정신병 약물 (APD) 인간의 보존 목표를 포함한다. APD 노출은 농도 의존적​​으로 발달 지연 및 / 또는 선충의 치사율을 생성한다. 이러한 표현형 1,2 펌핑 인두의 APD 유발 억제에 의해 부분적으로 발생합니다. 따라서, 개발 표현형 진통제의 약물 유전 학적 연구에 유용하고, 신경 근육 학 기초를 가지고 있습니다. 여기에서 우리는 선충 개발 및 인두 펌핑에 APD의 효과를 시험하기위한 세부 절차를 보여줍니다. 개발 분석의 경우, 동기화 된 배아는 선충의 성장 매체 APDS를 포함 (NGM) 접시, 및 애니을 개발하는 단계에 배치됩니다MALS 그런 다음 매일 채점됩니다. 인두 펌핑 속도 분석을 위해, 젊은 성인 동물 APDS를 포함 NGM 플레이트에 시험 무대. 단위 시간당 인두 펌프의 개수가 기록되고, 펌핑 속도가 계산된다. 이러한 분석법은 작은 분자 또는 큰 분자의 다른 많은 유형을 연구를 위해 사용될 수있다.

1. 발달 지연 / 주죠 분석 결과 발달 지연 / 주죠 분석의 전형적인 결과는도 1a에서 설명하고 1B된다. 컨트롤 그룹에있는 야생 형 동물이 임신하는 성인 단계 (그림 1a)로 성장했을 때, 클로자핀에 노출 된 야생 형 동물은 죽은 (그림 1b). 그림 1C는 비교 대표적인 결과를 보여줍니다 젊은 유생 단계에 아직도 또는이다 억제 돌연변이...

Watch this Scientific Journal Video about Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans at JoVE.com

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

예쁜 꼬마 선충에서 항 정신병 약물 (APDS)의 효과를 테스트하기위한 접근 방법을 설명합니다. 분석법 개발과 생존에와 인두 펌핑 속도에 약물 효과를 테스트하기 위해 설명되어 있습니다. 이러한 방법은 APDS 이외의 다른 약 종류와 약물 유전 학적 실험에 적용 할 수 있습니다.

에서 항 정신병 약물의 작용 메커니즘을 공부하는 방법<em&gt; 예쁜 꼬마 선충</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

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JoVE Journal

Neuroscience

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

11.4K 조회수.  Harvard Medical School.  예쁜 꼬마 선충에서 항 정신병 약물 (APDS)의 효과를 테스트하기위한 접근 방법을 설명합니다. 분석법 개발과 생존에와 인두 펌핑 속도에 약물 효과를 테스트하기 위해 설명되어 있습니다. 이러한 방법은 APDS 이외의 다른 약 종류와 약물 유전 학적 실험에 적용 할 수 있습니다. 

비디오: Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

마우스 Utero에 Electroporation : 통제 Spatiotemporal 진 Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

연구

신경과학

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

<em&gt; Caenorhabditis의 엘레</emAmylopathy 연구에 대한&gt; 모델 시스템

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

에있는 후각 수용체 뉴런의 전체 마운트 Immunolabeling<em&gt; 초파리</em&gt; 안테나

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

의 로코 속도에 에탄올의 효과를 측정하는 분석<em&gt; 예쁜 꼬마 선충</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

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 ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

소설 메트릭은 선충의 배아 신장의 특성을<em&gt; 예쁜 꼬마 선충</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

에이<em&gt; Caenorhabditis elegans</em&gt; 영양 상태 기반 구리 혐오 검정

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

꼬마 선 충 을 행동에 개별 뉴런의 비율 칼슘 이미징

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

Caenorhabditis elegans의 수지상 가시 이미징

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...