R01AA016837 (JCB)와 P20AA017828 (AGD와 JCB) :이 연구는 국립 보건원, 국립 알코올 중독과 알코올 남용 연구소에서 보조금에 의해 지원되었다.

분석하기 전에 날에 대해 수행 할 1 단계 <ol><li> 신선한 선충 성장 매체에 L4 무대 웜을 선택 (NGM) 판 OP50 E.의 잔디 파종 20 주 / N에서 대장균, 문화를. 각각의 분석 조건 (10) 웜이 필요합니다; 웜의 O / N 손실을 허용하는 웜의 과잉을 선택합니다. <ol><li> 첫날 성인들만 분석 동물; 많은 돌연변이가 야생형보다 느린 속도로 성장한다. 테스트 한 모든 동물이 처음 일 성인 있도록 발달 지연이 균주에 대한 따기의 타이밍을 조정합니다. </li></ol></li></ol> 2 단계 분석의 날에 수행하는 <ol><li> 분석을위한 준비 : <ol><li> 표준 시드 화 60 X 15mm NGM 판에 분석을 수행합니다. 판 모두 뚜껑 오프로, 2 시간 동안 37 ℃에서 사용하는 건조. 각각의 실험 시험의 경우, 네 건조 NGM 판을 사용한다; 이들은 0 mM의 400 mM의 에탄올 분석 플레이트 및 동반 적응 판을 것입니다. 참고 :NGM 플레이트의 사용은 여기에 중요하다 판의 조성의 차이가, 특히 그들의 삼투압에 강하게 행동에 에탄올 용량 반응에 영향을 미칠 수 있으며, 이는 동물 (20)에 의해 축적 된 에탄올의 양의 변화에 적어도 부분적으로 기인한다. NGM 한천 160 mOsm입니다. </li><li> 건조 후에, 시드 화 NGM 판 각각이 분석에서 사용 중량을 유의하여야 무게. 빈 접시의 중량을 기준으로 번호판 미디어의 볼륨을 결정한다. 볼륨 한천의 중량의 변환을 근사, 미디어가 같은 부피의 물과 동일한 무게가 있다고 가정한다. 주 : 당점 일관된 결과가 원래의 10 ml의 용적이 8.3-8.9 ml이다 건조 후의 부피가 충분히 탈수했다 NGM 매체와 함께 밝혀졌다. 2 시간 건조 시간에 대한 대안은 플레이트 044의 차이를 설명하기 위해이 범위의 부피에 도달 할 때까지 건조한다. </li><li> 4 구리 반지 (1 내경 용융.6cm) 판의 표면은 약 3 염에 다른 유전자형 또는 worms.Grasp 각각의 집게 링, 열 치료 그룹에 대한 가축 우리 (분젠 버너에서 강한 불꽃이 잘 작동)로 작동으로 초. 여전히 &#39;건너 뛰기&#39;에서 그것을 방지하기 위해 집게로 반지를 잡고 즉시 접시에 반지를 배치합니다. <ol><li> 링 반지의 여러 지점에서 집게로 가볍게 아래로 눌러 한천의 표면에 평평하게 달려 있는지 확인합니다. 반지를 배치 할 때, 추가로 3 반지를위한 공간을 확보하기 위해주의해야합니다. </li><li> 네 개의 모든 카메라의 시야가 맞춰 지도록 함께 가깝게 가능한 올려 놓으주의하면서 판의 표면에 구리 세 개의 추가 고리를 녹아. 참고 : 한천과 좋은 도장을 만드는 것은 분석하는 동안 링에 벌레를 유지하는 것이 필수적입니다. 접시에 건너 뛸 반지는 한천과 올바른 도장을 할 가능성이 있으며벌레가 파고 할 수 있으며 벌레를 시각화 방해 한천을, 흉터 수 있습니다. </li><li> 각 분석 판 건조한 시드되고 더 에탄올을받을 수 없습니다해야 첨부 &quot;적응&quot;판을 준비합니다. 이 판에 네 개의 구리 반지를 놓습니다. </li></ol></li><li> 링 자체의 시야 쓰지주의하면서 옆 링에 사용되는 웜 균주 각각 링 플레이트 하단 레이블. 그 동반 순응 플레이트 분석 플레이트에 레이블을 맞 춥니 다. </li><li> 최종 농도가 0 mm로부터 400 mM의 에탄올 (부피 중량)가되도록 각각의 분석 플레이트에 추가 100 % 에탄올의 양을 계산한다. 각각의 실험을 위해 (N = 1), 하나의 0 mM 내지 400 mM의 하나의 에탄올 플레이트있을 것; 순응 판은 에탄올을받지 않습니다. 판의 표면 주위를 피펫 팅, 에탄올을 추가합니다. 플레이트를 밀봉하기 위해 실험실 막을 사용하고 2 시간 동안 벤치 탑에서 평형을 허용한다. </li><li> 1.5 시간이 경과하면, 상기 분석 단계 2.2 순응하는 단계를 시작한다. </li></ol></li><li> 운동 분석을 수행 분석 판을 <ol><li> 조심스럽게 순응 접시에 구리 반지의 중심에 각 실험군의 10 웜을 전송합니다. 웜 픽으로 조심스럽게 긁어서이 시점에서 한천에서 볼 수있는 모든 음식을 제거합니다. 동일한 시험 균주에서 동일한 순서로 플레이트 상에 배치되지 않도록 실험군은 실험에 걸쳐 시험 플레이트 상에 배치되는 순서를 다양하다. 주 : 목표 순응 접시에 식품 분석 플레이트에 전달되면 벌레 음식과 운동에 약물의 효과 가려한다 주위 응집되므로, 식품의 최소량으로 플레이트에 동물을 전송하는 것이다. </li><li> 이 벌레가 굴을 할 수 있도록하고, 분석을 방해하므로, 선택과 한천의 표면을 파괴하지 않도록주의하십시오. 실온에서 30 분 동안 인큐베이션 웜. </li><li> 각 판의 입문 사이에 적절한 간격이 있는지 확인하십시오. 숙련 된 실험자 (40) 동물, 4 반지 &lt;1.5 분 동안 10 / 반지를 이동할 수 있지만, 2 분까지의 간격은 허용됩니다. 표준 분석은 10 ~ 12 분 0 mM의 400 mM의 에탄올 모두 노출의 30 ~ 32 분에서 기록 영화가 있습니다. </li><li> 0 mM의 노출에 대한 순응 판과 약 2 분 30 초 떨어져 두 번째 영화가 시작해야합니다 전에 첫 번째 동영상 파일의 저장이 가능하도록 400 mM의 노출에 대한 적응 판을 시작합니다. </li><li> 30 분의 적응 기간 후, 분석 플레이트에 순응 판에서 벌레를 전송합니다. 그들은 각 플레이트의 사이 완료 타이밍을 추적, 순응 플레이트에 첨가하는 것과 동일한 순서로 분석 플레이트에 웜 (0 MM 또는 400 mM의 에탄올)를 전송합니다. 에탄올을 증발 손실을 최소화하기 위해 실험실 필름 : 봉쇄 판. </li><li> 유평평해진 선택의 상단에 벌레를 수집하는 얇은 가장자리가 평평 웜 골라 함께 떠 운동을 SE는. 이 선택에 벌레를 붙 도움으로 일반적으로 웜을 전송에 사용되는 박테리아를 사용하지 않고 분석 플레이트에 시드 화 순응 플레이트 웜의 전송을 수행합니다. 주 : 접시에 제 웜이 플레이트에 첨가 마지막 웜 이상 에탄올에 노출되기 때문에, 동물이 단계에서 전달되는 속도가 매우 중요하고, 이전 시점이 약간의 시간 의존적 효과를 보여줄 수있다. 이 균주는 실험 반복 실험을 통해 처음으로 접시에 배치되는 회전의 주요 이유입니다. </li></ol></li><li> 로코 분석을 수행 얇은 껍질을 <ol><li> 등으로 2048x2048 7.4와 0.5 배 현미경 목적, 0.8 배의 배율 카메라로 시야 (약 42x42 mm 2 광장)에서 네 개의 링을 동시에 이미징 할 수있는 현미경 / 카메라 조합을 사용하여81; m 화소 CCD. </li><li> 강도 임계치 단계에서 물체 인식 에이즈 시야에 걸쳐 심지어 조명을 사용하여 (아래 참조). 3 &quot;X3&quot;백라이트가 잘 작동합니다. </li><li> 이미지 광원 전송되는 기존의 현미경에 비해 백라이트를 사용하는 경우 손실 대비를 생성하는 판에 배치 미디어 측 최대의 벌레 (덮개 아래). </li><li> 이동 벌레의 시간 경과 영화를 캡처 이미지 분석 소프트웨어를 준비합니다. 2 분 (120 프레임)의 동영상으로, 12 비트 그레이 스케일 이미지 1 초를 캡처 할 수있는 소프트웨어를 설정합니다. 여전히 충분한 이미지 해상도를 유지하면서, 출력 파일의 크기를 줄이기 위해, 1024 × 1024 화소의 이미지를 캡처하는 2 × 2 비닝 모드를 사용한다. </li><li> 동영상을 기록합니다. 마지막 웜 후 10 분은 그 판에 배치 된 제 1 플레이트 (0 mM의 에탄올)의 120 프레임 동영상 촬영을 시작합니다. 동영상 파일을 저장합니다. </li><li> 제 2 플레이트 (400 mM의 에탄올)을 기록한다. 이 과정을 반복마지막 웜 후 30 분부터 두 플레이트는 각각 노광용 30-32 분 시점을 캡처하는 제 1 플레이트 (0 mM의 에탄올)에 배치했다. 참고 : 분석 할 다음 판으로 녹음을 시작하기 전에 각 캡처 세션 후 이미지 파일을 저장하기에 충분한 시간을 준다. </li><li> 보관 된 영화의 미래 교정 및 8 비트 256 그레이 스케일 TIFF 파일에 각 영화의 사본을 변환,이 영화가 개방 및 ImageJ에 (28)와 같은 다른 공개 오픈 소스 이미징 소프트웨어 프로그램에 의해 분석 될 수 있도록. 주 : 여기에서 설명 된 이미지 분석 소프트웨어 독자적인 파일 포맷을 사용한다. </li></ol></li><li> ImagePro 소프트웨어를 사용하여 영화의 분석. <ol><li> 촬영 후에는 이미지-Pro 소프트웨어 또는 그와 동등한 Plus를 사용하여 영화를 분석 할 수 있습니다. 주의 : 다른 물체 추적 소프트웨어의 다양한 여러 성공적 C.을 추적하는 데 사용 된 것을 사용할 한 번에 29 등의 엘레소프트웨어는 합리적 오브젝트의 식별 단계가 유사한 원리에 기초하여 여기서 설명 된 바와 해당 대체 할 수있다. 설명하는 방법은 약간의 차이가있을 수 있습니다 이미지 - 프로 플러스 소프트웨어 버전 6.0-6.3 및 7.0, 이미지 - 프로 플러스 소프트웨어의 새 버전에 관한 것이다. </li><li> 2 분 (120 프레임) 세그먼트에서 영화를 분석 할 수 있습니다. 먼저, 배경 평탄화 및 웜 물체의 콘트라스트를 향상시키기 위해 화상에 필터를 적용한다. 다음과 같이 필터를 선택 : 메뉴&gt; 프로세스&gt; 필터&gt; 향상&gt;는 평평 : 매개 변수 : 배경 = 다크; 폭 = 20 픽스 기능이 있습니다. </li><li> 필터링 단계 후 영화를 다시 저장하지만 항상 필터링되지 않은 형태로 원본 동영상을 유지합니다. <ol><li> 배치 및 구리 반지와 중복 할 수있는 크기이자 (ROI)의 원형 영역과 별도로 각 링에 동물의 운동을 분석한다. 확인하고 메뉴&gt; 측정과 벌레를 추적&gt; 트랙 ... 명령 개체; 이 추적 D를 불러옵니다ATA는 표 창. 추적 옵션 버튼은 특정 트랙을 제외하고 모든 실험 유물을 제한 할 수 있습니다. </li></ol></li><li> 자동 추적 탭에서 다음 매개 변수를 사용 : 트랙 매개 변수 : 속도 제한 (검색 반경) = 400 μm의 / 프레임, 가속 한계 = 자동, 최소 총 트랙 길이 = 400 μm의, 우위 모션 유형 = 혼란; 트랙 매개 변수의 객체는 : 부분 트랙 = 예, 최소 길이 = 21 프레임, 추적 예측 깊이 = 1 프레임을 허용합니다. </li><li> 추적 프로세스를 시작하려면, 자동으로 모든 트랙을 찾아 개수 / 크기 옵션 대화 상자와 추적 대화 상자를 표시하는 버튼을 기능을 클릭합니다. 분석 용 웜 오브젝트를 강조하고 밝은 배경 픽셀을 무시하도록 웜 화소의 계조 강도를 사용하는 중요한 임계 공정을 제공 개수 / 크기 대화 상자의 강도 범위 선택을위한 수동 옵션을 선택한다. <ol><li> 강도 임계 값을 조정 SLI폰더 모든 어두운 오브젝트를 강조 포괄적 범위를 만들기 위해 (하나의 하한을 설정하기 위해, 하나의 상한을 설정하기 위해). 0-4,095의 규모에 0-1,500의 범위는 미세한 튜닝을위한 좋은 출발점이 될 것입니다. </li><li> 이러한 구리 링과 접시에 이물질 같은 단일 웜 개체보다 크고 작은 개체를 제외 할 크기의 필터를 적용합니다. 측정과 자세의 다양한 개별 웜에 대한 크기를 덮이 작업을 수행하는 두 개의 크기 매개 변수를 설정&gt; 카운트 / 크기 옵션 대화 상자에서 선택 측정 메뉴 항목을 선택합니다. (지역 범위 : 28,000-120,000 μm의 2 및 경계 범위 : 600-2,500 μm의). </li><li> 변이주가 분석 될 접시에 상당히 작거나 다른 동물보다 크면 설정을 변경할 필요가 없도록, 제 동물을 추적하기 전에 균주의 다른 크기를 수용하기 위해 객체 인식 용 필터 설정의 범위를 확장 중간 분석. </li></ol> &lt;/ 리&gt; <li> 추적 대화 상자에서 계속을 클릭하여 추적 프로세스를 완료합니다. 시각적으로 자동 필터 설정에 따라이를 제외 할 타당한 이유가없는 한 모든 웜이 표시되어 있는지 확인하기 위해 영화의 각 웜의 진행과 출력 트랙을 비교합니다. 수동 필터 크기 기준을 만족 확인 비 웜 물체의 존재에 의해 생성 된 트랙을 삭제. </li><li> 각 프레임 사이의 각각의 웜의 속도를 계산하는 소프트웨어를 사용하여 (거리는 프레임 간의 1 초당 물체의 무게 중심에 대해 이동) 및 웜의 인구 모든 트랙에 걸쳐 각 트랙에 대한 평균 속도와 평균 속도를 표시 구리 반지. 마지막 평균이 실험 시험의 관점에서 N = 1로 고려한다. 통계 분석 및 데이터 보관을위한 스프레드 시트 프로그램에 데이터를 보냅니다. </li><li> 데이터가 플레이트에 제 1 링에 대해 기록 된 후, 그 다음에 링 ROI를 놓고매개 변수를 변경하지 않고도, 추적 처리를 반복한다. </li><li> 운동을 통해의 상대 속도를 계산 : </li><li> 상대 속도 (%) = 처리 치료 (400 mM의) 속도 / (0 mM의) 속도 × 100. 참고 : 다른 유전 적 조작은 종종 동물의 기저 운동 속도를 변경. 동물의 운동 속도에 대한 에탄올의 효과를 결정하는 다른 유전자형 또는 증상에 걸쳐 이러한 효과를 비교할 수 있도록하기 위해, 상대 속도를 계산한다. </li></ol></li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+assess+initial+sensitivity+and+acute+functional+tolerance+to+hypnotic+effects+of+ethanol.">A novel method to assess initial sensitivity and acute functional tolerance to hypnotic effects of ethanol.</a> J. Pharmacol. Exp. Ther. 302, 257-263 (2002).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9, 671-675 (2012).</li><li>Husson, S. J., Costa, W. S., Schmitt, C., Gottschalk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+track+of+worm+trackers.">Keeping track of worm trackers.</a> WormBook. , (2012).</li></ol>

The authors have nothing to disclose.

C.에서 사용할 수있는 간단한 신경 생물학 및 유전 적 도구 엘레 웜 행동에 에탄올의 효과의 분자 기초를 공부하는 우수한 모델합니다. 여기서는 8,10,20,24,25을 에탄올 급성 행동 반응의 여러 환경 매개체 분자를 식별하는 데 사용 된 분석법을 설명한다. 이 방법은 함께 포유 동물에서 응답의 수준의 복합 표현형 모델, 분화 및 두 개의 서로 다른 에탄올 응답 행동 표현형, 초기 ...

Alcohol use disorders (AUD) are widespread and produce serious health, social, and economic problems. In humans, the susceptibility to developing an AUD is heavily influenced by both genetics and the environment1,2. A strong physiological predictor of abuse liability is the initial level of response (LR) to alcohol (ethanol) that is exhibited by na&#239;ve drinkers3-5. This LR phenotype is influenced by genetics and non-genetic components6. Determining the molecular mechanisms that influence the LR to ethanol is an important goal of the study of ethanol response behaviors.
The nematode, Caenorhabditis elegans, has been increasingly used as a model for studying the effects of ethanol on behavior7-9. There is strong molecular conservation in the machinery of nervous system function between worms and mammals, and several genes that have been shown to influence the LR to ethanol in worms have been shown to influence LR to ethanol in mammals10-16, and have been implicated in abuse liability in humans17-19. 
Ethanol intoxicates worms, which is reflected in a decrease in their locomotion speed. Several different laboratories have developed behavioral assays that differ in several ways, for example, the locomotion behavior that they study (crawling versus swimming11,12,14,20,21) or in the composition of the solutions in which the assays are performed (nematode growth medium versus Dent&#8217;s saline20,22). Interestingly, these diverse assays have yielded somewhat different dose response profiles for the effects of ethanol. These results have pointed to important differences in the underlying behaviors of crawling and swimming9,23, as well as a role for the environmental variable osmolarity in ethanol responses20, and have highlighted the importance of describing experimental detail of the various assays.
An assay to measure the acute effects of ethanol on crawling behavior is presented here. This assay has been used extensively to study the genetic and environmental influences on the LR to ethanol8,10,20,24,25. The mammalian LR phenotype is a composite of at least two components, initial sensitivity to ethanol and acute functional tolerance to ethanol26,27. In worms, the LR phenotype has been shown to be separable into these two components through the use of this behavioral assay. The influences of genetic and environmental manipulations on both phenotypes can be examined using this single assay. Importantly, these two phenotypes are genetically separable.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>C. elegans strains</td>
			<td>Caenorhabditis Genetics Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 x15 mm Petri plates, triple vented</td>
			<td>Greiner Bio-One</td>
			<td>628161</td>
			<td>Other plate brands will suffice.</td>
		</tr>
		<tr>
			<td>NGM agar</td>
			<td>Various</td>
			<td></td>
			<td>NaCl (3g/L), agar (17g/L), peptone (2.5g/L), 1 mL cholesterol (5mg/mL in ethanol), 1 mL (1M) MgSO4, 1 mL (1M) CaCl2, 25 mL (1M) KPO4, pH=6, 975 mL H2O</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Various</td>
			<td></td>
			<td>e.g. Fisher Scientific #10300</td>
		</tr>
		<tr>
			<td>37&#176;C Incubator</td>
			<td>Various</td>
			<td></td>
			<td>For drying agar</td>
		</tr>
		<tr>
			<td>Digital balance</td>
			<td>Various</td>
			<td></td>
			<td>For determining plate weights and agar volume</td>
		</tr>
		<tr>
			<td>Copper rings</td>
			<td>Plumbmaster</td>
			<td>STK#35583 (48 cap thread gasket)</td>
			<td>1.6 cm inner diameter, 1.8 cm outer diameter copper rings</td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>QImaging</td>
			<td>RET-4000R-F-M-12</td>
			<td>This camera has a large field of view.</td>
		</tr>
		<tr>
			<td>Stereomicroscope with C-mount and 0.5X objective</td>
			<td>Leica</td>
			<td>MZ6</td>
			<td>Discontinued model, M60 is current equivalent.</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Schott</td>
			<td>A08923</td>
			<td>3&#8221;x3&#8221;&#160; backlight for even illumination across the field of view</td>
		</tr>
		<tr>
			<td>Imaging and tracking software</td>
			<td>Media Cybernetics</td>
			<td>ImagePro-Plus v6.0-6.3</td>
			<td>Newer versions of the software have tracking functions.</td>
		</tr>
	</tbody>
</table>

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.

여러 가지 유전자형과 짝을 컨트롤에서 대표 데이터 (그림 1)는 8,24을 제시; 데이터는 구체적으로 분석 동물에서 그 하이라이트 차이를 선정 하였다. 노출 10 분으로 영향 정도는도 1B-G의 좌측 축 상에 도시되어 균주의 초기 감도 여겨진다. 10 분으로 제어보다 큰 상대 속도와 변이주가, 에탄올 (도 1F, G) 내성 인 것으로 간주되는 반면 (도 1D ...

Watch this Scientific Journal Video about An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans at JoVE.com

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 ...

an-assay-for-measuring-effects-ethanol-on-locomotion-speed

Research

JoVE Journal

Neuroscience

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

8.5K 조회수.  Virginia Commonwealth University. 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. 

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

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

가스 크로마토 그래피에 의한 operant 에탄올 자기 관리 및 에탄올 결정시 에탄올의 Microdialysis

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

연구

신경과학

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

Zebrafish 배아 Neuroepithelium의 투과율에 대한 분석

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

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 ...

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

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

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

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

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.  ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

에탄올 진정 감도 및 신속한 공차 측정을위한 저렴하고 확장 가능한 행동 분석<em&gt; 초파리</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

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. ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

의 기록 세포 내 전압 응답에 대한 전기 생리 방법<em&gt; 초파리</em&gt; 빛 자극에 광수와의 interneurons<em&gt; 생체</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

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 ...