Summary

의 로코 속도에 에탄올의 효과를 측정하는 분석<em> 예쁜 꼬마 선충</em

Published: April 09, 2015
doi:

Summary

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.

Abstract

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. 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. elegans.

Introduction

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 naïve 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’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.

Protocol

분석하기 전에 날에 대해 수행 할 1 단계 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. 첫날 성인들만 분석 동물; 많은 돌연변이가 야생형보다 느린 속도로 성장한다. 테스트 한 모든 동물이 처음 일 성인 있도록 발달 지연?…

Representative Results

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 …

Discussion

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 …

Disclosures

The authors have nothing to disclose.

Acknowledgements

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

Materials

C. elegans strains Caenorhabditis Genetics Center
60 x15 mm Petri plates, triple vented Greiner Bio-One 628161 Other plate brands will suffice.
NGM agar Various 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
Forceps Various e.g. Fisher Scientific #10300
37°C Incubator Various For drying agar
Digital balance Various For determining plate weights and agar volume
Copper rings Plumbmaster STK#35583 (48 cap thread gasket) 1.6 cm inner diameter, 1.8 cm outer diameter copper rings
100% ethanol Various
Parafilm M Bemis PM996
CCD camera QImaging RET-4000R-F-M-12 This camera has a large field of view.
Stereomicroscope with C-mount and 0.5X objective Leica MZ6 Discontinued model, M60 is current equivalent.
Light source Schott A08923 3”x3”  backlight for even illumination across the field of view
Imaging and tracking software Media Cybernetics ImagePro-Plus v6.0-6.3 Newer versions of the software have tracking functions.

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Cite This Article
Davies, A. G., Blackwell, G. G., Raabe, R. C., Bettinger, J. C. An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans. J. Vis. Exp. (98), e52681, doi:10.3791/52681 (2015).

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