Summary

测量乙醇的影响上的步态速度的测定<em>秀丽隐杆线虫</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

步骤1.在分析之前执行的日挑L4阶段蠕虫新鲜线虫生长培养基(NGM)板接种OP50 E.的草坪大肠杆菌 ,并对其进行培养,在20 CO / N。每个实验条件要求10蠕虫;挑过量蠕虫允许O / N损失的蠕虫。 只有实验动物,是第一天的成年人;许多突变体生长以较慢的速度比野生型。调整拾取对于具有发育迟缓,使所有测试的动物是第一天的成人菌株的定时。 <p class="jove_title"…

Representative Results

从几个不同的基因型和它们的配对控制代表数据( 图1)呈现8,24;数据特别选择在检测动物亮点的差异。的效果,在暴露10分钟的程度被认为是一个菌株,其上示出在图1B-G的左侧轴的初始灵敏性。突变菌株与相对速度比在10分钟的控制较大被认为是乙醇抗性( 图1F,G),而突变菌株具有相对速度小于被认为是过敏乙醇( 图1D的控制,…

Discussion

简单的神经生物学和遗传学工具C.提供线虫使蠕虫病毒在其中研究乙醇对行为影响的分子基础的极好的模型。在这里,我们描述了已被用于识别急性行为反应的几种分子和环境介质为乙醇8,10,20,24,25的测定。这种方法允许分化和同时检查两个不同乙醇响应行为表型,初始灵敏度和急性官能耐受的发展,它们一起模拟响应在哺乳动物的水平的复合型。相同的测定,可以很容易地?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

R01AA016837(JCB)和P20AA017828(AGD和JCB):这些研究是由美国国立卫生研究院酒精中毒和酒精滥用国家研究院​​的资助。

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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