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

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 environment^1,2. A strong physiological predictor of abuse liability is the initial level of response (LR) to alcohol (ethanol) that is exhibited by naïve drinkers^3-5. 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 el

Protocol

1. Steps to Perform on the Day before the Assay

1. Pick L4 stage worms to fresh nematode growth medium (NGM) plates seeded with a lawn of OP50 E. coli, and culture them at 20 ˚C O/N. Each assay condition requires 10 worms; pick an excess of worms to allow for O/N loss of worms.
   1. Only assay animals that are first-day adults; many mutants grow at a slower speed than wild type. Adjust the timing of picking for strains that have developmental delays so that all animals tested are first-day adults.

2. Steps to Perform on the Day of the Assay

1. Preparation for the assay:

Results

Representative data (Figure 1) from several different genotypes and their paired controls are presented^8,24; data were specifically chosen that highlight differences in the assayed animals. The degree of effect at 10 min of exposure is considered the initial sensitivity of a strain, which is shown on the left axes in Figure 1B-G. Mutant strains with a relative speed larger than the control at 10 min are considered to be ethanol resistant (Figure 1F, G), w...

Discussion

The simple neurobiology and genetic tools available in C. elegans make the worm an excellent model in which to study the molecular bases of the effects of ethanol on behavior. Here, we describe an assay that has been used to identify several molecular and environmental mediators of the acute behavioral response to ethanol^{8,10,20,24,25}. This method allows the differentiation and simultaneous examination of two different ethanol response behavior phenotypes, initial sensitivity and the development of ac...

Materials

Name	Company	Catalog Number	Comments
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.