测量乙醇的影响上的步态速度的测定<em>秀丽隐杆线虫</em
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
Representative Results
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. |
References
- Prescott, C. A., Kendler, K. S. Genetic and environmental contributions to alcohol abuse and dependence in a population-based sample of male twins. Am. J. Psychiatry. 156, 34-40 (1999).
- Schuckit, M. A. Genetics of the risk for alcoholism. Am. J. Addict. Article Review. 9, 103-112 (2000).
- Heath, A. C., et al. Genetic differences in alcohol sensitivity and the inheritance of alcoholism risk. Psychol. Med. 29, 1069-1081 (1999).
- Rodriguez, L. A., Wilson, J. R., Nagoshi, C. T. Does psychomotor sensitivity to alcohol predict subsequent alcohol use. Alcohol. Clin. Exp. Res. 17, 155-161 (1993).
- Schuckit, M. A., Smith, T. L. An 8-year follow-up of 450 sons of alcoholic and control subjects. Arch. Gen. Psychiatry. 53, 202-210 (1996).
- Kalu, N., et al. Heritability of level of response and association with recent drinking history in nonalcohol-dependent drinkers. Alcohol. Clin. Exp. Res. 36, 522-529 (2012).
- Davis, S. J., Scott, L. L., Hu, K., Pierce-Shimomura, J. T. Conserved single residue in the BK potassium channel required for activation by alcohol and intoxication in. C. elegans. J. Neurosci. 34, 9562-9573 (2014).
- Raabe, R. C., Mathies, L. D., Davies, A. G., Bettinger, J. C. The Omega-3 Fatty Acid Eicosapentaenoic Acid Is Required for Normal Alcohol Response Behaviors in C. elegans. PLoS ONE. 9, e105999 (2014).
- Topper, S. M., Aguilar, S. C., Topper, V. Y., Elbel, E., Pierce-Shimomura, J. T. Alcohol disinhibition of behaviors in C. elegans. PLoS ONE. 9, e92965 (2014).
- Bhandari, P., et al. Chloride intracellular channels modulate acute ethanol behaviors Drosophila,Caenorhabditis elegans and mice. Genes Brain Behav. 11, 387-397 (2012).
- Davies, A. G., et al. A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans. Cell. 115, 655-666 (2003).
- Davies, A. G., Bettinger, J. C., Thiele, T. R., Judy, M. E., McIntire, S. L. Natural variation in the npr-1 gene modifies ethanol responses of wild strains of C. elegans. Neuron. 42, 731-743 (2004).
- Kapfhamer, D., et al. Loss of RAB-3/A in Caenorhabditis elegans and the mouse affects behavioral response to ethanol. Genes Brain Behav. 7, 669-676 (2008).
- Speca, D. J., et al. Conserved role of unc-79 in ethanol responses in lightweight mutant mice. PLoS Genet. 6, e1001057 (2010).
- Thiele, T. E., Badia-Elder, N. E. A role for neuropeptide Y in alcohol intake control: evidence from human and animal research. Physiol. Behav. 79, 95-101 (2003).
- Treistman, S. N., Martin, G. E. BK Channels: mediators and models for alcohol tolerance. Trends Neurosci. 32, 629-637 (2009).
- Han, S., et al. Integrating GWASs and human protein interaction networks identifies a gene subnetwork underlying alcohol dependence. Am. J. Hum. Genet. 93, 1027-1034 (2013).
- Kendler, K. S., et al. Genomewide association analysis of symptoms of alcohol dependence in the molecular genetics of schizophrenia (MGS2) control sample. Alcohol. Clin. Exp. Res. 35, 963-975 (2011).
- Schuckit, M. A., et al. Autosomal linkage analysis for the level of response to alcohol. Alcohol. Clin. Exp. Res. 29, 1976-1982 (2005).
- Alaimo, J. T., et al. Ethanol metabolism and osmolarity modify behavioral responses to ethanol in C. elegans. Alcohol. Clin. Exp. Res. 36, 1840-1850 (2012).
- Morgan, P. G., Sedensky, M. M. Mutations affecting sensitivity to ethanol in the nematode, Caenorhabditis elegans. Alcohol. Clin. Exp. Res. 19, 1423-1429 (1995).
- Mitchell, P. H., et al. The concentration-dependent effects of ethanol on Caenorhabditis elegans behaviour. Pharmacogenomics J. 7, 411-417 (2007).
- Vidal-Gadea, A., et al. Caenorhabditis elegans selects distinct crawling and swimming gaits via dopamine and serotonin. Proc. Natl. Acad. Sci. U S A. 108, 17504-17509 (2011).
- Bettinger, J. C., Leung, K., Bolling, M. H., Goldsmith, A. D., Davies, A. G. Lipid environment modulates the development of acute tolerance to ethanol in Caenorhabditis elegans. PLoS ONE. 7, e35129 (2012).
- Davies, A. G., et al. Different genes influence toluene- and ethanol-induced locomotor impairment in C. elegans. Drug Alcohol Depend. 122, 47-54 (2012).
- Newlin, D., Thomson, J. Alcohol challenge with sons of alcoholics: a critical review and analysis. Psychol. Bull. 108, 383-402 (1990).
- Ponomarev, I., Crabbe, J. C. A novel method to assess initial sensitivity and acute functional tolerance to hypnotic effects of ethanol. J. Pharmacol. Exp. Ther. 302, 257-263 (2002).
- Schneider, C. A., Rasband, W. S., Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 9, 671-675 (2012).
- Husson, S. J., Costa, W. S., Schmitt, C., Gottschalk, A. Keeping track of worm trackers. WormBook. , (2012).
