“Drinking in the Dark” (DID): A Simple Mouse Model of Binge‐Like Alcohol Intake

Todd E. Thiele1, John C. Crabbe2, Stephen L. Boehm3

1 Department of Psychology, University of North Carolina, Chapel Hill, North Carolina, 2 Portland Alcohol Research Center, Department of Behavioral Neuroscience, Oregon Health & Sciences University, and VA Medical Center, Portland, Oregon, 3 Department of Psychology, Indiana University—Purdue University Indianapolis, Indianapolis, Indiana
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 9.49
DOI:  10.1002/0471142301.ns0949s68
Online Posting Date:  July, 2014
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Abstract

One of the greatest challenges that scientists face when studying the neurobiology and/or genetics of alcohol (ethanol) consumption is that most preclinical animal models do not voluntarily consume enough ethanol to achieve pharmacologically meaningful blood ethanol concentrations (BECs). Recent rodent models have been developed that promote binge‐like levels of ethanol consumption associated with high BECs (i.e., ≥100 mg/dl). This unit describes procedures for an animal model of binge‐like ethanol drinking which has come to be called “drinking in the dark” (DID). The “basic” variation of DID involves replacing the water bottle with a bottle containing 20% ethanol for 2 to 4 hr, beginning 3 hr into the dark cycle, on cages of singly‐housed C57BL/6J mice. Using this procedure, mice typically consume enough ethanol to achieve BECs >100 mg/dl and to exhibit behavioral evidence of intoxication. An alternative two‐bottle (ethanol and water) procedure is also described. Curr. Protoc. Neurosci. 68:9.49.1‐9.49.12. © 2014 by John Wiley & Sons, Inc.

Keywords: ethanol; binge‐like; drinking‐in‐the‐dark; consumption; limited‐access

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Four‐Day DID Procedure
  • Alternate Protocol 1: Two‐Bottle Choice Preference Drinking in the Dark (DID)
  • Support Protocol 1: Construction of the Sipper Tubes
  • Support Protocol 2: Preparing the Ethanol Drinking Solution
  • Support Protocol 3: Determination of Blood Ethanol Concentration
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Four‐Day DID Procedure

  Materials
  • Adult C57BL/6J mice (8 week old, male or female depending on experimental requirements; Jackson Laboratory)
  • 20% (v/v) ethanol solution (see protocol 4)
  • Clear polycarbonate, standard shoebox mouse cage (dimensions: 10.5 × 6 × 5 in.)
  • Electropolished stainless steel wire bar lid (dimensions: 10.5 × 6 in.) with food hopper
  • Balance or scale
  • Water bottle with sipper tube
  • Constructed ball‐bearing sipper tubes (see protocol 3)
  • Binder clip (1.587 cm capacity; Acco brand medium binder clips)
  • Flashlight with red lens cover
  • Heparin‐coated capillary tubes and associated supplies for blood sampling (see protocol 5 for determination of blood ethanol concentration)

Alternate Protocol 1: Two‐Bottle Choice Preference Drinking in the Dark (DID)

  Additional Materials (see protocol 1Basic Protocol)
  • One additional ball‐bearing sipper tube per cage (see protocol 3)
NOTE: Some modification of the cage lid may be adopted to accommodate the extra bottle.

Support Protocol 1: Construction of the Sipper Tubes

  Materials
  • Sterile utility knife blade
  • Stainless steel ball‐bearing sipper tubes (2.5 in. long, 5/16 in. diameter; Ancare Corp.)
  • Heat‐shrink tubing (3/8 in. diameter; 3M)
  • Heat gun (Master Heat Gun, model HG‐301A; Master Appliance Corp.)
  • Silicone stoppers (European size 10D; Fisher)

Support Protocol 2: Preparing the Ethanol Drinking Solution

  Materials
  • 190 proof ethanol (Pharmco)
  • Tap water
NOTE: The 20% ethanol drinking solutions should be made fresh daily. The 190 proof grain ethanol is composed of approximately 95% ethanol and 5% water.NOTE: Depending on the goals of the experiment, and the mouse genotype tested, the experimenter may wish to try higher or lower ethanol concentrations. However, concentrations greater than 20% have an increasing tendency to leak. For a discussion of the various ethanol preparations available, see UNIT 9.26.

Support Protocol 3: Determination of Blood Ethanol Concentration

  Materials
  • Sterile surgical grade razor blades
  • 20 to 50 μl heparinized capillary tubes
  • Sterile gauze
  • 5 ml microcentrofuge tubes
  • Analox alcohol analyzer with Analox reagent and Analox ethanol standard (Analox Instruments) or gas chromatograph
NOTE: Immediately upon conclusion of the final ethanol access period, fluid volumes should be read and recorded, and a blood sample taken for assessment of blood ethanol concentration.
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Figures

Videos

Literature Cited

Literature Cited
  Barkley‐Levenson, A.M. and Crabbe, J.C. 2012. Ethanol drinking microstructure of a high drinking in the dark selected mouse line. Alcohol Clin. Exp. Res. 36:1330‐1339.
  Barkley‐Levenson, A.M. and Crabbe, J.C. 2013. High Drinking in the Dark mice: A genetic model of drinking to intoxication. Alcohol PII:S0741‐8329(13)00203‐6. doi:10.1016/j.alcohol.2013.10.007.
  Blednov, Y.A., Ponomarev, I., Geil, C., Bergeson, S., Koob, G.F., and Harris, R.A. 2012. Neuroimmune regulation of alcohol consumption: behavioral validation of genes obtained from genomic studies. Addict. Biol. 17:108‐120.
  Cox, B.R., Olney, J.J., Lowery‐Gionta, E.G., Sprow, G.M., Rinker, R.A., Navarro, M., Kash, T.L., and Thiele, T.E. 2013. Repeated cycles of binge‐like ethanol (EtOH)‐drinking in male C57BL/6J mice augments subsequent voluntary EtOH intake but not other dependence‐like phenotypes. Alcohol Clin. Exp. Res. 37:1688‐1695.
  Crabbe, J.C., Metten, P., Rhodes, J.S., Yu, C.H., Brown, L.L., Phillips, T.J., and Finn, D.A. 2009. A line of mice selected for high blood ethanol concentrations shows drinking in the dark to intoxication. Biol. Psychiatry 65:662‐670.
  Crabbe, J.C., Harris, R.A., and Koob, G.F. 2011a. Preclinical studies of alcohol binge drinking. Ann. N.Y. Acad. Sci. 1216:24‐40.
  Crabbe, J.C., Spence, S.E., Brown, L.L., and Metten, P. 2011b. Alcohol preference drinking in a mouse line selectively bred for high drinking in the dark. Alcohol 45:427‐440.
  Crabbe, J.C., Metten, P., Huang, L.C., Schlumbohm, J.P., Spence, S.E., Barkley‐Levenson, A.M., Finn, D.A., Rhodes, J.S., and Cameron, A.J. 2012. Ethanol withdrawal‐associated drinking in the dark: Common and discrete genetic contributions. Addict. Genet. 1:3‐11.
  Crabbe, J.C., Metten, P., Belknap, J.K., Spence, S.E., Cameron, A.J., Schlumbohm, J.P., Huang, L.C., Barkley‐Levenson, A.M., Ford, M.M., and Phillips, T.J. 2013. Progress in a replicated selection for elevated blood ethanol concentrations in HDID mice. Genes Brain Behav. 13:236‐246.
  Didion, J.P. and de Villena, F.P. 2013. Deconstructing Mus gemischus: Advances in understanding ancestry, structure, and variation in the genome of the laboratory mouse. Mamm. Genome 24:1‐20.
  Giardino, W.J. and Ryabinin, A.E. 2013. CRF1 receptor signaling regulates food and fluid intake in the Drinking‐in‐the‐Dark model of binge alcohol consumption. Alcohol Clin. Exp. Res. 37:1161‐1170.
  Kamdar, N.K., Miller, S.A., Syed, Y.M., Bhayana, R., Gupta, T., and Rhodes, J.S. 2007. Acute effects of naltrexone and GBR 12909 on ethanol drinking‐in‐the‐dark in C57BL/6J mice. Psychopharmacology 192:207‐217.
  Kaur, S. and Ryabinin, A.E. 2010. Ghrelin receptor antagonism decreases alcohol consumption and activation of perioculomotor urocortin‐containing neurons. Alcohol Clin. Exp. Res. 34:1525‐1534.
  Linsenbardt, D.N. and Boehm, S.L., II. 2013. Alterations in the rate of binge ethanol consumption: implications for preclinical studies in mice. Addict Biol. doi:10.1111/adb.12052.
  Linsenbardt, D.N., Moore, E.M., Griffin, K.D., Gigante, E.D., and Boehm, S.L., II. 2011. Tolerance to ethanol's ataxic effects and alterations in ethanol‐induced locomotion following repeated binge‐like ethanol intake using the DID model. Alcohol Clin. Exp. Res. 35:1246‐1255.
  Lyons, A.M., Lowery, E.G., Sparta, D.R., and Thiele, T.E. 2008. Effects of food availability and administration of orexigenic and anorectic agents on elevated ethanol drinking associated with drinking in the dark procedures. Alcohol Clin. Exp. Res. 32:1962‐1968.
  Moore, E.M., Serio, K.M., Goldfarb, K.J., Stepanovska, S., Linsenbardt, D.N., and Boehm, S.L., II. 2007. GABAergic modulation of binge‐like ethanol intake in C57BL/6J mice. Pharmacol. Biochem. Behav. 88:105‐113.
  Moore, E.M., Mariani, J.N., Linsenbardt, D.N., Melon, L.C., and Boehm, S.L., II. 2010. Adolescent C57BL/6J (but not DBA/2J) mice consume greater amounts of limited‐access ethanol compared to adults and display continued elevated ethanol intake into adulthood. Alcohol Clin. Exp. Res. 34:734‐742.
  Mulligan, M.K., Rhodes, J.S., Crabbe, J.C., Mayfield, R.D., Harris, R.A., and Ponomarev, I. 2011. Molecular profiles of drinking alcohol to intoxication in C57BL/6J mice. Alcohol Clin. Exp. Res. 35:659‐670.
  NIAAA. 2004. National Institute on Alcohol Abuse and Alcoholism Council approves definition of binge drinking. http://pubs.niaaa.nih.gov/publications/Newsletter/winter2004/Newsletter_Number3.htm#council
  Parasuraman, S., Raveendran, R., and Kesavan, R. 2010. Blood sample collection in small laboratory animals. J. Pharmacol. Pharmacother. 1:87‐93.
  Rhodes, J.S., Best, K., Belknap, J.K., Finn, D.A., and Crabbe, J.C. 2005. Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice. Physiol. Behav. 84:53‐63.
  Rhodes, J.S., Ford, M.M., Yu, C.H., Brown, L.L., Finn, D.A., Garland, T., Jr., and Crabbe, J.C. 2007. Mouse inbred strain differences in ethanol drinking to intoxication. Genes Brain Behav. 6:1‐18.
  Zorrilla, E.P., Inoue, K., Fekete, E.M., Tabarin, A., Valdez, G.R., and Koob, G.F. 2005. Measuring meals: Structure of prandial food and water intake of rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 288:R1450‐R1467.
Key Reference
  Rhodes et al., 2005. See above.
  In this paper, the authors describe basic features of the DID model and report several experiments identifying the most effective test parameters.
Internet Resources
  http://www.scripps.edu/california/research/inia/methodology.html
  The standard operating procedure (SOP) for DID can also be viewed at the “Methodologies” section of the Integrative Neuroscience Initiative on Alcoholism (INIA) website.
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