Bennett3 project protocol

Drug study: Acute functional tolerance to ethanol in 74 ILSXISS recombinant inbred strains of mice   (2007)

Bennett B, Johnson TE
With: Downing C, Carosone-Link P, Ponicsan H, Ruf C


Project protocol — Contents
Reagents, supplies, and solutions




  • Testing apparatus: Plexiglas box, 31 cm on a side and 47 cm high, with a 2 cm thick layer of bedding material on the floor; the dowel 36 cm above the floor
  • 1.6 cm (5/8 inch) diameter wooden dowel
  • Centrifuge
  • Balance scale
  • Timer
  • Freezer
  • pH meter
  • Spectrophotometer

Reagents, supplies, solutions  

  • Heparinized capillary tubes
  • Needles and syringes
  • Glass centrifuge tubes
  • 1.75 g/kg ethanol (15% w:v)
  • 2.0 g/kg ethanol (15% w:v)
  • 0.55 M perchloric acid
  • 0.6 M KOH
  • 500 mM Tris-HCl buffer
  • 2 mM nicotine adenine dinucleotide (NAD, Boeringer)
  • 8-10 U of yeast alcohol dehydrogenase (ADH, Sigma)


I. Acute functional tolerance test (AFT)
a. Tests are conducted between 08:00 a.m. and 02:00 p.m. using a similar paradigm to the one used to select the HAFT (high AFT) and LAFT (low AFT) mice.
Mice are weighed, tail marked, and then trained to stay on a 1.6 cm (5/8 inch) diameter wooden dowel for 1 min.
Each mouse is trained on the testing apparatus for only 10 tries. A mouse that fails to learn the task after the given trials is not used.
Each mouse is administered an initial dose of 1.75 g/kg ethanol (15% w:v) i.p. and is immediately placed on the dowel and tested only once, to avoid practice effects.
Soon after the mouse falls off the dowel, a retro-orbital blood sample is obtained (BEC0, see Figure 1 below). If fall time is greater than 10 min, a blood sample is taken, but the injection is considered misplaced and the mouse is not tested further.
Ten to 15 min after the initial loss of balance, and at 5-min intervals thereafter, the mouse is tested for its ability to remain on the dowel for 1 min.
At the first regain of balance (shown by its ability to remain on the dowel for 1 min), a 2nd retro-orbital blood sample (BEC1) is obtained, followed by a second i.p. injection of 2.0 g/kg EtOH.
Approximately 30 to 60 min postinjection, testing for the 2nd regain of balance is conducted. It is about this same time that a mouse shows signs of consciousness and begins moving around.
Mice are placed on the dowel and tested at 5-min intervals thereafter.
A final retro-orbital blood sample is obtained (BEC2) when a mouse is able to stay on the dowel again for 1 min.

Figure 1. Schematic illustration of the acute functional tolerance test to alcohol.

II. Blood collection and EtOH concentration determination
Approximately 10 µL of blood is collected via retro-orbital sinus using heparinized capillary tubes.
Collected blood is added to 200 µL of ice-cold 0.55 M perchloric acid to precipitate blood solids.
c. Samples are vortexed and then centrifuged at 1,900 X g for 10 min.
The supernatant is then removed from the pellet and an equal volume (200 µL) of 0.6 M KOH is added to neutralize the perchloric acid.
The samples are then vortexed and stored in the freezer until analysis.
BEC is determined by spectrophotometer analysis of an enzyme assay.
A 50 µL sample is aliquoted into duplicate assay tubes, consisting of 2 mM NAD, 8-10 U of yeast alcohol dehydrogenase (ADH), and 500 mM Tris-HCL buffer, pH 8.8, at a final volume of 400 µL, plus a blank tube (without ADH).
The reaction mixture is allowed to incubate at room temperature for 30 min.
After incubation the absorbance of enzymatically formed NADH is measured at 340 nm using a spectrophotometer.
j. Blood ethanol content is calculated from linear regression analysis of a standard curve prepared on the same day.

Data collected by investigator

• blood ethanol concentration at first loss of balance (ataxia), first regain of balance, and second regain of balance
• acute functional tolerance to two-stage injections of EtOH as a function of first regain of balance and as a function of first loss of balance computed using blood ethanol concentrations at first loss of balance (ataxia), first regain of balance, and second regain of balance (see definitions)

Definitions and calculations

AFT: acute functional tolerance

BEC: Blood ethanol concentration (i.e. BEC0 at time "0" or first loss of balance (ataxia), BEC1 at time "1" or first regain of balance, BEC2 at time "2" or second regain of balance, see Figure 1 above)

AFT1= The primary dependent variable calculated as BEC2 - BEC1

AFT2= A secondary dependent variable calculated as BEC2 - BEC0

Where BEC0, taken at the initial loss of balance, was used as the measure of initial sensitivity because BEC1 (taken at the first regain of balance) incorporates some early development of acute tolerance (see Bennett et al 2007).


    Bennett B. Congenic strains developed for alcohol- and drug-related phenotypes. Pharmacol Biochem Behav. 2000 Dec;67(4):671-81. PubMed 11166057

    Bennett B, Carosone-Link PJ, Lu L, Chesler EJ, Johnson TE. Genetics of body weight in the LXS recombinant inbred mouse strains. Mamm Genome. 2005 Oct;16(10):764-74. Epub 2005 Oct 29. PubMed 16261418

    Bennett B, Carosone-Link P, Zahniser NR, Johnson TE. Confirmation and fine mapping of ethanol sensitivity quantitative trait loci, and candidate gene testing in the LXS recombinant inbred mice. J Pharmacol Exp Ther. 2006 Oct;319(1):299-307. Epub 2006 Jun 27. PubMed 16803863

    Downing C, Carosone-Link P, Bennett B, Johnson T. QTL mapping for low-dose ethanol activation in the LXS recombinant inbred strains. Alcohol Clin Exp Res. 2006 Jul;30(7):1111-20. PubMed 16792557

    Hu W, Saba L, Kechris K, Bhave SV, Hoffman PL, Tabakoff B. Genomic insights into acute alcohol tolerance. J Pharmacol Exp Ther. 2008 Sep;326(3):792-800. Epub 2008 Jun 11. PubMed 18550690   FullText