Strains were tested on two doses of alcohol (see details below). A strain of mice grouped within a cage was randomized into four groups.
Group A dose: 2.75g EtOH /kg bw
Group B dose: 3.0g EtOH /kg bw
Group C dose: 3.25g EtOH /kg bw
Group D dose: 3.5g EtOH /kg bw
Each mouse was tested before and after EtOH treatment as shown in workflow table.
Mice were injected sequentially at 75 s intervals by a single technician.
Behaviors were rated by others (see details below) who, at the time of scoring, were blinded to treatment groups.
Step Duration Injection Tested Data collected 1 1 h None No None during acclimation 2 45 s None Yes Baseline observations/measurements 3 75 s EtOH No Body weight before i.p. injection 4 5 min None No None, 1st interval before 1st measurements 5 45 s None Yes 1st interval measurements 6 10 min None No None, 2nd interval before 2nd measurements 7 45 s None Yes 2nd interval measurements 8 15 min None No None, 3rd interval before 3rd measurements 9 45 s None Yes 3rd interval measurements 10 30 min None No None, 4th interval before 4th measurements 11 45 s None Yes 4th interval measurements
Dose 17.5 to 22.25 mL EtOH per kg body weight Delivery intraperitoneal injection (i.p.)
Ataxia assessment scale
Definitions of ataxia behaviors are described in Metten et al., 2000.
Splayed Hind Legs Rating Scale Wobbling Rating Scale Score Splay= hindlimbs unable to support bw Associated activity Score Wobbling= erratic gate 0 standing; no splaying moves freely 0 no wobbling 1 standing; brief splaying movement slightly impaired 1 wobbles; no loss of balance 2 recurrent splaying; can stand able to move in straight line 2 wobbles; occasional loss of balance 3 persistently splayed uncoordinated movements 3 wobbles with repeated falls 4 persistently splayed unable to move effectively 4 falls unable to move 5 persistently splayed no deliberate movement - -
- Open field arena is described in detail in Metten et al., 2004.
- Briefly, the open field consisted of a smooth, non-shiny, gray polyvinyl chloride surface (50 cm x 50 cm) surrounded by a 5-cm wall enclosure.
- A stationary Sony hi-resolution black and white video camera positioned directly above the enclosure recorded behavior throughout the procedure. The video monitor screen was divided into 9 equal quadrants, and used to assess changes in activity over time.
- Test agent: ethanol (EtOH), Pharmoco Inc, Brookfield CT, USA; EtOH solution was prepared at 20% v/v in saline.
- Saline (physiological saline): 0.9%, Baxter Healthcare Corp, Deerfield IL, USA.
- Isopropyl alcohol: 10% for wipe downs between tests.
Acclimation to test conditions
Mice were acclimated to testing environment for 1 h.
a. Following 45 s of baseline measurements in an open field square enclosure, each mouse is injected with one of two doses of alcohol, and placed into holding cages until ready for measurement at 5, 10, 15, 30 min intervals post-injection.
b. Mice are monitored via video camera for 45 s before and after injection, and after each subsequent interval. Activity and motor function is measured by counting the number of line crossings, or frequency a quadrant is traversed in the video monitor screen at that time.
c. To correlate behavioral presentation with alcohol intoxication, videotapes are examined and scored for wobbling and splaying behaviors during each 45 s-test according to ataxia assessment scale. The observer-rater is blinded to the dose of alcohol given, to the particular mouse strain, and to the sex of the mice.
d. The apparatus is cleared and wiped with 10% isopropyl alcohol after each test.
Line crossings and score on alcohol-induced intoxication or ataxia score.
Crabbe JC, Metten P, Yu CH, Schlumbohm JP, Cameron AJ, Wahlsten D. Genotypic Differences in Ethanol Sensitivity in Two Tests of Motor Incoordination. J Appl Physiol. 2003 Oct;95(4):1338-51. Epub 2003 Apr 18.
Kamens HM, Phillips TJ, Holstein SE, Crabbe JC. Characterization of the parallel rod floor apparatus to test motor incoordination in mice. Genes Brain Behav. 2005 Jun;4(4):253-66.