Metten1 project protocol

Drug study: Assessment of ethanol-induced intoxication using a standard rating scale, in 9 inbred strains of mice   (2004)

Metten P, Crabbe JC, Wahlsten D
With: Best KL, Cameron AJ, Saultz AB, Zuraw JM, Yu C-H




Metten1 Protocol

Project protocol — Contents
 Workflow, sampling, and experimental treatment
Equipment
Reagents, supplies, and solutions
Procedure
Data
References



Workflow, sampling, and experimental treatment

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.

Workflow

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

Treatment

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
-
-

Equipment

  • 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.

Reagents, supplies, solutions

  • 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.

Procedure

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.

Data collected by investigator

Line crossings and score on alcohol-induced intoxication or ataxia score.



References

    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. PubMed 12704090

    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. PubMed 15924557