Lake3 project protocol
Behavioral and neuromuscular phenotypes, C57BL/6J-Chr#A/J/NaJ mouse chromosome substitution panel (2005)
Lake J, Donahue L, Davisson MTProject protocol — Contents
Workflow and sampling
Equipment
Reagents, supplies, and solutions
Procedure for testing motor coordination and balance using an accelerating Rotarod
Procedure for testing grip strength using a force gauge transducer
Procedure for testing exploratory activities using a hole-board
Data
References
Workflow (the same set of mice were tested in Lake2)
Data collected Behavioral and physical assessments Latency to fall Overall grip strength (3 trials) • Accelerating Rotarod, Economex (Columbus Instruments, Columbus, OH)
Figure 1. A. The 4-lane accelerating rotarod; B. Each lane is equipped with a built-in automatic timer; C. The speed of the rotating rod is adjusted manually.• Grip-strength transducer: Chatillon-Ametek digital Force Gauge model DFIS 2 (Columbus Instruments, Columbus, OH).
Figure 2. A. The Chatillon-Ametek Digital Force Gauge, DFIS 2, system; B. The triangular metal transducer (8 x 8 x 8 cm; ~0.0625" gauge wire) is situated 25 cm above an insulated platform; C. The Force Gauge digital display.• Exploratory hole-board (Ugo Basile, Comerio, Italy)
The exploratory hole-board consists of a piece of opaque Plexiglas, 24-inch x 24-inch in total area, and positioned 12 inches above the floor. See Figure 3 below.
Figure 3. A. Modified Exploratory Board with Perimeter. B. Dimensions of the exploratory board
• The board is separated into 4-inch perimeter (outlined in red) and 20-inch open arena (shaded).
• The perimeter is further divided into six 4-inch boxes along each side to determine the total number of line crossings and locomotor activity of the mouse.
• The open arena has 16, 1-inch holes, each containing infrared photobeam detectors.
• A black, matte Plexiglas box (30-inch high) surrounds the hole-board and contains the mouse within the arena.• Cleaning supplies: paper towels, 70% alcohol.
• Masking tape: for securing vertical wire-mesh and tail weights.
Acclimation to test conditions
Mice are allowed to acclimate to testing room over the weekend.
Procedures for behavioral and neuromuscular phenotyping
I. Procedure for testing motor coordination and balance using an Accelerating Rotarod
The Economex Accelerating Rotarod is used to analyze motor coordination and balance.
a. Mice are placed on a rod 3.5 cm in diameter, which is situated 40 cm above a foam platform.
b. Four mice are tested simultaneously, with each mouse being separated from the next by a 30 cm wide x 60 cm high opaque Plexiglas wall.
c. The mice are initially acclimated to the instrument by being placed on the stationary rod for three consecutive trials with a 1 min limit.
d. Each mouse is then replaced onto the rod at a constant speed of 4 rpm for three consecutive trials- each trial is limited to 1 min.
e. Finally, to measure balance performance, the mice are re-positioned on the rod, starting at a constant speed of 4 rpm, with an acceleration of 1 rpm/s over a 1 min period, and ending at about 64 rpm for three successive trials.
f. Latency to fall is recorded for each trial.II. Procedure for testing grip strength using a force gauge transducer
The Chatillon-Ametek Digital Force Gauge, DFIS 2 is used to determine the strength exerted by the forelimbs of a mouse in response to a constant downward force of 1.1N.
a. The grip-strength meter is positioned vertically, with the triangular metal transducer situated 25 cm above a foam platform.
b. A 4-oz/113.4 g weight is attached to the base of the tail with masking tape, which allows for the consistent application of a known downward force.
c. The mouse is then raised toward the triangular transducer and it instinctively grasps for the bar.
d. Care is taken to ensure the mouse is holding the grip transducer properly, with and only with both front paws.
e. Once an appropriate grip is assumed, the mouse and the weight are released simultaneously and peak force is measured in kg.
f. Three consecutive trials are performed and a mean peak tension over 3 trials is calculated for each animal.
g. A maximum peak tension over 3 trials is also calculated.III. Procedure for testing exploratory activities using a hole-board
The exploratory hole-board and open field is used to assess anxiety, exploratory, risk assessment, locomotor activity, and arousal behaviors.
a. Each mouse is placed in the center of the open arena for observation and evaluation.
b. While in the arena the behavior and performance of the mouse are recorded over a 5 min test session.
c. A Sony digital video camera is used to capture all events, and allows for further analysis by an unbiased observer.
d. When a mouse dunks its head into any of the holes, a beam break is recorded.Data collected by investigator
Rotarod
• Latency to fall from stationary rod in 3 trials
• Latency to fall from constant 4 rpm rod in 3 trials
• Latency to fall from 1 to 4 rpm acceleration rod in 3 trialsGrip strength: Overall grip strength for 3 trials.
Exploratory hole-board:
• stretch attends
• grooming episodes
• fecal boli
• urine puddles
• arena entries
• head dunks
• hole visits
• arena rearings
• perimeter rearings
• line crossings
Definitions & formulas
Total locomotor activity = (line crosses + arena rearings + perimeter rearings)
References
Bearzatto B, Servais L, Cheron G, Schiffmann SN. Age dependence of strain determinant on mice motor coordination. Brain Res. 2005 Mar 28;1039(1-2):37-42.
PubMed 15781044 Binder E, Droste SK, Ohl F, Reul JM. Regular voluntary exercise reduces anxiety-related behaviour and impulsiveness in mice. Behav Brain Res. 2004 Dec 6;155(2):197-206.
PubMed 15364478 Brooks SP, Pask T, Jones L, Dunnett SB. Behavioural profiles of inbred mouse strains used as transgenic backgrounds. I: motor tests. Genes Brain Behav. 2004 Aug;3(4):206-15.
PubMed 15248866 Buitrago MM, Schulz JB, Dichgans J, Luft AR. Short and long-term motor skill learning in an accelerated rotarod training paradigm. Neurobiol Learn Mem. 2004 May;81(3):211-6.
PubMed 15082022 Crabbe JC, Cotnam CJ, Cameron AJ, Schlumbohm JP, Rhodes JS, Metten P, Wahlsten D. Strain differences in three measures of ethanol intoxication in mice: the screen, dowel and grip strength tests. Genes Brain Behav. 2003 Aug;2(4):201-13.
PubMed 12953786 Li X, Mohan S, Gu W, Wergedal J, Baylink DJ. Quantitative assessment of forearm muscle size, forelimb grip strength, forearm bone mineral density, and forearm bone size in determining humerus breaking strength in 10 inbred strains of mice. Calcif Tissue Int. 2001 Jun;68(6):365-9.
PubMed 11685425 Ohl F, Roedel A, Binder E, Holsboer F. Impact of high and low anxiety on cognitive performance in a modified hole board test in C57BL/6 and DBA/2 mice. Eur J Neurosci. 2003 Jan;17(1):128-36.
PubMed 12534976 Ohl F, Sillaber I, Binder E, Keck ME, Holsboer F. Differential analysis of behavior and diazepam-induced alterations in C57BL/6N and BALB/c mice using the modified hole board test. J Psychiatr Res. 2001 May-Jun;35(3):147-54.
PubMed 11461710 Smith JP, Hicks PS, Ortiz LR, Martinez MJ, Mandler RN. Quantitative measurement of muscle strength in the mouse. J Neurosci Methods. 1995 Nov;62(1-2):15-9.
PubMed 8750080
