Biological evaluation of a Mice in Space housing system, JCPB-00-0611 (supplement C) Page 2
Description of behavioural tests
Following the experimental phase of 25 days, animal pairs from the MIS and MISg cage group (n=8 each) denoted as “B” (cf. experimental design in supplement A) and, additionally, one animal pair from the MIS group previously denoted as “T” (n=2; Trial 4) were subjected separately to a battery of fundamental tests according to the following schedule and order:
Motor performance: Motor testing consisted of tests for grip strength, motor coordination, and cage activity. Grip strength was measured using a commercial grip strength meter consisting of a T-shaped bar connected to a digital dynamometer (Ugo Basile, Comerio, Italy). Mice were placed before the bar, which they usually grabbed spontaneously, and gently pulled backwards until they released the bar (the peak force was measured). In a first series, the mice were allowed to grasp the grid with the fore limbs only; in a second series they grasped the grid with the four legs. In each series, the individual performance, measured by the best maximal value among ten graspings, was normalised with reference to the animal weight (mN/g). Two tests were repeated with three days interval. Motor coordination and balance were tested using a commercial accelerating rotarod (Med. Associates, St. Albans,VT). The mice were placed on the rod, rotating at an initial speed of 4 rpm; the speed gradually increased from 4 to 40 rpm over a 5 min session. The mice were given three successive trials, and the test was repeated on two consecutive days. The performance scored the latency to fall. Cage activity was recorded using a lab-build activity logger connected to three infrared (IR) photo beams (D'Hooge et al. 2005). Mice were put separately in 20 x 30 cm2 transparent cages, placed between the photo beams, and activity was expressed as beam crossings for each 30 min, during a 24 h interval.
Anxiety: Emotionality testing consisted of two tests of anxiety (light/dark [L/D] box and elevated plus-maze [EPM]) and a measure of depression state with Porsolt forced swimming test. L/D box testing consisted of a 30 x 30 cm2 square arena with a 10 x 30 cm² dark box placed inside along one of the walls of the arena. The box had one entry hole. The illumination was 500 lux in the light compartment and 12 lux in the dark compartment. The mouse was placed in the centre of the light arena. The delay to the first entry in the dark box, the number of transitions between the light and dark compartments, the total time spent in the light area, and the number of rearings were recorded during 10 minutes. The elevated plus-maze (EPM) consisted of a plus-shaped maze with two arms (5 cm wide) closed by side walls (light 4 lux) and two arms without walls (light 200 lux). The mice were placed at the centre of the maze and were allowed to explore it freely for 10 min. Exploration activity in the arena (i.e., area was subdivided by 4x4 squares [5 cm2] in center surrounded by a peripheral border area [5 cm wide] around the inner wall) was recorded by four infrared (IR) beams connected to a computerized activity logger. In addition, movements between area squares / borders were recorded on line with the Ethovision (Noldus) video analysis of movement. The frequency and the time spent in the open arms, closed arms and in the central area was counted. The frequency of rearings was noted. For the Porsolt test, the mice were placed in a glass tank (height 20 cm, diameter 140 cm) filled with 12 cm of water at a temperature of 22°C. The total swimming time measured during 6 min provided an indication of the level of depressive state.
Exploration testing was carried out in an open field consisting of a 50 x 50 cm² arena illuminated with white light (340 lux). Each mouse was individually placed in the centre of the open field and the movements were recorded using the EthoVision video tracking system (Noldus, Wageningen, The Netherlands) for 10 min. For the analysis of exploration behaviour, the arena was virtually divided in a central zone (20 x 20 cm²) and a peripheral zone (5 cm wide area around the wall). The following parameters were analysed: total distance travelled; duration of movement; time in the central zone; time in the border zone. Rearings were also manually counted.
Cognition and memory was evaluated by the passive avoidance test and T-Maze alternation task. Passive avoidance learning was tested in a step-through box. During training, dark-adapted mice were put in the small illuminated compartment (1500 lux) of the box. After 5 seconds, a sliding door to the larger dark compartment (4 lux) was opened, and entry latency was recorded. The door was closed as soon as all four feet were on the grid floor, and a slight foot shock (0.3 mA, 1 sec) was delivered using a constant current shocker (Medical Associates). Retention was tested 24 h later using the same procedure, and entry was recorded up to 300 s cut-off. The latency to enter the dark compartment on second day was a measure of memory capability (D'Hooge et al. 2005). The set-up consisted of a T-maze with a starting arm and two opposed arms that can be individually closed with a door. The test consisted in a succession of one forced choice and 14 free choices, following the T-CAT procedure (Gerlai, 1998). At first trail one arm was closed, while the other was still open (forced choice). After five seconds in the starting arm, the mouse was free to explore the maze until it visited the open arm and returned to the starting zone. The mouse was restricted by a door for five seconds. Meanwhile the goal door was open. Then the mice could freely choose a goal arm. When it entered an arm (four feet inside), the other arm was closed, and the mouse was left free to explore the maze until it returned to the starting zone where it was restricted again for 5 seconds. The door of the goal arm was open and the procedure was repeated for 14 trails. The frequency of alternation over the 14 trails was recorded. The test was repeated for two days. In this situation the mice alternated spontaneously.
Aggressiveness: The experiment was carried out in a neutral arena (50 x 50 cm²) with a fresh layer of saw dust on the floor. Two unfamiliar mice were gently placed in the diagonal corners of the arena. The latency for the first contact was recorded, then the mice were video taped for 10 minutes. The video tapes were then viewed and the number of offensive (head, flank, tail or genitals sniffing; tail rattling, pursuit, biting, attack) behaviours, defensive (side rearing, flank exposition, lying down on the back, flight), and self-comfort behaviours (self-grooming, rearing, digging) were counted. The sawdust layer was changed after each pair interaction.