Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Elevated maze test
• Procedure: Open field test
• Procedure: Novelty-induced fear test
• Procedure: Plasma corticosterone levels
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Elevated maze test (5 min)	Elevated maze	Time spent in open arms, enclosed arms, middle platform, and total number of entries into the arms
2	Open field test under dim lighting conditions (5 min)	Opto-Varimex	Distance traveled, ambulatory time in the horizontal plane, and number of vertical movements (rearings)
3	Open field test under bright lighting conditions (5 min)	Opto-Varimex	Distance traveled, ambulatory time in the horizontal plane, and number of vertical movements (rearings)
4	Novelty-induced fear test (10 min)	Opto-Varimex	Number of approaches to food, latency to start eating, and time eating
5	Plasma corticosterone measured	Restraining cages	Plasma corticosterone levels

Equipment and supplies

• Elevated maze: constructed entirely of black Plexiglas, consisting of two open arms (10 x 5 cm) and two enclosed arms (30 x 5 x 15 cm) extending from a center platform (5 x 5 cm); a 0.5 cm high Plexiglas strip is attached to the open arms of the maze to prevent animals from falling; maze is elevated 31 cm off the floor; testing performed in a windowless room under dim light (two fluorescent 15 W bulbs directly facing the wall on a shelf 4 m away and 1 m above the center of the maze)
• Opto-Varimex computerized activity monitor (Columbus Instruments, Columbus OH) under dim lighting conditions (described above) or bright lighting conditions (two fluorescent 15 W bulbs located 70 cm over the center of the apparatus)
• Lucite restraining cages (Harvard Instruments)

Reagents and solutions

• Ethanol/water (50% v/v) for wiping down each apparatus between tests for each mouse

Acclimation period

One week after arrival at the test facility, mice were moved from the housing colony room to the testing area in their home cages and allowed to adapt to the new environment and light level for 1 h before testing.

Procedure: Elevated maze test

1. Group 1: naive mice
2. Scoring is for 5 min under dim light.
3. Each mouse is placed at the intersection of the four arms so that its head is in the center of the platform.
4. Mice are considered to be in open or closed arms only when they have all four legs within; otherwise they are scored as being on the middle platform.
5. Parameters are recorded using an Apple IIe computer.
6. Percentage of time spent in the open arms is calculated over the sum of time spent in the open and closed arms; the percentage of entries into the open arms is calculated based on the total arm entries.

Procedure: Open field test

1. Group 1: same animals tested in Elevated Maze one day prior; under dim lighting conditions
2. Group 2: naive mice; under bright lighting conditions
3. Mice are placed in the center of the Opto-Varimex open field arena and spontaneous activity scored for 5 min with a computerized activity monitor.

Procedure: Novelty-induced fear test

1. Group 3: naive mice; under bright lighting conditions.
2. Mice are food-deprived for 24 h.
3. Mice are placed in the corner of the Opto-Varimex open field that contains a pellet of rat chow in the center.
4. Scoring is for 10 min.

Procedure: Plasma corticosterone levels

1. Group 4: naive mice; no stress
2. Group 5: naive mice; 30 min of restraint stress
3. For determination of basal corticosterone values, mice are removed from their home cage, and rapidly transferred to a different room where trunk blood is collected (< 1 min).
4. For determination of stress-induced corticosterone values, mice are placed in individual lucite restraining cages for 30 min; after stress period, mice are rapidly transferred to a different room and trunk blood collected.
5. Basal and stress groups are performed concurrently.
6. Mice are euthanized in a predetermined order, randomized for both strain and treatment.
7. Plasma corticosterone levels are determined by radioimmunoassay.

Data collected by investigator

• Elevated Maze
  • time spent in open arms
  • time spent in closed arms
  • time spent in middle platform
  • total number of entries into the arms
• Open Field Test
  • distance traveled
  • ambulatory time
  • vertical movements
• Novelty-induced Fear Test
  • number of approaches to food
  • latency to start eating
  • time eating
• Plasma Corticosterone Levels
  • basal level
  • stress-induced level