Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Open field test
• Procedure: Radial maze test
• Procedure: Resident intruder test
• Procedure: Hippocampal histology and morphometry
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Open field test	Open field apparatus	Time and distance in center and in periphery; total activity; number of leanings, rearings, jumping; number of fecal boli; grooming
2	Weigh mice	Scales	Body weight
3	Radial maze test	Radial maze apparatus	Time to complete task; number of arms visited; number of errors made; mean duration per arm
4	Resident intruder test	Home cage, stopwatch	Aggression: whether an animal attacks (score) and attack latency
5	Histopathology	Microscope, ImageJ	Size of hippocampus and specific subfields of hippocampus

Equipment and supplies

• Open field apparatus
  • Rectangular cage (109 x 49 x 49 cm) with a clear Plexiglas front panel that is placed in a brightly lit room (~300 lux)
• Ethovision (v. 3 and upgraded to v. 8 over the course of the project), Noldus, Wageningen, The Netherlands
• Radial maze apparatus
  • Eight-arms; central part measures 22 cm in diameter and the arms are 25 cm long, 6 cm high and 6 cm wide
  • Arms are enclosed with transparent Plexiglas and the floor of the maze is gray
• The Observer XT (Noldus, Wageningen, The Netherlands)
• Stopwatch
• Microscope (Leica, DM6000 B x 10, Leica, St Jorioz, France)
• ImageJ (NIH v. 1.48)

Reagents and solutions

• Postfixative solution (3% glutaraldehyde, 20% sucrose)
• Timm's silver sulfide stain
• Disinfectant

Procedure: Open field test

1. Thirty minutes prior to testing, mice are taken to the experiment room (all tests take place between 0830 and 1730h).
2. Mice are placed in the center and, starting 5 s later, observed directly and continuously for 20 min.
3. Overall locomotor activity and time spent in the periphery (surface within 5 cm of the walls) are recorded automatically using Ethovision software.
4. Rearing (standing upright on hind legs, while forepaws are free), leaning (standing upright on the hind legs, one or both forepaws against the wall), jumping, and grooming frequency and duration are scored manually in Ethovision by using a computer keyboard with several keys coding for the different behaviors.
5. Defecation is quantified by counting the number of fecal boli deposited during a session.

Procedure: Radial maze test

1. Mice are housed individually and testing begins 4 days after the open field test.
2. At the end of each arm of the radial maze, some food pellets are deposited behind a perforated wall in order to prevent the mice from smelling the presence of absence of the food reward. A small fresh food pellet (~10 mg) is placed in each arm behind a low barrier preventing the animal from seeing whether a specific arm is still baited or not.
3. The maze is always oriented in space in the same way, but turned by 45° daily. Several extra maze cues are provided close to the arms in a fixed configuration.
4. A confinement procedure is used, consisting of transparent guillotine doors at the entrance of each arm. These doors are lowered and kept closed for 5 s after the mouse has returned to the central platform (this procedure is known to disrupt chaining responses and kinesthetic strategies in mice).
5. Two identical mazes are used, one for females, the other for males.
6. The maze is placed on the floor of a dedicated lighted procedure room in the animal facility.
7. On the first day, mice are subjected to a habituation session in which they are allowed to explore the maze freely for 10 min (arm doors remain open, and no food is accessible).
8. Mice are then subsequently deprived of food (but not water), and are maintained at about 85% of their initial body weight throughout the experiment.
9. The habituation trial is followed by 5 days of training with one trial per day, during which all eight arms contain a food reward. Mice are confined between arm visits for 5 s on the central platform by lowering the Plexiglas doors.
10. On the first 2 days of training, animals often enter an arm without eating the food reward; therefore, only results from the last 3 days of the training are included in the statistical analyses.
11. Trials end when an animal has found and eaten all 8 rewards except on the first 2 days of training, where trials are stopped after a maximum of 30 min, even if an animal has not yet eaten all rewards.
12. Errors are counted if an animal enters an arm that has been visited before or without eating the reward.
13. The experimenter is seated next to the maze, always in the same position.
14. Arm visits are recorded using The Observer XT software.

Procedure: Resident intruder test

1. Mice are housed individually and testing begins on the 5th day after the end of the radial maze test.
2. Tests take place on two subsequent days.
3. As standard opponents, males of the pacific A/JOlaHsd strain are used (never initiate attacks).
4. The observation starts when the intruder is introduced into the home cage of the test mouse and lasts until an attack occurs or a maximum of 10 min.
5. Attacks are defined as an animal lunging towards its opponent and attempting to bite it. Observations are immediately stopped at this point to avoid wounding.
6. Attack latency is measured directly using a stopwatch assigning a score of 600 s to animals that do not attack.
7. The test is repeated the next day with a different intruder mouse.

Procedure: Hippocampal histology and morphometry

1. Three days after the resident intruder test, mice are perfused intracardiacally with sodium sulfide followed by glutaraldehyde and their brains removed and placed in a postfixative solution of 3% glutaraldehyde and 20% sucrose for 24h.
2. Sections are developed for Timm's silver sulfide stain for heavy metals, which is associated with synaptic enzymes containing zinc and allows the visualization of the terminal fields of the hippocampal projections in the form of colored bands and patches.
3. For morphometry, the brains with the clearest histology/coloration are selected, for a maximum of five males and five females per strain.
4. Starting at the midseptotemporal level, immediately after the disappearance of the septal pole, 10 horizontal 40-µm sections are taken and measured every second one for a total of five sections.
5. Both left and right hippocampi are measured.
6. A microscope is used to make micrographs using an automated procedure to create high definition mosaic pictures. The morphometrical analysis is performed using Image J with macros developed by one of the investigators.

Data collected by investigator

• Open field test
  • Center and periphery distance and time
  • Total activity
  • Number of leanings, rearings, jumping
  • Number of fecal boli
  • Grooming frequency and duration
• Body weight
• Radial maze test
  • Time to complete task
  • Number of different arms visited
  • Number of errors made
  • Mean duration per arm visit
• Resident intruder test
  • Latency to attack
  • Whether an animal attacks or not
• Hippocampal histology and morphometry
  • Total hippocampus
  • Intra- and infrapyramidal mossy fibers
  • Hilus
  • Suprapyramidal mossy fibers
  • Stratum pyramidale
  • Stratum oriens
  • Stratum radiatum
  • Stratum lacunosum-moleculare