Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Open field test
• Procedure: Fear conditioning
• Procedure: Forced swim test
• Procedure: Sensorimotor gating
• Procedure: Blood glucose levels
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Mice assigned an arbitrary five-digit identification number that obscures their strain and genotype from experimenter	-	-
2	Mice weighed at 50 +/- 1 days of age	Scales	Body weight
3	Open field test followed by at least 4 days of rest	Open field arena	Exploratory activity and duration in center
4	Fear conditioning test followed by at least 4 days of rest	Fear conditioning chamber	Contextual fear learning and cued fear learning
5	Forced swim test followed by at least 4 days of rest	Swimming bucket	Duration of immobility
6	Sensorimotor gating followed by at least 4 days of rest	Acoustic startle chamber	Acoustic startle response and prepulse inhibition
7	Fasted (16 h) blood glucose followed by at least 4 days of rest	Glucometer	Fasted blood glucose level
8	Baseline blood glucose	Glucometer	Baseline blood glucose level

Equipment and supplies

• Scales
• Open field arena: Versamax (AccuScan Instruments, Columbus OH)
  • 40 x 40 x 30 cm
  • Housed within sound-attenuating chamber
  • 80 lux overhead lighting
  • Rear wall fan masking background noise
  • Center region defined as the inner 20 x 20 cm
• Fear conditioning chamber
  • 29 x 19 x 25 cm
  • Metal walls on each side, a clear plastic front, back walls and ceiling
  • Stainless steel bars on the floor (Med Associates, St. Albans VT)
  • 10 lux fluorescent lighting
  • Fan masking background noise
• FreezeFrame Software (Actimetrics, Evanston IL)
• Forced swim test
  • White polyethylene buckets (15.2 cm depth x 20.3 cm diameter)
  • Filled with 25°C water to 3.8 cm below the top of the bucket
  • Water maintained at 23-25°C
• Sensorimotor gating
  • 5-cm diameter plexiglas cylinder on a platform contained within a lighted, ventilated chamber (San Diego Instruments, Can Diego CA)
  • Cylinder connected to a piezoelectric accelerometer to measure the startle response
• Contour TS Glucometer (Bayer, Pittsburgh PA)

Reagents and solutions

• 10% isopropanol (for cleaning equipment between tests)
• 0.1% acetic acid (for cleaning fear conditioning chamber on day 3 of testing (altered context))

Procedure: Open field test

1. Mice are placed in the arena and allowed to freely explore for 30 min.
2. Positional data are automatically converted into the distance traveled by the software, which monitors infrared beam breaks within the arena.
3. Exploratory activity is defined as the total distance traveled during the test.

Procedure: Fear conditioning

1. Carried out in a three day paradigm; behavior is digitally recorded and is analyzed by FreezeFrame software; a 5-min trial occurs on each day of testing at the same time each day.
2. On day 1: mice are conditioned to associate a test chamber and a tone with a shock by being placed into the chamber and being exposed 180 s later to two conditioned stimuli consisting of an 85 dB, 3 kHz tone (the tone persists for 30 s) and is co-terminated with an unconditional stimulus, a 2 s, 0.5 mA foot shock.
3. On day 2: mice are re-exposed to exactly the same testing environment but no tones or shocks are given.
4. On day 3: mice are exposed to the conditioned stimulus in an altered context: the metal shock grid, chamber door and one wall are covered with white plastic; yellow film is placed over the overhead light; 0.1% acetic acid is used to clean the grid between mice; the fan is partially obstructed to change noise level; the holding cages contain no bedding; experimenter is a different individual who wears gloves of a different material.
5. The fear phenotypes are the average proportion of time spent freezing in the 30-180 s interval on day 2, and average proportion of time spent freezing to the tone during the 180-210 s and 240-270 s intervals on day 3.

Procedure: Forced swim test

1. Mice are placed in the bucket of water for 6 min.
2. Behavior is digitally recorded and immobility is automatically scored during the last 4 min of the test by Noldus Ethovision XT software.
3. The duration of immobility is defined as the number of seconds spent with a mobility threshold of <2% movement between frames.

Procedure: Sensorimotor gating

1. Mice are placed in the apparatus and experience 5 min of 70 dB white noise followed by 62 trials which occur with the 70 dB noise in the background.
2. Testing consists of pulse-alone trials (40 ms, 120 dB burst); no-stimulus trials; and prepulse trials (20 ms prepulse, 3, 6, or 12 dB above background noise) followed by 100 ms later by a 40 ms, 120 dB pulse.
3. Trials are arranged in four blocks: blocks 1 and 4 are pulse alone trials; blocks 2 and 3 contain pseudo-random combinations of pulse alone, no stimulus, and each type of prepulse trial (3, 6, and 12 dB).
4. Responses are recorded for 65 ms after the beginning of the 120 dB stimulus.
5. The inter-trial interval is 9-20 s (average 15 s) throughout the test.
6. The acoustic startle response is the average startle amplitude (SA) measured in the pulse-alone trials in testing blocks 2 and 3.
7. The prepulse inhibition (PPI) phenotype is defined as the difference of the average startle amplitude during the 6 dB prepulse trials and the average startle amplitude during the pulse-alone trials, normalized by the pulse-alone startle amplitude: PPI = (SA_pulse - SA_prepulse) / SA_pulse.

Procedure: Blood glucose levels

1. Fasted blood glucose levels are remeasured between 0900-1000 h after lab chow has been removed from the cage for 16 h overnight.
2. Baseline blood glucose levels are measured between 0900-1600 h.

Data collected by investigator

• Open field test
  
  • exploratory activity
  • duration in center
• Fear conditioning
  • contextual fear learning
  • cued fear learning
• Forced swim test
  • duration of immobility
• Acoustic startle test
  • acoustic startle response
  • prepulse inhibition
• Blood glucose levels
  • fasted
  • baseline