| Mice assigned an arbitrary five-digit identification number that obscures their strain and genotype from experimenter
||Mice weighed at 50 +/- 1 days of age
||Open field test followed by at least 4 days of rest
||Open field arena
||Exploratory activity and duration in center
||Fear conditioning test followed by at least 4 days of rest
||Fear conditioning chamber
|Contextual fear learning and cued fear learning
||Forced swim test followed by at least 4 days of rest
||Duration of immobility
||Sensorimotor gating followed by at least 4 days of rest
||Acoustic startle chamber
||Acoustic startle response and prepulse inhibition
||Fasted (16 h) blood glucose followed by at least 4 days of rest
||Fasted blood glucose level
||Baseline blood glucose
||Baseline blood glucose level
- 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)
- 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
- Mice are placed in the arena and allowed to freely explore for 30 min.
- Positional data are automatically converted into the distance traveled by the software, which monitors infrared beam breaks within the arena.
- Exploratory activity is defined as the total distance traveled during the test.
Procedure: Fear conditioning
- 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.
- 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.
- On day 2: mice are re-exposed to exactly the same testing environment but no tones or shocks are given.
- 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.
- 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
- Mice are placed in the bucket of water for 6 min.
- Behavior is digitally recorded and immobility is automatically scored during the last 4 min of the test by Noldus Ethovision XT software.
- The duration of immobility is defined as the number of seconds spent with a mobility threshold of <2% movement between frames.
Procedure: Sensorimotor gating
- 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.
- 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.
- 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).
- Responses are recorded for 65 ms after the beginning of the 120 dB stimulus.
- The inter-trial interval is 9-20 s (average 15 s) throughout the test.
- The acoustic startle response is the average startle amplitude (SA) measured in the pulse-alone trials in testing blocks 2 and 3.
- 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 = (SApulse - SAprepulse) / SApulse.
Procedure: Blood glucose levels
- Fasted blood glucose levels are remeasured between 0900-1000 h after lab chow has been removed from the cage for 16 h overnight.
- Baseline blood glucose levels are measured between 0900-1600 h.
- Open field test
- exploratory activity
- duration in center
- Fear conditioning
- contextual fear learning
- cued fear learning
- Forced swim test
- Acoustic startle test
- acoustic startle response
- prepulse inhibition
- Blood glucose levels