| Mice assigned an arbitrary five-digit identification number that obscures their strain and genotype from experimenter
||Light-dark test followed by at least 4 days of rest
||Percent time in light
||Open field test + methamphetamine sensitivity followed by at least 4 days of rest
||Open field arena
||Exploratory activity, duration in center, and methamphetamine sensitivity
||Forced swim test followed by at least 4 days of rest
|Duration of immobility
||Acoustic startle chamber
||Acoustic startle response
- Syringes and needles
- Light-dark box
- White plastic testing chambers (40 x 40 x 30 cm) containing a black plastic box insert (20 x 20 x 30 cm)
- Light side illuminated by fluorescent overhead light (~500 lux)
- Black insert has removable lid that blocks all light from entering
- A small doorway located on the insert wall facing the light side allows mice to freely pass between the light and dark sides
- Noldus Ethovision XT v 5.1 (Noldus Information Technology, Leesburg VA)
- 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
- 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
- 10% isopropanol (for cleaning equipment between tests)
- 0.9% sterile saline
- Methamphetamine hydrochloride (Sigma-Aldrich, St. Louis MO) dissolved in sterile 0.9% saline
Procedure: Light-dark test
- Mice are placed in the light side of the chamber and their behavior is video recorded for 5 min.
- Noldus Ethovision XT software is used to record and automatically score the time spent in the light vs. dark side.
Procedure: Open field test and methamphetamine sensitivity
- Methamphetamine sensitivity is assessed in a 3-day test that is given at the same time each day.
- Mice are transported from the vivarium to the testing room and are allowed at least 30 min to habituate in their home cages.
- On the first and second days of testing, mice are removed from their home cages, weighed, and placed in individual holding cages with clean bedding.
- Mice then receive an i.p. injection of sterile 0.9% saline solution and are immediately placed in individual activity chambers where locomotor activity is monitored for 30 min.
- On the third day of testing, mice receive i.p. injections of 2 mg/kg methamphetamine (all injections are administered in a volume of 10 mL/kg body weight).
- Following the appropriate treatment, mice are immediately placed in the activity chamber and their locomotor activity monitored for 30 min.
- Exploratory activity and duration in center phenotypes are assessed on day 1. The methamphetamine sensitivity phenotype is exploratory activity on day 3 minus the exploratory activity on day 2.
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.
- Light-dark box
- percent time in the light box
- Open field test
- exploratory activity
- duration in center
- methamphetamine sensitivity
- Forced swim test
- Acoustic startle test
- acoustic startle response