Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Light-dark test
• Procedure: Open field test and methamphetamine sensitivity
• Procedure: Forced swim test
• Procedure: Sensorimotor gating
• 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	Light-dark test followed by at least 4 days of rest	Light-dark box	Percent time in light
3	Open field test + methamphetamine sensitivity followed by at least 4 days of rest	Open field arena	Exploratory activity, duration in center, and methamphetamine sensitivity
4	Forced swim test followed by at least 4 days of rest	Swimming buckets	Duration of immobility
5	Sensorimotor gating	Acoustic startle chamber	Acoustic startle response

Equipment and supplies

• Scales
• 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

Reagents and solutions

• 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

1. Mice are placed in the light side of the chamber and their behavior is video recorded for 5 min.
2. 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

1. Methamphetamine sensitivity is assessed in a 3-day test that is given at the same time each day.
2. Mice are transported from the vivarium to the testing room and are allowed at least 30 min to habituate in their home cages.
3. 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.
4. 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.
5. 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).
6. Following the appropriate treatment, mice are immediately placed in the activity chamber and their locomotor activity monitored for 30 min.
7. 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

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.

Data collected by investigator

• Light-dark box
  • percent time in the light box
• Open field test
  
  • exploratory activity
  • duration in center
  • methamphetamine sensitivity
• Forced swim test
  • duration of immobility
• Acoustic startle test
  • acoustic startle response