Mooney2 project protocol

Drug study: Nicotine effects on fear conditioning in males of 21 inbred strains of mice   (2021)

Mooney-Leber S, Gould TJ
With: Zeid D, Garcia-Trevizo P, Seemiller LR, Peltz G




Project protocol - Contents

Workflow and sampling

Step Description Equipment Data collected
1 Mice exposed to saline or nicotine for 12 days (chronic) Subcutaneous osmotic mini pump -
2 Mice trained Training/context chamber Percent freezing (baseline)
3 Contextual fear conditioning Training/context chamber Percent freezing during period between CS and US and during a context testing trial
4 Cued fear conditioning Cued fear chamber Percent freezing for pre-CS exposure and CS exposure in a novel context

Procedures

Procedure 1: Nicotine administration

Equipment, software, and supplies

  • Subcutaneous osmotic mini pump (Alzet Inc., #1002, Cupertino, CA, USA)

Reagents and solutions

  • Isoflurane
  • Nicotine

Steps

  1. Mice are anesthetized with 3.5% isoflurane.
  2. Subcutaneous osmotic mini pumps are surgically implanted using aseptic procedures.
  3. Nicotine in saline is administered through pumps at 0 (baseline), 12, and 18 mg/kg/day for 12 days.

Procedure 2: Fear conditioning

Equipment, software, and supplies

  • Chamber for fear conditioning training and contextual fear learning, noise-attenuating, with metal bar grid flooring (18.8 x 20 x 18.3 cm), with side-mounted speakers and fans to provide ventilation and background noise (65 dB), with ceiling-mounted camera (MED Associates, St. Albans, VT, USA)
  • Chamber for cued fear learning, noise attenuating with solid plastic flooring (20.32 x 22.86 x 17.78 cm), with side-mounted speakers and fans to provide ventilation and background noise (65 dB), with ceiling-mounted camera, with vanilla background odor (MED Associates, St. Albans, VT, USA)
  • Cameras (Ikegami, Tokyo, Japan)
  • EthoVision XT (Noldus, Wageningen, Netherlands)
  • Med-PC software and hardware (MED Associates, St. Albans, VT, USA)

Steps

  1. Following nicotine exposure for 12 days, mice are trained and tested in both contextual and cued fear conditioning.
  2. For training, mice are placed in an operant chamber for a total of 5 min. The first 2 min consists of a stimulus-free period (baseline) followed by 2 conditioned stimulus (CS; 30 s 85 dB white noise) - unconditioned stimulus (US; 2 s 0.45 mA foot shock) pairings presented two minutes apart, in which the US overlapped with the last 2 s of the CS.
  3. The 2-min period between the two CS-US pairings serves as the immediate or post-shock period.
  4. Two associations are formed during the training trial and are used to assess fear conditioning: 1) Between the tone CS and footshock US (cued fear conditioning) and 2) Between the footshock US and the context/environment (contextual fear conditioning).
  5. To assess the strength of these unique forms of learning and memory, mice are tested for both contextual and cued learning 24h after training.
  6. To test contextual fear learning, mice are placed back inside the training chamber over a 5-min trial with no stimulus presentation.
  7. Cued testing occurred at least one hour after context testing, for which mice are placed in a novel chamber for a 3-min baseline assessment (pre-cue), followed by a 3-min CS exposure (cued). Novel cues (plastic flooring and vanilla scent) are present during cued testing to minimize generalization to the training chambers.
  8. All sessions are video recorded. Freezing behavior is tracked during all sessions via EthoVision XT.