B Spontaneous Discrimination with Enrichment - michelletytong/TRGProtocols GitHub Wiki

Odours

An “odor set” consists of a habituation odor (Ohab), one test odor. All odorants will be diluted in mineral oil to an approximate vapor partial pressure of 1.0 Pa (See Odour Dilutions for specific mixture amounts for the specific odors used)

Pre- and Post-test parameters

  1. Inter-trial Interval (ITI) will remain at a constant value of 5 mins
  2. Each odor (including mineral oil) will be presented for a total of 50sec
  3. Mineral Oil (MO) will be presented first (1 trial) – experimenter times “investigation time” using a Seiko stop watch a. Investigation is any time a mouse’s nose is with 1 cm of the teaball
  4. The Ohab will be presented 4 times (4 trials) – experimenter times investigation time
  5. T1, the test odor, (1 trial) – experimenter times investigation time
  6. There will be a total of 6 trials (Total: ~ 30 minutes)

Pre- and Post-test Steps

  1. Lay out the number of clean teaballs needed per mouse (1 teaball/odor; each mouse will require 6 teaballs)
  2. Cut out 1 inch squares of filter paper (#1; Whatman, Florham Park, NJ) on which odor will be pipetted – fold it twice into a square
  3. Place 1 square in each open teaball
  4. Before each trial, pipette 60 μL of the diluted odor directly onto filter paper and latch the teaball.
  5. To “present” the odor, hang the teaball in the corner of the test cage to allow mice to investigate (approximately same level each time and same corner each trial for every mice) – experimenter will be timing and recording
  6. Each cage should have 2 stopwatches: one to measure the trial duration and ITI; another to record investigation times
  7. Record time mouse spends sniffing teaball (active sniffing <1 cm away from teaball)

Running multiple mice at once (by one person) What people have done before:

  • Staggering: one person can work on a maximum 4 mice by staggering the mice 1 min apart (e.g. Mouse 1 is presented with odor at time 0 min, undergoes 50s of trial & ITI of 5 mins begins. Mouse 2 presented with odor at time 1 min, undergoes 50s of trial & ITI of 5 mins begins etc.)
  • We’ll need to work this out as we go along and get more familiar with the process. Coordinating with the brain tissue harvesting time will also affect this.
  1. When finished with experiment for the day, teaballs need to be soaked in soapy tub and then washed, and rinsed. Lay out on flat surface to dry.

Enrichment Steps

All this can be done in the Mouse Housing Room.

  1. Lay out the number of clean teaballs needed per cage (2 teaball/cage).
  2. Cut out 1 inch squares of filter paper (#1; Whatman, Florham Park, NJ) on which odor will be pipetted – fold it twice into a square
  3. Place 1 square in each open teaball
  4. Before each trial, pipette 60 μL of the diluted odor directly onto filter paper and latch the teaball. In the Summer 2019 experiment, the odor depends on the Experimental Group. They will either get two teaballs of 1.0Pa +limonene or two teaballs of mineral oil.
  5. Hang the two teaballs in the opposite corners of the home cage for an hour.
  6. Remove the teaballs after the hour, soak in tub of soapy water, wash, and rinse. Lay out on flat surface to dry.

Enrichment will occur one hour per day for 13 consecutive days. The one-hour period should occur at roughly the same time each day.