Chesler4 project protocol

Behavioral and nociception traits in a Diversity Outbred (DO) mouse population and 8 founder inbred strains   (2013)

Logan RW, Robledo RF, Recla JM, Chesler EJ
With: Philip VM, Bubier JA, Jay JJ, Harwood C, Wilcox T, Gatti DM, Bult CJ, Churchill GA

Chesler4 Protocol

Project protocol - Contents

Workflow and sampling

Data collected *
Open field test
Open field arena
Distance traveled; time in corners, periphery, and center; time immobile
Light-dark box test
Light-dark box
Distance traveled, transitions, time in light
4 Visual-cliff avoidance test Modified open field boxes
Distance traveled, velocity, transitions
5 Hot-plate test Hot plate Latency of hindpaw withdrawal
9 Tail-suspension test Ring stand Frequency climbing, time immobile, latency immobile

* Additional data available Chesler4_suppdata.xlxs

Equipment and supplies

  • Ethovision XT (Noldus Information Technology, Wageningen, The Netherlands)
  • Open-field arena (see full description below)
  • Light-dark box (see full description below)
  • Visual-cliff arena (see full description below)
  • Hot plate analgesia meter (IITC Life Science Inc, catalog #39, Woodland Hills CA)
  • Tail suspension apparatus (see full description below)
  • Paper cones
  • Masking tape

Reagents and solutions

  • Ethanol for disinfecting

Procedure: General

    1. Mice are tested in order of perceived stressfulness of the test (see Workflow).
    2. Mice are randomly assigned to testing groups such that an equal number of male and female mice are tested each day (n=24 per sex).
    3. Mice are allowed to acclimate at least 6 wks after shipping from vendor.
    4. For the open-field, light-dark box, and visual-cliff tests, mice are habituated to test room for 1 h prior to testing; for the hot plate and tail-suspension tests, mice are habituated 30 min.
    5. For each assay, mice are removed by the tail then returned to the clean side of a duplex home cage until each cage mate completes testing.
    6. A single experimenter handles mice for each test.
    7. Behavioral measures are recorded and analyzed by real-time video tracking using Ethovision XT.

Procedure: Open-field test

    1. Apparatus: opaque Plexiglas box (39x39x39 cm) with a dark gray floor, illuminated at 43 +/- 4 lux in a 10x15 ft room; zones of arena are delineated as follows -- center: 10x10 cm; corners: 4x4 cm; periphery: 31x4 cm.
    2. Each mouse is placed into the center of the arena.
    3. Behaviors are recorded for 20 min.

Procedure: Light-dark box test

    1. Apparatus: open-field apparatus with an insert evenly dividing the apparatus into light and dark compartments (light side illuminated at 17 +/- 2 lux; compartments separated by a sliding door).
    2. Mice are placed into the dark compartment when sliding door is closed.
    3. Sliding door is open and the lid is closed.
    4. Behaviors are recorded for 20 min.

Procedure: Visual-cliff avoidance test

    1. Apparatus: open-field box with clear Plexiglas bottom that is secured so that half of the floor overhangs the table-top to create an appearance of a ledge drop-off; checkerboard table cloth is draped from the table-top to the floor (serves to enhance the visual appearance of the cliff); vertical distance between box floor and testing room floor is 93 cm; an opaque neutral zone (10x10 cm) is located in the center of the box floor; entire field-of-view is covered with black and white checkerboard to emphasize changes in depth. 'Top' and 'bottom' refer to areas of the visual cliff, where 'top' is over the supported half of the apparatus and 'bottom' is over the suspended half.
    2. Mice are placed onto the neutral center region.
    3. Behaviors are recorded for 20 min.

Procedure: Hot-plate test

    1. Apparatus: hot plate (11x10.5 cm) surrounded by a clear acrylic cage (15 cm tall, 10 cm diameter, open top with clear acrylic lid) to constrain locomotion.
    2. The surface of the hot plate is set to 55°C as measured by a built-in digital thermometer.
    3. Each mouse is placed on the hot plate and timed until it performs either of two behaviors regarded as indicative of nociception: hindpaw lick or hindpaw shake/flutter.
    4. Latency to respond is measured to the nearest 0.01 s.
    5. If there is no response within 30 s, mice are removed from hot plate and time recorded as 30 s.

Procedure: Tail-suspension test

    1. Apparatus: horizontal ring-stand bar elevated ~30 cm above the floor of the apparatus.
    2. A paper cone is placed on the tail of each mouse to limit climbing during the test session.
    3. Using masking tape, mice are suspended by a point near the tip of the tail on the ring-stand bar.
    4. Behaviors are recorded in 2 test sessions (consecutive days, 5-min trials).

Data collected by investigator

  • Open field test
    • distance traveled in first 4 min (response to novelty)
    • distance traveled in first 10 min (response to novelty)*
    • total distance traveled (general locomotor activity)
    • distance traveled (slope)*
    • total time in corners*
    • percent time in corners
    • percent time in corners (slope)*
    • total time in periphery*
    • percent time in periphery
    • percent time in periphery (slope)*
    • total time mobile*
    • total time immobile*
    • percent time mobile*
    • percent time immobile
    • percent time mobile (log)*
    • total time in center*
    • total time in center (log)*
    • percent time in center
    • percent time in center (log)*
    • total time in center (slope)*
    • number of fecal boli*
  • Light-dark box test
    • distance traveled in light compartment (habituation)
    • number of light-dark transitions
    • time in light compartment first 4 min (response to novelty)
    • time in light compartment*
    • percent time in light compartment first 4 min (response to novelty)*
    • percent time in light compartment
    • percent time in light compartment (slope)*
    • time in light compartment (slope)*
    • distance traveled in light compartment (slope)*
  • Visual-cliff avoidance test
    • distance traveled in top during first 4 min (response to novelty)
    • time in top during first 4min (response to novelty)
    • total distance traveled in top
    • total time in top
    • percent time in top*
    • number of top entries*
    • time immobile in top*
    • mean velocity in top
    • distance traveled in bottom during first 4 min (response to novelty)
    • time in bottom during first 4 min (response to novelty)
    • total distance traveled in bottom
    • total time in bottom
    • percent time in bottom
    • number of bottom entries*
    • time immobile in bottom*
    • mean velocity in bottom
    • number of top to bottom transitions
    • total distance traveled*
    • total time immobile*
    • total mean velocity*
    • top to bottom ratio for time spent during first 4 min (response to novelty)*
    • bottom to top ratio for time*
    • bottom to top ratio for time (log)*
    • top to bottom ratio for time*
    • bottom to top ratio for distance*
    • top to bottom ratio for distance*
    • total distance traveled*
    • total time in bottom*
    • percent time in neutral*
    • total to bottom ratio for time immobile*
    • bottom to total ratio for distance*
  • Hot-plate test
    • latency of hindpaw withdrawal
    • latency of hindpaw withdrawal (log)*
  • Tail-suspension test
    • climbing*
    • time spent climbing*
    • average frequency climbing
    • latency to first climb*
    • time spent not climbing*
    • frequency not climbing*
    • average time immobile
    • frequency immobile*
    • average latency to first immobility episode
    • time mobile*
    • frequency mobile*
    • time moving at centerpoint*
    • time not moving at centerpoint*
    • mean velocity*
    • latency to first climb (log)*
    • frequency immobile (square root)*
    • latency to first immobile (log)*
  • Body weight at dissection

* Additional data available Chesler4_suppdata.xlxs