Procedure: Modified SHIRPA

1. Behavior observed in the viewing jar:
   1. The viewing jar is placed upright on top of the plastic sheet.
   2. A mouse is removed from its homecage by gripping the tail between the thumb and forefinger and placed in the viewing jar.
   3. The following behaviors are recorded without disturbing the mouse. Incidents of bizarre behavior, stereotypy and convulsions are recorded as well.
      1. Body position: inactive, active, excessively active
      2. Tremor: trembling (not to be confused with breathing)
      3. Palpebral closure: note
      4. Lacrimation: note
      5. Defecation: note
2. Behavior observed in the arena:
   1. Each mouse is transferred to the center of the arena inside the viewing jar, using the plastic sheet underneath the jar to contain the mouse. The plastic sheet is carefully removed at ~25 cm above the arena floor.
      1. Transfer arousal: freeze, move immediately, move head, look around, etc.
      2. Locomotor activity: total number of squares the mouse enters with all four feet within 30 s.
      3. Gait: retropulsion, uncoordinated movement, etc.
      4. Pelvic elevation: extent pelvis is elevated
      5. Tail elevation: dragging, horizontally extended, elevated
      6. Startle response: response of mouse following 90 dB tone from click box ~30 cm above arena
      7. Touch escape: response of mouse following a stroke on the back of the neck with a bent index finger (mouse approached from front).
3. Behavior observed outside (above) the arena
   1. Each mouse is removed from the arena by gripping the tail between the thumb and forefinger to observe the following:
      1. Positional passivity: record immediate response to a sequence of handling positions (once a response is recorded, the sequence is terminated and no more positions are used):
         1. Held by tail: record (if mouse does not struggle, move to next test; if mouse struggles, move to trunk curl, below).
         2. Held by neck (loose scruff between the forefinger and thumb): record (if mouse does not struggle, move to next test; if mouse struggles, move to trunk curl, below).
         3. Held in supine position: record if mouse struggles or not.
      2. Trunk curl: note forward curling of each mouse when held by tail.
      3. Limb grasping: note whether mouse grasps its limbs when held by tail.
4. Behavior observed above the arena on the overlying grid
   1. Pinna reflex: place each mouse gently on the grid and touch proximal part of inner canthus lightly with the tip of a fine cotton probe; record ear retraction.
   2. Corneal reflex: touch cornea lightly with the tip of a fine cotton probe; record eye-blink response.
5. Behavior observed in the clear plastic tube: place each mouse into the tube and roll the tube slowly until the mouse is upside down; record righting reflex.
6. Other behaviors to record during the entire session:
   1. Evidence of biting: note whether mouse attempts to bite handler or equipment.
   2. Vocalization: note whether mouse was vocal at any time.