Workflow (the same set of mice were tested in Lake3)
Test Category Procedure performed Data collected 1 Primary screen SHIRPA Behavioral and physical assessments 2 Motor learning and memory Accelerating rotarod (ROD) (Lake3) Latency to fall 3 Neuromuscular function Grip strength (Lake3) Overall grip strength (3 trials) 4 Exploratory behavior Hole-board test (Lake3) Open arena, latency to perimeter, latency to hole, arena entries, stretched attends, head dunks, hole visits, arena rearings, perimeter rearings, line crossings, grooming episodes, fecal boli, urine puddles
Figure 3. SHIRPA tools. A. 1) vertical woven grid (30 x 8 cm; wire ~0.050" gauge), 2) horizontal woven grid (30 x 8 cm; wire ~0.050" gauge), 3) rubber bulb, 4 & 5) plastic probes, 6) curve tweezers; B. stand with horizontal grid; C. holding board with vertical grid.
- Cleaning supplies: paper towels, 70% alcohol.
- Masking tape: for securing vertical wire-mesh and tail weights
Acclimation to test conditions
Mice are allowed to acclimate to animal room over the weekend.
• The SHIRPA primary behavioral screen provides a behavioral and functional profile by observational assessment of mice. (SHIRPA was first described by Rogers et al. (1977) and stands for: SmithKline Beecham, Harwell, Imperial College School of Medicine, Royal London Hospital, Phenotype, Assessment.) This screen has been modified and evaluates gait or posture, motor control and coordination, changes in excitability and aggression, salivation, lacrimation, piloerection, defecation, and muscle tone. All parameters are scored to enable comparison of results both over time and between different laboratories.
• Mice are assessed for various behaviors as described below in a view jar, observation arena, horizontal and vertical grids, as well as during tail suspension and supine restraint.
• All equipment is arranged in the same order for each test.
• The entire test area, including Arena, View Jar, and Grids are wiped down with ethanol and allowed to dry completely before the beginning of each test.
Tests performed in sequential order
a. A mouse is placed in the View Jar for 30s, and observed and scored for body position, spontaneous activity, tremor, twitches, defecation, and urination (see Figure 1, Table 1).
b. The mouse is then removed from View-Jar and placed in the viewing Arena by tipping the jar and allowing the mouse to drop into the wean cage.
c. While transferring, the mouse is evaluated and scored for transfer arousal. In the Arena, the mouse is evaluated for palpebral closing, piloerection, startle response, gait, pelvic elevation, tail elevation, hindlimb splay, touch escape, finger approach, positional passivity, defecation, and urination (see Figure 2, Table 2). A fear assessment is made based on the overall response in the arena. The mouse is then handled sequentially for eliciting a response (called positional passivity) while transferring to the next test.
d. Next, the mouse is suspended via its tail and moved from the Arena to the horizontal wire-grid.
e. While descending toward the grid, the mouse is evaluated and scored for visual placement (see Figure 3A, Table 3).
f. Once the mouse is has on all four limbs on the grid, it is evaluated for body tone, pinna reflex, corneal reflex, withdrawal reflex, and crossed extensor reflex (see Figure 3 A4, Table 3).
g. Subsequently, from a standing position the mouse is held by tail suspension for 15s.
h. While in mid-air the mouse is assessed for hind-limb splay, trunk-curl, and limb-grasp (see Table 4).
i. After being suspended the mouse is lowered via its tail again towards the horizontal wire-grid. Once the mouse has grasped the wire with its forelimbs (only) it is rotated horizontally by the tail.
j. The ability of the mouse to maneuver and to negotiate its position in the horizontal wire platform is scored based on hindlimb response (see Figure 3B, Table 5).
k. Next, the mouse is handled and placed in a supine restraint, along its cervical-thoracic dorsal skin and gently scuffed.
l. While securely restrained in a recumbent position, the mouse is further evaluated for skin color, limb tone, abdominal tone, lacrimation, salivation, biting, irritability, provoked biting, irritability, aggression, and vocalization (see Table 6).
m. The mouse is kept recumbent or in supine restraint for the examination of its righting reflex (see Table 7).
n. Then the mouse is placed on a vertical wire-grid with its head upward, then downward, and evaluated and scored for catalepsy and negative geotaxis (see Figure 3C, Table 8).
o. The mouse is returned to its home-cage.
p. All equipment is cleaned with ethanol and allowed to evaporate completely.
No. Tests Score Table Measurement category 1 Body position 1-5 Neuromuscular function 2 Activity level 1-3 Activity & motor function 3 Tremor 0-2 Activity & motor function 4 Twitches 0-2 Activity & motor function 5 Defecation (jar) Behavior-anxiety 6 Urination (jar) Behavior-anxiety 7 Transfer arousal 0-4 Behavior-anxiety 8 Palpebral closing 0-2 Autonomic function 9 Piloerection 0-1 Autonomic function 10 Air puff startle reflex 0-4 Autonomic function 11 Gait 0-3 Neuromuscular function 12 Pelvic elevation 0-3 Coat color & appearance 13 Tail elevation 0-4 Coat color & appearance 14 Fear 0-1 Behavior-anxiety 15 Hindlimb pattern 0-2 Coat color & appearance 16 Touch escape 0-3 Sensorimotor function 17 Finger approach 0-4 Sensorimotor function 18 Positional passivity 0-4 Neuromuscular function 19 Defecation (arena) Behavior-anxiety 20 Urination (arena) Behavior-anxiety 11 Visual placing 0-3 Sensorimotor function 12 Body tone 0-2 Neuromuscular function 13 Pinna reflex 0-2 Sensorimotor function 14 Corneal reflex 0-2 Sensorimotor function 15 Toe pinch Sensorimotor function 16 Crossed extensor reflex Sensorimotor function 17 Suspended hindlimb splay 0-2 Coat color & appearance 18 Trunk curl 0-1 Autonomic function 19 Hindlimb grasp 0-1 Autonomic function 20 Horizontal rotation response 0-4 Sensorimotor function 21 Skin color 0-2 Coat color & appearance 22 Hindlimb tone 0-4 Neuromuscular function 23 Abdominal tone 0-2 Neuromuscular function 24 Lacrimation 0-1 Autonomic function 25 Salivation 0-2 Autonomic function 26 Provoked biting 0-1 Behavior-wildness 27 Irritability 0-1 Behavior-wildness 28 Aggression 0-1 Behavior-wildness 29 Vocalization 0-1 Behavior-wildness 30 Righting reflex (held/released) 0-3 Sensorimotor function 31 Righting reflex (flipped) 0-3 Sensorimotor function 32 Catalepsy 0-4 Neuromuscular function 33 Negative geotaxis 0-4 Neuromuscular function
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Rogers DC, Peters J, Martin JE, Ball S, Nicholson SJ, Witherden AS, Hafezparast M, Latcham J, Robinson TL, Quilter CA, Fisher EM. SHIRPA, a protocol for behavioral assessment: validation for longitudinal study of neurological dysfunction in mice. Neurosci Lett. 2001 Jun 22;306(1-2):89-92.