Modified-SHIRPA v3 1/AUG/2023 RIKENMPP_001_003 test 2 1 testSt test modification

The "SHIRPA" method is a package developed by the Medical Research Council (MRC) in the United Kingdom for comprehensive testing of mouse behaviors. The SHIRPA method allows primary screening for 40 test items by visual inspection and manual handling and can evaluate characteristic behaviors of mice in a relatively short period of time. We focused on these primary screens, added items related to morphological characteristics of the mice, and named the modified procedure the "modified SHIRPA" method. This modified method is compatible with the original "SHIRPA" method. The new Ver.3.20 of the modified SHIRPA method includes the items from the original modified SHIRPA (Ver. 1.00), which makes it compatible with "pipeline 1" from the Japan Mouse Clinic. In this version, the times and testing criteria were improved and new items related to eye morphology were added. The skilled staff members can test the morphological and behavioral characteristics of a mouse in 15 minutes using a package of tests comprising 56 items. Details of the reconstruction of the items are discussed in the "History review" section.

These procedures are applicable to the mouse phenotyping platform in the RIKEN BRC. The tests should be performed by trained staff members who understand the safety and hygiene rules required by RIKEN. Any queries, comments, or suggestions, relating either to this SOP in general or to a specific problem encountered during a procedure, should be addressed to the Morphological, Hearing, and Behavioral Neuroscience Unit Leader. Any deviation from this protocol must be reported to the Morphological, Hearing, and Behavioral Neuroscience Unit Leader.

Testing is carried out in accordance with common animal handling procedures. Coveralls (in the rearing facility) or laboratory coats (in the laboratory facility) must be worn with gloves at all times in the work area. If animals are dropped on the floor, treat them in accordance with the safety and hygiene guidelines from the rearing facility at RIKEN. If something unexpected happens during the primary screening, it must be quickly reported to the Morphological, Hearing, and Behavioral Neuroscience Unit Leader and recorded.

Image: Equipments for modified-SHIRPA

Considerations before the tests Mice behavior can be affected by the rearing environment. Therefore, it is important that the environmental influences are consistent as possible. For example, because mice reared in a group may not be subjected to a uniform level of stress, our laboratory raises the mice to be examined with the modified-SHIRPA method in separate cages after weaning. Illumination Although a desk lamp can be used to illuminate the mice and facilitate observation, such illumination should be avoided, especially when the behavioral test items are being measured. It is also advisable not to illuminate the mice directly for the general observations. Cleaning equipment When a number of mice are tested continuously, residual odors from the equipment used in the preceding test may affect the test results. The floor and walls of the arena, ruler, and metal net should be wiped clean before introducing the next mouse. To prevent infection, the equipment should be washed with water at the completion of the day's tests. Some specific pathogen-free facilities use ultraviolet irradiation when tests are not being performed. Care needs to be taken, however, to ensure that ultraviolet irradiation does not crack any acrylate equipment covered with residual alcohol. Recording comments and phenotypes and the scoring system Because test scores alone do not completely capture behavioral phenotypes, written records containing other comments should be kept. To facilitate detailed phenotypic analyses based on the modified-SHIRPA method, recording behaviors and phenotypes with a digital video camera is recommended.

Arena (clear Perspex arena) The arena is a transparent acrylate box (55 cm length, 33 cm width and 18 cm height). The arena floor is a white plate marked with 15 (5 x 3) squares (11 cm2). A meshed (12 mm2) grid (40 cm x 20 cm) is secured across the top of the arena. Viewing jar The transparent acrylate jar is a cylinder 11 cm in diameter and 25 cm in height. Grid floor A 40 x 12 cm metal net about 12 mm in mesh size is used as the bottom plate of the viewing jar. Supports for grid floor The supports elevate the grid floor. They are a cylinder 2.5 cm in diameter and 3 cm in height. Our laboratory uses a base plate, on which the supports are glued. Wire attached to the end of a dowel A wire (0.15 cm thickness) is attached to a dowel and used to examine the pinna reflex and corneal reflex. Plastic rod The rod is about 5 mm in diameter and as long as a pencil. The tip is trimmed to resemble a round pencil. Forceps with bent tips The forceps are used to deliver a toe pinch. The tip should be relatively fine but not so sharp that it can injure the mice. Stopwatch A multi-channel timer is better. IHR click box The click box is used to test auditory sense. It emits sound about 20 KHz in frequency and about 90 dB in acoustic pressure at a distance of 30 cm. Scale Ruler The ruler is used to measure the body length and tail length of mice. A transparent ruler about 20 cm in length would be easy to use. Transparent tube The tube is used to test the contact righting reflex. It is a transparent acrylate cylinder 30 cm in length. Tubes with various diameters (e.g. 2.5-3.5 cm) should be equipped to match mouse size. A mouse should be able to enter the tube easily and stay close to the internal wall of the tube.

Paper towels Disinfecting ethanol Digital camera A digital video camera with a high frame rate (approximately 30 fps) to record high-quality videos and pictures should be used, if available. Ear muffs Ear muffs should be used to protect the experimenter's ears from the IHR click box during the startle response test.

Overview The Arabic numeral of each test almost corresponds to number for the test in the modified SHIRPA method (v.1). In this chapter, the tests are listed according to the screening order. Equipment installation Install the pieces of equipment introduced in the previous section such that they are easy to use. Viewing jar Place a paper towel on the grid floor supports. Place the grid floor on the scaffold. Place the viewing jar on the grid floor. Arena Place the arena at a steady table where it is easy to handle. For convenience, other equipment can be placed within reach. In the Viewing Jar First, transfer a mouse from a rearing cage to the viewing jar. Take the utmost care that the mouse is not frightened (do not peek in). Allow the mouse to get used to the emvironment for five minutes. Q1: Coat color Determine the hair color of the mouse. Q1.1: Major color Determine the hair color of the large part of the mouse's body. 0 = Agouti 1 = Diluted agouti 2 = Black 3 = Diluted black 4 = Brown 5 = Diluted brown 6 = White 7 = Brown agouti 8 = Diluted brown agouti 9 = Yellow agouti 10 = Diluted yellow agouti 11 = Other Q1.2: Minor Color Determine the hair color of the small part of the mouse's body. 0 = No minor color 1 = Slight dilution of the major color 2 = Agouti 3 = Diluted agouti 4 = Black 5 = Diluted black 6 = Brown 7 = Diluted brown 8 = White 9 = Brown agouti 10 = Diluted brown agouti 11 = Yellow agouti 12 = Diluted yellow agouti 13 = Multicolor 14 = Other Q1.4: Pattern in the region with a different color Determine the pattern in the region with a different color. 0 = No minor color 1 = Small spots (total area: 0-10 cm^2) 2 = Large spots (total area: more than 10 cm^2) 3 = Tabby coat 4 = Mottled coat 5 = Other Q1.5: White belly Determine the kind of white belly. 0 = No 1 = White 2 = Tan 3 = Other Q2: Hair length Examine hair length. 0 = Normal 1 = Alopecia 2 = Short 3 = Long Q3: Hair morphology Examine the morphological characteristics of the mouse's body hair. 0 = Normal 1 = Curly 2 = Other Q5: Tremor Determine whether the mouse shakes. 0 = No shaking 1 = Mild shaking 2 = Marked shaking In the Arena After completing "In the Viewing jar section," transfer the mouse from the Viewing jar to the arena, gently insert the stainless steel plate between the Viewing jar and the Grid floor and place the mouse in the Viewing jar onto the stainless steel plate (take care not to pinch the fingers of the mouse). Bring the Viewing jar and the stainless steel plate right above the center of the arena to hold still at a height of 30 cm. Turn the stainless steel plate downward to drop the mouse to the arena. Start the stopwatch simultaneously to begin the tests described below. Q10: Time elapsed before the Mouse Starts to Move (seconds) Make a note of the time elapsed before the mouse starts to move after dropping it onto the floor of the arena (seconds) Q11: Locomotor activity Make a note of the total number of squares entered by any of its four feet in a 30-second period (Count the number of squares, assuming that the mouse turned at a right angle if it moved in a diagonal direction). Q12: Transfer arousal Evaluate the behavior of the mouse in the arena. 0 = Coma 1 = Prolonged freeze, then slight movement 2 = Extended freeze, then moderate movement 3 = Brief freeze (a few seconds), then active movement 4 = Momentary freeze, then swift movement 5 = No freeze, immediate movement Q15: Startle Response Use the IHR click box placed 30 cm above the floor of the arena to present an auditory stimulus, and evaluate how the mouse responds on the following scale. 0 = No response 1 = Preyer’s reflex (backward flick of the pinna) 2 = Jump less than 1 cm 3 = Jump more than 1 cm Q16: Gait Observe the gait of the mouse. 0 = Normal 1 = Fluid but abnormal 2 = Limited movement only 3 = Incapacitated (unable to walk) Q18: Tail elevation Determine how the position of the tail. 0 = Dragging 1 = Extended horizontally 2 = Elevated / Straub tail (more than 45 degrees) Q19: Touch escape Approach the mouse from the side with a finger, and stroke the mouse, if possible. Rate the response. 0 = No response 1 = Mild (escape response to a firm stroke) 2 = Moderate (escape response to a light stroke) 3 = Vigorous (escape response as the finger approaches) Above the Arena: Part 1 Place the metal net over the arena. Hold the mouse by the tail and place it on the metal net to make the observations. Q20: Positional passivity Hold the mouse by the tail on the arena, and see if it resists being held. If the mouse does not resist, hold the mouse by the neck. If the mouse does not resist, place it on its back and hold it there. If the mouse still does not resist, hold it by its hindlimb. Determine whether the mouse resists being held at each step, and stop when the mouse resists. 0 = Struggles when held by the tail 1 = Struggles when held by the neck (a loose scruff between the forefinger and thumb) 2 = Struggles when laid on its back 3 = Struggles when held by its hindlimb 4 = Never struggled Q21: Trunk curl Determine whether the mouse raises its upper body by lowering its abdomen and sitting up (trunk curl) when dangled by the tail. Twisting the upper body to the side is different from the trunk curl. 0 = Trunk curl absent 1 = Trunk curl present Q22: Limb grasping Determine whether the mouse holds its forelimbs and hindlimbs together or either of them when dangled by the tail. 0 = Limb grasping absent 1 = Limb grasping present On the Arena Manipulate the mouse and observe the responses using the metal net mounted on the arena. Q23: Visual placing Dangle the mouse by the tail and lower it onto the metal net on the arena. Determine the distance between the mouse and the metal net when it extends its forelimbs to grasp the metal net. The eyes of the observer should be at the level of the metal net. 0 = Does not extend its forelimbs 1 = Upon nose contact 2 = Upon vibrissa contact 3 = Before vibrissa contact (18 mm) 4 = Early vigorous extension (25 mm) Q26: Head morphology Observe the morphology of the mouse's head. 0 = Normal 1 = Abnormal Q27: Pinna reflex Stimulate the base of the pinna with the wire probe (do not prick the mouse’s skin). Record how the pinna moves. Only one pinna needs to be tested. 0 = No movement 1 = Active retraction, moderately brisk flick 2 = Hyperactive, repetitive flick Q28: Pinna morphology ( R ) Examine the morphology of the right pinna. 0 = Normal 1 = Abnormal Q29: Pinna morphology ( L ) Examine the morphology of the left pinna. 0 = Normal 1 = Abnormal Q30: Corneal reflex Stimulate the cornea of the mouse softly with the curved part (not the tip) of the wire attached to the dowel and observe whether the mouse blinks. 0 = Does not blink 1 = Single blink 2 = More than one blink Q31: Toe pinch Use curved forceps to squeeze some fingers of the hindlimb (do not squeeze strongly enough to injure the mouse), while allowing the mouse to hold onto the metal net on the arena. Observe the response. 0 = None 1 = Slight withdrawal 2 = Moderate withdrawal, not brisk 3 = Brisk, rapid withdrawal 4 = Very brisk repeated extension and flexion of the hindlimbs Above the Arena: Part 2 Following the Above the arena section, manipulate the mouse and observe. Most of the manipulation is performed on the metal net mounted on the arena. Q32: Body length Restrain the mouse by the hand of the observer, and with the tail pulled straight, measure the distance from the tip of the nose to the tail base (hairline) with a ruler (mm). Do not pull the tail excessively. Q33: Tail length Measure tail length (the distance from the tail base to the tip of the tail) with the ruler while allowing the mouse to hold onto the metal net on the arena (mm). Q34: Tail morphology Examine the morphology of the tail. 0 = Normal 1 = Kinked 2 = Curled 3 = Other abnormality Q35: Lacrimation Restrain the mouse and determine whether tears are secreted. 0 = None secreted 1 = Tears secreted 2 = Other Q14: Palpebral closure Examine both eyes and their degree of closure. 0 = Both eyes wide open 1 = One eye 1/2 closed 2 = One eye closed 3 = Tending to close the eye 4 = Other Q62: Palpebral fissure length Observe the length of the palpebral fissures. 0 = None 1 = Short 2 = Normal 3 = Long Q59: Eye Observe the eye of the mouse. Q59.1: Cornea Observe the cornea of the mouse. 0 = Normal 1 = Opacity 2 = Other Q59.2: Pupil Observe the pupil of the mouse. 0 = Normal 1 = Leukocoria 2 = Other Q59.3: Eye size Observe the eye size of the mouse. 0 = Normal 1 = Microphthalmos 2 = Macrophthalmos 3 = Other Q59.4: Eye color Observe the eye color of the mouse. 0 = Black 1 = Pink 2 = Other Q59.5: Eye shape Observe the shape of eye of the mouse. 0 = Normal 1 = Abnormal Q36: Whisker morphology Examine the morphology of the whiskers (sensory hairs). 0 = Normal 1 = Abnormal Q37: Tooth morphology Open the mouse's lips with a plastic rod or the fingers of observer and examine the morphology of the incisor. 0 = Normal 1 = Abnormal Q38: Provoked biting Approach the mouth of the mouse with a plastic rod held sideways and observe whether the mouse bites the rod. 0 = No (Does not bite the rod) 1 = Yes (Bites the rod) Q39: Salivation Examine the plastic rod used in Q38 (place the plastic rod into the mouth of the mouse, if it did not bite the rod) to determine how much saliva adhered to the rod. 0 = None 1 = Saliva in an area that represents the margin of the submaxillary area 2 = Wet zone represents the entire submaxillary area Q40: Heart rate Place a forefinger on the chest of the mouse and feel the heartbeat. Assess the relative heart rate, because the heart rate of mice is too rapid to count. 0 = Slow, bradycardia 1 = Normal 2 = Fast, tachycardia Q41: Abdominal tone Gently press on the abdomen of the mouse with a forefinger and evaluate the degree of resistance. 0 = Flaccid, no return of cavity to normal 1 = Slight resistance 2 = Extreme resistance, board-like Q42: Skin color Examine the color of the ventral aspects of the footpads. 0 = Pale 1 = Pink 2 = Bright, deep red 3 = Mild pigmentation 4 = Dark, pigmented Q43: Limb morphology (FR) Examine the shape of the right forelimb, including the positions of the pads and the shapes of the digits. 0 = Normal 1 = Polydactyly 2 = Ectrodactyly 3 = Abnormality in the upper limb 4 = Other abnormality Q44: Limb morphology (FL) Examine the shape of the left forelimb, including the positions of the pads and the shapes of the digits. 0 = Normal 1 = Polydactyly 2 = Ectrodactyly 3 = Abnormality in the upper limb 4 = Other abnormality Q45: Limb morphology (HR) Examine the shape of the right hindlimb, including the positions of the pads and the shapes of the digits. 0 = Normal 1 = Polydactyly 2 = Ectrodactyly 3 = Abnormality in the upper limb 4 = Other abnormality Q46: Limb morphology (HL) Examine the shape of the left hindlimb, including the positions of the pads and the shapes of the digits. 0 = Normal 1 = Polydactyly 2 = Ectrodactyly 3 = Abnormality in the upper limb 4 = Other abnormality Q47: Limb tone Press against the sole of the mouse's hindlimbs with a forefinger and middle finger and evaluate how strong the mouse makes resistance (kicks back) against the tactile stimulus. 0 = No resistance 1 = Slight resistance 2 = Moderate resistance 3 = Marked resistance 4 = Extreme resistance Q50: Contact righting reflex When a transparent tube large enough to hold the mouse is drawn near the head of a mouse that is being dangled by its tail, the mouse will enter the tube. After a mouse has done so, turn the tube so that the mouse is on its back, and determine whether the mouse tries to turn over onto its abdomen. 0 = Does not try 1 = Tries Q51: Negative geotaxis Place the metal net onto the arena floor. Dangle the mouse by the tail, and allow the mouse to hold onto the metal net (tail toward the observer). When released, the mouse should move forward. Turn the metal net 90 degrees vertically so that the mouse is oriented with its head down. Observe how the mouse moves in response to this orientation. 0 = Turns and climbs the grid 1 = Turns, then freezes 2 = Moves, but fails to turn 3 = Does not move within 30 seconds Additional Comments and Body Weight A scale is used to measure the body weight of the mouse. Additionally, it is advisable to record as many written comments as possible. Q52: Fear Determine if the mouse freezes (complete lack of movement, including its whiskers), whenever it is transferred from one place to another. 0 = Does not freeze 1 = Freezes during transfer arousal Q53: Irritability Determine whether the mouse violently resists being restrained during the tests. 0 = Does not violently struggle 1 = Violently struggles Q54: Aggression Determine whether the mouse frequently defied and bit the handler during the tests. 0 = None or low aggression 1 = Provoked biting or attack frequently Q55: Vocalization Examine the degree the mouse squeaked with during the test. 0 = None or moderate vocalization 1 = Repeated vocalization with stimulus 2 = Repeated vocalization without stimulus Q58: Body weight Weigh the mouse on a scale (g). Additional Body Ratio Parameters Q60: Body mass index (BMI) Calculated using the following formula: w = Body weight (g) l = Body length (mm) BMI = w/l^2*1000 Q61: Tail ratio (Tr) Calculated using the following formula: t = Tail length (mm) Tr = t/l

There is another method called Modified SHIRPA. The Modified SHIRPA published in EMPReSS (European Mouse Phenotyping Resource of Standardised Screens) is another protocol. Masuya H, Inoue M, Wada Y, Shimizu A, Nagano J, Kawai A, Inoue A, Kagami T, Hirayama T, Yamaga A, Kaneda H, Kobayashi K, Minowa O, Miura I, Gondo Y, Noda T, Wakana S, Shiroishi T. Implementation of the modified-SHIRPA protocol for screening of dominant phenotypes in a large-scale ENU mutagenesis program. Mamm Genome. 2005 16:829-837.

[2017.7] For v3.20, "Q62: Palpebral fissure length" was added. Options were changed, from "0=No" and "1=Yes" to "0=None or low aggression" and "1=Provoked biting or attack frequently" in "Q54: Aggression;" from "0=No" and "1=Yes" to "0=None or moderate vocalization," "1=Repeated vocalization with stimulus" and "2=Repeated vocalization without stimulus" in "Q55: Vocalization." Q10, Q11 and Q12 were returned. Version history of the modified SHIRPA method [2011.6] For v3.10, "Q1.3: Characteristic of the region with a different color"was deleted. Only title was changed from "Q1.4: Pattern in the region with a different color" to "Q1.4: Characteristic of the region with a different color" in Q1.4. "3 = Tending to close the eye" and "4 = Other" were added as a choice in Q14. "4 = Falls off the metal net" was deleted from Q51. [2008.4] A new shortened version of the modified SHIRPA method (Ver. 3.00) was added. This version involves 53 subtests: Q1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 2, 3, 5, 14, 15, 16, 18, 19, 20, 21, 22, 23, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59.1, 59.2, 59.3, 59.4, 59.5, 60, and 61. Details of the 42 subtests from Q2 to Q58 except for Q42 are identical to those of v1.04. Ver. 3.00 of the modified SHIRPA method contains a different set of items than those found in the "V2shirpa" version. Improved Tests "Q1: Coat Color" was deleted, and "Q1.1: Major Color," "Q1.2: Dilution," "Q1.3: White Spot," "Q1.4: Albino," "Q1.5: Color Change," and "Q1.6: White Belly" were added. The criteria for "Q42: Skin Color" was changed and the new choice "moderately dark footpad, pigmentation" was added. The choices for Q42 include five possibilities: "0 = Blanched," "1 = Pink," "2 = Bright, deep red flush," "3 = Moderately dark footpad, pigmentation," and "4 = Dark footpad, pigmentation." Deleted tests "Q10: Time Elapsed Before the Mouse Starts to Move After It Is Dropped onto the Floor of the Arena (Seconds)," and "Q11: Locomotor Activity" were deleted. In the Japan Mouse Clinic, these tests were substituted with the open-field test, which is performed when the mice are 7 weeks old. "Q56: Bizarre Behavior" and "Q57: Convulsions" were also deleted. Maintained Tests "Q18: Tail Elevation," "Q20: Positional Passivity," "Q30: Corneal Reflex," "Q31: Toe Pinch," "Q38: Provoked Biting," "Q39: Salivation," "Q40: Heart Rate," "Q47: Limb Tone," "Q50: Contact Righting Reflex," "Q53: Irritability," "Q54: Aggression," and "Q55: Vocalization" were maintained in this version. Newly Added Tests "Q59.1: Cornea," "Q59.2: Pupil," "Q59.3: Eye Size," "Q59.4: Eye Color," and "Q59.5: Eye Shape" were added to this version. "Q60: Body Mass Index" and "Q61: Tail Ratio" were also added. Modification of the procedure and equipment The tests for Q1, Q2, Q3, and Q5 were performed in the arena instead of a viewing jar. The equipment required to observe the mice in the viewing jar, such as the jar, the supports for the grid floor, and the stainless steel plate, were not used. [2004.4.1] A new shortened version of the protocol "V2shirpa" was added. This shortened version includes 35 subtests: Q1, 2, 3, 5, 10, 11, 14, 15, 16, 19, 21, 22, 23, 26, 27, 28, 29, 32, 33, 34, 35, 36, 37, 38, 41, 42, 43, 44, 45, 46, 51, 52, 56, 57, and 58. Details of the subtests are identical to those used in v1.04. [2003.11.1] The "Set Name" field was added to the database to allow discrimination between the versions of the modified SHIRPA method. Descriptions of the Set Names are as follows. "BL and BW" This shortened version involves only 3 subtests: Q15, Q32, and Q58. Details of subtests are identical to those used in v1.04. "v1.0x Early" This version is identical to v1.04. [2002.3.8] For v1.04, "3: dark footpad or pigmentation" was added as a choice in Q42. [2000.6]