Modeling Autism Spectrum Disorders Motor Deficits in Mice

Fig. 1

Nesting cage

2.2 Sensorial and Motor Development Measurement

Fox published the first development test battery for rodents [29]. The test has be substantially modified by several authors [30] who preserved the original idea of Fox that was to model in rodents the major features of the baby development. The following sections describe the test most of them being illustrated by pictures. The range of age of appearance of the adult pattern of response is indicated. As a general rule, the appearance of a pattern of adult response in a mouse should be confirmed by the observation of the same response the day after. The reliability was estimated as indicated in [30]. The first estimate is the test retest correlation (Spearman ρ across 15 inbred strains of laboratory mice) and the second is the communality obtained from a factor analysis including the development measures [28]. Three scoring methods are available, all being applied individually. (1) A binary scoring can be performed. The adult pattern of response is observed as present (score 1) or absent (score 0) on a given day. The groups are compared with an appropriate nonparametric statistics. (2) The score corresponds to the day when the pattern of adult response is present. The response to stimulation is absent when tested on day 7 and 8 but it appears on day 9 in the pup. We observe that the response remains present on day 10. The pup is subsequently scored 9 in the mouse. (3) The strength of the response can be scored every day and some responses only are quantifiable. For example, the strength of the righting response can be evaluated by the time to right. The responses are generally distributed on a small number of classes and the treatment requires nonparametric statistics.

A long separation from the mother inducing a drop in temperature are not recommended. The measurements are done on a heating table (38 °C). Half of the litter is transferred into a cage on the heating table and it is replaced by the other half-litter when the observations are over.

Specific material is described for each test and the dimensions are indicated. A stopwatch is necessary for several measurements. We suggest a silent chronometer. It can be monitored by a computer or standing on a table like a clock.

Righting pattern appears between day 1 and 3 days of age. The reliability was .72 when estimated by test–retest and .69 when estimated by communality. The response is obtained when the pup is placed on its back (Fig. 2a) tries immediately to straighten up (Fig. 2b) and get onto all four paws (Fig. 2c). The strength of the response is measured by the time to turn over. The maximum allowed was 20 s.

Fig. 2

Righting response. The pup is placed on its back (a), tries to straighten up (b) and get onto its four paws (c)

Cliff drop aversion (between day 1 and 3; test retest reliability .63; communality .61). A 5 mm thick plastic slice was put on a smooth surface generating a cliff (Fig. 3a). The forepaws of the pup were placed on the edge of the cliff its head being over the cliff. Figure 3a, b shows wrong positioning of the forepaws. The forepaws are well positioned in Fig. 3c. Note that the edge is positioned at the joint between metatarsals and ulna, at the navicular lunate place. The pup straights up the head and trunk leaning on forelimbs (Fig. 3d) then turns (Fig. 3e, f) and crawls away (Fig. 3g). Each step can be scored.

Fig. 3

Cliff drop aversion as described in the text (note the wrong positioning in a and in b)

Forepaw grasping pattern (between day 1 and 3; test retest reliability .72; communality .67). When the inside of one forepaw is scratched with the sharp tip of a pencil (Fig. 4a) the paw flexes (Fig. 4b, scored 1) to grasp the object (Fig. 4c and c bis, scored 2). The two forepaws are tested and the two scores are summed.

Fig. 4

Forepaw grasping response with zoom on the right-paw grasping (b and c bis). The inside of one forepaw is scratched (a) the paw flexes (b) to grasp the object (c and c bis)

Forelimb placing (between day 3 and 6; test–retest reliability .80; communality .53). The dorsum of the forepaw is brought into contact with the flat surface of a thin object (Fig. 5a). The pup raises the forepaw (scored 1) and places it on the object (Fig. 5b, scored 2). The right and left limbs rate of development might differ. The two forepaws are tested therefore and the two scores are summed. Administration of growth factors increases the precocity of the response.

Fig. 5

Forepaw placing. Dorsum of the forepaw brought into contact with the flat surface of an object (a); the forepaw is raised and placed on the object (b)

Adult walking pattern (between day 4 and 8, test–retest reliability of the age when the adult pattern is observed .73). The pup is motion less during the first days of life. The hind limbs look like non-innervated (Fig. 6a). A pivoting walk follows during which the body is arced. The pup seems to pivot on one of the hind limb because the hind limbs are motion less the displacement being produced by the movement of the forepaws (Fig. 6c–e). The pivoting displacement is scored 1. Later the pup develops a linear displacement. It is scored 2 (Fig. 6f). Straight walk longer than 5 cm is scored 2.

Fig. 6

(a)–(f): Walking patterns as described in the text

Adult paw position (between day 7 and 9, test–retest reliability of the age when the adult pattern is observed .82). The adult pattern of response is present when the four paws are put all four flat on the ground (scored 1 for each paw, Fig. 7).

Fig. 7

Adult paw position at day 7. Note that fingers and toes lay flat on the ground

Hind limb placing (between day 4 and 7; test–retest reliability .79; communality .56). The dorsum of the hind paw is brought into contact with the flat surface of a thin object (Fig. 8a and a bis). The pup raises the forepaw (scored 1) and places it on the object (Fig. 8b and b bis, scored 2). The two forepaws are tested therefore and the two scores are summed.

Fig. 8

Hind paw placing with zoom on the right paw placing (a bis and b bis). The procedure was similar to forepaw placing (Fig. 5)

Rooting response (between day 6 and 11; test–retest reliability .80; communality .62). The bilateral stimulation (between the thumb and the index) of the face at the level of the vibrissae (Fig. 9) causes the pup to crawl forwards (scored 1) and pushing the head (scored 2) in a rooting manner (scored 3). The age when the behavior disappears is generally scored (beyond day 8 of age). The Adr ^egene that contributes to C57BL/6J and Balb/c difference for the age of disappearance of the rooting response was one of the first identified genetic correlates of a behavioral trait [31, 32]. The response is mediated by the 5th sensory cranial nerve that mediates proprioceptive information from neck, head, face, and masticatory muscles particularly. It appears in reaction to the bilateral stimulation of the face. The response disappears when the development of the central nervous system allows others sensorial information to compete.

Fig. 9

Rooting response at day 4 (a) and at day 8 (b)

Age of appearance of the crossed extensor pattern (between day 6 and 11; test–retest reliability .62; communality .62). When pinched, the hind limb stimulated flexed (Fig. 10a, scored 1) while the opposite hind limb extended. The adult pattern consists in flexing the two limbs (Fig. 10b, scored 2). The age when the adult pattern appears is generally scored. The response is mediated at the spinal level and the appearance of the response indicates the myelination of the spinal chord. The two limbs are tested and the scores are cumulated.

Fig. 10

Appearance of the crossed extensor pattern

10.

Motor reaction to slope, usually labeled “geotaxis” (between day 4 and 8; test–retest reliability .68; communality .45). It is difficult to distinguish the contribution of the perception of inclination and the development of motor behavior in the response of the mouse. Our version avoids dramatic slopes that use most of the protocols. The pup is placed on a rough surface, facing downwards on a 30° angle slope surface and the head pointing down the incline (Fig. 11a). The surface must be rough to avoid sliding. The pup is scored 1 when it turns up the head, 2 when it turns the body up 90°, 3 when he turns the body up 180° and 4 when he walks up (Fig. 11c). The progressive appearance of the response seems to be due to the maturation of semicircular canals. Several factors including bone morphogenetic protein 2 are known for their contribution in the maturation of the canals [33].

Fig. 11

Motor reaction to slope

11.

Trunk curling test and vibrissae placing (between day 5 and 8; test–retest reliability .85; communality .79 and .90 and .64 respectively for vibrissae placing). The mouse is held by tail. It is scored 0 if it curves towards its belly [34] (Fig. 12a), 1 if it curves moderately and 2 if it arches its back (Fig. 12b). We approach a fin stick of the vibrissae (Fig. 12c) when the mouse presents a lordosis, the back being arched. When the vibrissae touched the stick the pup raised its head (scored 1) and extends the forelimb (scored 2) to grasp the tip of the pencil (scored 3) (Fig. 12e). An exaggerated fore limb bilateral extension may indicate a hyperreflexia from cerebellum origin.

Fig. 12

Trunk curling test (a and b). Note the curling trunk (a) and the arched back (b). Vibrissae placing (c) observe the position of the fin stick on the vibrissae; the stick touching the vibrissae is moved from up to down, (d) the pup does not try to grasp the stick (e) the pup attempts to grasp the stick

12.

Hearing onset (between day 9 and 13; test–retest reliability .81; communality .77). The ability to hear is progressively developed. At the end of the first week, the middle ear cavity is full of liquid, the ear canal is close and the pinna is not developed [35, 36]. The development of the external ear starts with lifting of the upper edge of the pinna from the head between 7 and 8 days of age and follows with the opening of the ear canal after 9 days of age. The functionality of the auditory system can be detected in two manners: (1) by auditory average potential recordings in the inferior colliculus [37] and (2) by inducing Preyer response. It consists in pinna twitching and going flat backwards against the head as reaction to sounds [38]. This simple and noninvasive method is sufficient to detect the onset of capacity to hear since its median age of apparition correspond to the age of appearance of the averaged evoked auditory potential [39]. Sophisticated devices as those used for the prepulse inhibition study can be used to induce the Preyer response. We suggest here a more economic manner for triggering the response. We use a cricket toy to elicit the response. It produces a composite sound (including 10–30 kHz frequencies). The cricket is placed 4 cm above the head of the pup (Fig. 13a) and the sound is produced once. The response (Fig. 13b) is scored 1 for partial response (ear startling) and 2 for full response (pinna going flat backwards against the head). It is also possible to refine the measure of development of the auditory function by scoring also the days of pinna development and. The Preyer response can be considered as a startle response since it includes not only pinna twitching but limb contractions [35]. The strength of the startle response can be visually scored but its measurement with a device dedicated to prepulse measurement should be preferred. This complex response requires the development of a pathway beginning by the auditory pathway that comes out to the cochlear nucleus. The fibers of the lateral Lemniscus transfer the information from the cochlear nucleus towards the caudal pontine reticular nucleus. The motor neurons of the reticular formation transmit the information to the skeletal muscles.

Fig. 13

Induction of the Preyer response in the Mouse. (a) Typical pina position, (b) the use of a “cricket” to produce a sound and (c) twitching and flattening pina against the head

13.

Bar holding (between day 9 and 12; test–retest reliability .92; communality .88). The forepaws are placed on a round wooden bar (8 mm in diameter). Hanging for 10 s using the forepaws only is scored 1 (Fig. 14a). Hanging with its hind paw on the bar ensures a longer period of stability (scored 2, Fig. 14b). The response requires a composite set of capabilities including muscular strength, equilibrium, and motor coordination.

Fig. 14

Bar holding

14.

Vertical clinging (between day 9 and day 12; test–retest reliability .65; communality .70). The pup is held against a vertical grid (wire: 0.6 mm in diameter, mesh: 6 mm wide, Fig. 15). Clinging for 10 s or more is scored 1.

Fig. 15

Vertical clinging

15.

Vertical climbing (between day 11 and 14; test–retest reliability .81; communality .54). The pup is scored 1 per mesh passed.

16.

Age at eyelid opening (between day 12 and day 16; test–retest reliability .91; communality .87). The score is the age in days when the eyelids of the two eyes are opened. The response seems to be growth factor dependent since it is correlated with the quantity of epidermal growth factor [17, 40, 41] Visual placing (between day 13 and 17; test–retest reliability .94; communality .48). We follow here the vibrissae placing procedure. The observer takes gently the pup between the thumb and the forefinger and lifts it. When the pup is suspended the tip of a pencil is approached to the eyes, without touching the vibrissae. The pup raises the head, it expends the forelimbs and it grasps or it tries to grasp the pen (Fig. 16). The score is the number of days after visual placing. The response requires not only visual development but also a high level of sensorial and motor coordination.

Fig. 16

Vertical climbing

17.

Visual cliff aversion (between day 18 and 22; test–retest reliability .81). We use a plate of glass half of which being transparent and half being opaque. The mouse is placed on the edge of the two parts of the plate, the body being in the axis of the edge. The response can be scored as binary (the juvenile turns to the opaque part is scored 0 and 1 when it turns to the transparent part). Another solution is measuring the time spent in the two regions during a limited period. We have drawn a virtual limit of 1 cm wide on either side of the edge and we measured the time spent on the opaque, transparent and limit zone during 5 min. The Videotrack (Viewpoint-Behavior technologies: http://www.viewpoint.fr/news.php) setup was employed to measure the time and the length of the path. Measuring the time spent in the three zones with a stopwatch is suitable.

18.

Markers of physiological growth should be moreover considered. We list here the most frequently recorded.

Body mass: recorded daily. It requires a precision scale (1 mg) during the first 10 days.
Body length: measured daily. The pup is laid onto graph paper.
Fur apparition: it can be detected by rubbing the hairs on the back of the pups in the wrong way.
Teeth development: bulging of gum and appearance of the two incisors of lower mandible are easily observed and the day of appearance is recorded.

2.3 Recommendation

The conclusion of the section needs two remarks. The first refers to the computation of a global index of development and the second refers to the handling effect.

It could be appealing to pool these measures or to compute a composite index compute a global index of development. The strategy would imply the existence of a general factor of development but we failed to obtain a general factor when we performed a factor analysis including most of the variables listed above [28]. The genome wide scan of the same variables confirmed the conclusion. Very few developmental indexes shared common chromosomal region [28].
The repeated disturbance of the mother by the daily observation could modify the quality and quantity of cares to pups although we never observed such a phenomenon. Handling on the contrary has a well-known effect (see Chap. 13 for more information). It is possible to limit such an effect by substituting a transverse-longitudinal method to the longitudinal method. A group of pups is used for a limited number of test another group for further set of tests and so on [28].

3 Measuring Deficit of Motor Behavior in Adulthood

We consider the estimation of tonus, the measure of coordination deficits, of balance deficit and gait deficit.

3.1 Measurement of Tonus in Mice

Tonus permits to muscles to maintain their tension that is required for balance, coordination, and movement. Hypotonia in ASD has been reported for the first time by the Courchenne group that was pioneering in neurological investigation of ASD [42]. Several other groups confirmed a lower tonus in ASD: associated with SHANK3 [43, 44], NRXN1 [45, 46], in Potocki–Lupski syndrome [47, 48], in MECP2 Rett associated mutations [49], Smith–Lemli–Opitz syndrome [50] and in several microduplications or microdeletions associated with autism [51].

Hypotonia was estimated in mouse model of Prader–Willi syndrome by rapidity to right when put on the back [52]. The age at righting response in newborn mice as previously defined is an estimate of early hypotonia.

A non-expensive device has been suggested and used to measure the forelimb strength in different pathologies targeting the motor system in mice [53]. The rational is to measure the weight that a mouse can bear and how long. A 20 g weight is tied to a sponge. The mouse is taken by the middle of the tie and approached to the bench so that the mouse can grasp the sponge. The mouse is gently lifted when it has grasped the sponge. Time until the mouse releases the weight is measured. The operation is renewed with a 25 g weight and so on. The born weight or the duration of bearing is measured.

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