wild-type transgenic mice with the SHIRPA primary screen and tests of sensorimotor function and anxiety

Brain Research Bulletin 64 (2004) 251–258

Characterization of hemizygous SOD1/wild-type transgenic mice with the SHIRPA primary screen and tests of sensorimotor function and anxiety R. Lalondea,b,∗ , M. Dumonta , E. Palyc , J. Londonc , C. Strazielled a

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Facult´e de M´edecine et de Pharmacie, Universit´e de Rouen, INSERM U614, Bˆatiment de Recherche, 22 bld Gambetta, Salle 1D18, 76183 Rouen, Cedex, France b CHUM/St-Luc, Montr´ eal, Canada H2X 3J4 c Universit´ e de Paris 7 Denis-Diderot, EA 3508 Paris, France Laboratoire de Pathologie Mol´eculaire et Cellulaire des Nutriments, Facult´e de M´edecine, Universit´e Henri Poincar´e, Nancy I, INSERM EMI 0014 and Service de Microscopie Electronique, 54500 Vandoeuvre-les-Nancy, France Received 12 September 2003; received in revised form 21 June 2004; accepted 21 July 2004 Available online 7 September 2004

Abstract SOD1 is one of several overexpressed genes in Down’s syndrome. In order to dissect genetic causes of the syndrome, hemizygous human wild-type SOD1 transgenic mice were compared to FVB/N non-transgenic controls at 3 months of age in the SHIRPA primary screen of neurologic function as well as in tests of motor activity and coordination. The responsiveness of SOD1/wt transgenic mice to visual and somatosensory stimuli was reduced in placing, pinna, corneal, and toe-pinch tests. In addition, SOD1/wt transgenic mice crossed fewer segments on a stationary beam. On the contrary, there was no intergroup difference for motor activity and anxiety in open-field and emergence tests and for latencies before falling on the stationary beam, coat-hanger, and rotorod. These results indicate mild deficits in sensorimotor responsiveness in a mouse model expressing human SOD1 and that the overexpressed gene may be responsible for some Down symptoms. © 2004 Elsevier Inc. All rights reserved. Keywords: Motor activity; Motor coordination; Stationary beam; Rotorod; SHIRPA screening; Anxiety

1. Introduction The discovery of SOD1 mutations in a subset of patients with familial amyotrophic lateral sclerosis (ALS) [42] has spurred interest in examining the role of the encoded protein (superoxide dismutase-1) in this disease. Although no relation was established between SOD-1 aggregates and cell death [31], abnormal protein interactions caused by mutated SOD1 may be involved in ALS pathogenesis [27]. SOD1 mutations associated with ALS caused novel protein interactions [27]. A role for this protein in ALS pathogenesis is also supported by findings of paresis and motoneuron degeneration ∗

Corresponding author. Tel.: +33 2 35 14 82 81; fax: +33 2 35 14 82 37. E-mail address: [email protected] (R. Lalonde).

0361-9230/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2004.07.011

in transgenic mice with SOD1 mutations [23,35], probably mediated by a gain-of-adverse function, as no such effect was found with the endogenous SOD1 null mutation [40]. In addition to ALS, SOD1 may be involved in the pathogenesis of Down’s syndrome, characterized by trisomy of human chromosome 21, causing overexpression of a large set of genes including SOD1. The SOD1 gene was the first chromosome 21 gene to be cloned [15]. The mouse homologue for several of these genes is situated on chromosome 16 and several transgenic mouse models that mimic part of this disorder are available [43]. Transgenic mice overexpressing wild-type (wt) SOD1 were the first genetic model modelling trisomy 21 [12]. The role of overexpressed SOD1 has been linked both to protective and adverse actions [56]. On one hand, transgene-

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induced SOD1/wt expression (KT line) promoted survival of mouse cortical neurons exposed to neurotoxins [3]. Likewise, cultured midbrain neurons of SOD1/wt transgenic mice (SF-218 line) were less susceptible to decay over time than those of non-transgenic controls [38]. Moreover, SOD1 overexpression induced by an adenovirus-based vector protected superior cervical ganglion neurons grown in culture from nerve growth factor withdrawal [22]. In SOD1/wt transgenic rats, ventral horn motor neurons were more protected than wild-type from programmed cell death after traumatic spinal cord injury [48] and CA1 hippocampal neurons were relatively spared after global ischemia/reperfusion [6,47]. On the other hand, there is evidence that SOD1/wt expression promotes neuropathology. For example, SOD1/wt transgenic mice from the KT line developed mitochondrial vacuolization in ventral horn, cranial motor nuclei, and subiculum, argyrophilic degeneration in ventral horn, spinocerebellar tracts, cerebellar white matter, ventrolateral caudal medulla, and fornix, together with late-onset (24 months) ␣-motoneuron loss [21]. The same effect but with an earlier onset was found in the N29 Gurney line [21]. Although the ␣-motoneuron loss did not result in paresis, the SOD1/wt transgenic mice had poorer sensorimotor performances than non-transgenic controls in the rotorod test at 14 months of age. A detrimental effect of overexpressed SOD1 is also indicated by findings of hypoactivity in an open-field and deficient spatial learning in the Morris water maze in two other SOD1/wt transgenic mouse lines (Tg-51 and Tg-69) [13]. In the present study, we sought to determine whether sensorimotor functions and anxiety are altered in SOD1/wt transgenic mice from the KT line [3,21,37]. These mice were generated by injecting human wild-type SOD1 into fertilized eggs of the FVB/N strain under the control of its own promoter. The 3-month-old hemizygous SOD1/wt transgenic mice were compared to non-transgenic FVB/N controls in the SHIRPA (SmithKline Harwell/Imperial College/Royal Hospital/Phenotypic Assessment) primary screen of neurologic function [17,41]. The SHIRPA differentiated several transgenic mouse models from controls, including overexpression of a NFH-␤-galactosidase fusion protein [30]. The FVB/N strain is characterized by relatively high motor activity in the open-field [32,34] and retinal degeneration caused by the rd1 allele of the Pdeb gene [14,49], a likely cause of their poor spatial learning abilities [52]. Exploratory activity and anxiety were estimated by openfield [53] and emergence [18] tests. In the open-field, the distance travelled in the periphery was dissociated from the more anxiogenic central sector [29]. In the emergence test, the tendency of mice to remain inside a small, safe compartment and to avoid a larger and anxiogenic one was measured, a test sensitive to null mutations of several neurotransmitter receptors in mice [10,44,51] and medial frontal cortex lesions in rats [19]. The evaluation was completed by stationary beam, coathanger, and rotorod tests of motor coordination, sensitive to genetic and experimentally-induced lesions of motor-related pathways in mice [5,28,29].

2. Materials and methods 2.1. Animals SOD1/wt transgenic mice from the KT line were generated by injecting a 11.5 kb EcoRI–BamHI fragment containing wild-type SOD1 into fertilized eggs of the FVB/N strain [3,21,37]. Approximately 15 transgene copies were inserted in tandem on chromosome 15 under the control of its own promoter. Hemizygous SOD1/wt transgenic mice (n = 29, 13 males, 16 females; male/total ratio = 45%) and FVB/N nontransgenic littermates (n = 14, 7 males, 7 females; male/total ratio = 50%) were bred at the University of Paris 7 DenisDiderot and shipped by truck to the University of Nancy at 3 months of age (range: 62–105 days). During development, an equivalent number of toe-clippings was made in each group for the purpose of identification. The mice were kept inside group cages with woodchip bedding in a room with a 12 h light–dark cycle (lights on at 7:00). The experimenters were blinded as to group attribution, with the genotype determined at the end of the experiment by SOD1-induced inhibition of nitroblue tetrazolium reduction [37]. The entire research protocol adhered to the guidelines of the European Council Directive (86/609/EEC). 2.2. Apparatus and procedure After a week-long adaptation period to their new surroundings and to handling, the mice were evaluated in the SHIRPA primary screen (day 1), followed on day 2 by open-field and stationary beam tests, by emergence and coat-hanger tests on day 3, and ending with the rotorod test on days 4–6. The SHIRPA primary screen comprises reflex and sensorimotor tests with standard equipment purchased from Harrow, England. The battery is described in detail at the ENU Mutagenesis Programme web site (www.mgu.har.mrc.ac.uk). In compliance with previously described protocol guidelines [41], the mice were first placed under a viewing jar for 5 min, where body position and activity were estimated, as well as any unusual sign such as respiratory difficulties and body tremor. The number of fecal loci was also counted. The jar was then lifted over an open-field made of perspex with four transparent walls (height: 18 cm) and a white opaque floor measuring 55 cm × 33 cm, separated by black line drawings into 121 cm2 squares (5 rows and 3 columns) and the mice dropped in it. The degree of transfer arousal was evaluated and the number of segment crosses (4-paw criterion) was counted in a 30 s period. At the same time, eye opening, piloerection, a startle response to a 90 db auditory tone, and whole-body gait, as well as pelvic and tail elevation were checked. The experimenter then reached for the mouse with one hand and the degree of willingness to escape was judged (touch escape). Once captured, the extent of struggling was estimated. On being lowered by the tail toward a horizontal grid, the mice were evaluated for trunk curl and visual or vibrissae placing responses. The mice were then gently pulled

R. Lalonde et al. / Brain Research Bulletin 64 (2004) 251–258

by the tail and the degree of force exerted on the grid (grip strength) was estimated. Body tone was judged by pressing on the animals’ side. Pinna and corneal reflexes provoked by a probe were then checked. Their sensitivity to toe pinching with a hand-held forceps was also noted. The forepaws of the mice were then placed on a horizontal bar and their ability to lift their hindlimbs and remain on it (wire maneuver) was evaluated. When held by hand in a supine position, the mice were evaluated for skin color, heart rate, abdominal and hindlimb tone (by pressing), lacrimation, and salivation, together with a biting response provoked by a plastic probe. Sensorimotor tests related to body position were then evaluated. In the contact-righting test, the mice were placed inside a small plastic tube and then rotated, the normal response being a return to their original body position. In the negative geotaxis test, the mice were placed face down on a vertically-oriented grid, and their willingness to turn and climb toward the top was noted. The battery was completed with general estimations of fear, irritability, aggression, vocalization, and unusual neurological signs, such as paw clasping, together with body weight and length (from nose to anus). In the open-field test, the same apparatus was used as in the SHIRPA screen, except that the length of the session was extended to 5 min and the number of crossings in the periphery (12 squares) and in the center (3 squares) of the openfield was differentiated. In the emergence test, the mice were placed inside a small toy object (orange-colored plastic shoe, length: 13 cm, width: 6 cm, height: 7.5 cm) perforated with 3 holes (diameter: 3 cm) and situated in the middle of one of 2 larger enclosures made either of opaque white plastic (25 cm × 40 cm, height of walls: 15 cm) or a wooden table (62.5 cm × 25 cm open to all sides except for a 21 cm high wall). The mice had not been previously exposed either to the toy object or the enclosures. The latencies before emerging with either 2 or 4 paws were determined for 4 5 min trials (alternating between the 2 enclosures), with a 1 h intertrial interval. The mice were allowed to explore either enclosure for 10 s after leaving the object or placed there for the same amount of time when the cut-off period had ended. The stationary beam was made of wood covered with masking tape and measured 2 cm in diameter. The beam was separated by line drawings into eleven 10 cm segments and placed at a height of 65 cm from the padded floor. The number of segments crossed (4-paw criterion), and the latencies before falling were evaluated for 4 1 min trials with a 15 min intertrial interval. The coat-hanger was triangular-shaped and consisted of a horizontal steel bar 2 mm in diameter and 40 cm in length. The bar was placed at a height of 60 cm from a cushioned table and was flanked by 2 side-bars 19 cm in length and oriented at 35◦ from the horizontal axis. The mice were placed upside-down in the middle of the wire and were not released until all four paws gripped it. A series of 4 1 min trials was given with a 15 min intertrial interval. Seven movement times (MTs) were compiled, namely latencies before reaching (snout criterion)

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the first 10 cm segment or the extremity of the horizontal bar, latencies before grasping either side-bar with 2, 3 or 4 paws, and latencies before reaching either the midway or the top of the side-bar. The latencies before falling were also tabulated. The trial ended whenever the mice fell or reached the top of the side-bar, from which they were retrieved and the maximal score of 1 min given for latencies before falling. The accelerating rotorod (Letica Rota-Rod, Panlab, Bioseb, France, model 8200) consisted of a beam (diameter: 3 cm, width: 5 cm) made of ribbed plastic and flanked by round plates on either side for preventing any escape. The rod was suspended at a height of 17 cm above the plastic surface. The mice were placed on top of the revolving beam (4 rpm) facing away from the experimenter’s view in the orientation opposite to that of its rotation, so that forward locomotion was necessary for fall avoidance. The rotorod accelerated gradually without jerks from 4 to 40 rpm over the 2 min trial. Latencies were compiled for 3 daily sessions of 8 trials with a 15 min intertrial interval. Whenever the mice clung to the rod without moving (passive rotation) for 2 complete revolutions in succession, it was considered to have fallen. Since no mouse appeared to jump deliberately, a valid estimation of motor skills could be obtained on every trial. 2.3. Statistical analyses Group comparisons were assessed by the unpaired t-test or the non-parametric Mann–Whitney U-test, depending on their level of homogeneity. An analysis of variance (ANOVA) with 12 repeated measures (2-trial blocks) was used for the rotorod. Results are described as means ± S.E.M. Correlations were undertaken in the experimental group between body weight and length, on one hand, and other variables, on the other, with the Pearson product-moment correlation test.

3. Results 3.1. SHIRPA primary screen Despite the absence of overt neurologic dysfunction, hemizygous SOD1/wt transgenic mice differed from nontransgenic controls on certain measures of the SHIRPA primary screen. As shown in Table 1, SOD1/wt transgenic mice could be distinguished from controls on the basis of elevated body weight (t(41) = 3.47, p < 0.01) and length (t(41) = 2.05, p < 0.05). In addition, the sensitivity to visual placing (t(41) = 2.52, p < 0.05), pinna (t(41) = 3.24, p < 0.01), corneal (t(41) = 2.13, p < 0.05), and toe pinch (t(41) = 2.92, p < 0.01) responses was lower in SOD1/wt mice than controls. For the placing response, a value of 2 represents limb extension to vibrissae contact and a value of 3 limb extension to visual cues. The SOD1/wt group mean was nearer that of the former, indicating a loss of sensory responsiveness. In the pinna reflex test, a value of 1 repre-

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Table 1 Mean ± S.E.M. values from the SHIRPA primary screen by SOD1/wt transgenic mice and non-transgenic controls Tests

Controls

SOD1/wt

sents an active response and a value of 2 multiple flicks to a probe placed in the ear. The SOD1/wt group mean was closer to an active response than the multiple flicks displayed by many controls. Likewise, the SOD1/wt group mean was closer to an active response than the multiple blinks displayed by many controls. In the toe pinch test, the SOD1/wt group mean was closer to that of a slight (value of 1) as opposed to a moderate response (value of 2) seen in many controls. No other intergroup difference was found in regard to motor activity and coordination and emotional reactivity. In view of intergroup differences for body weight and length, we determined whether these two variables were linearly correlated in the SOD1/wt group with the other significant variables. The results indicate no significant association between body parameters and other variables. The only significant correlation to emerge was between corneal and pinna reflex scores (r = +0.39, p < 0.05), as would be expected of two tests measuring somatosensory reactivity.

Viewing jar Body position Activity Respiration rate Tremor Fecal boli

3.0 ± 0 2.0 ± 0 2.0 ± 0 0±0 2.9 ± 0.6

3.0 ± 0 2.0 ± 0 2.0 ± 0 0±0 3.7 ± 0.4

Open-field Arousal Activity Eye opening Coat status Startle response Gait Pelvic elevation Tail elevation Touch escape

4.9 ± 0.1 16.2 ± 1.3 0±0 0±0 0.8 ± 0.1 0±0 2.0 ± 0 1.0 ± 0 0.4 ± 0.1

4.9 ± 0.1 14.3 ± 0.9 0±0 0±0 1.1 ± 0.1 0±0 2.0 ± 0 1.0 ± 0 0.6 ± 0.1

Tail-lifting Struggling Trunk curl Visual placing Limb grasping

0±0 1.0 ± 0 2.4 ± 0.1 1.0 ± 0

0±0 1.0 ± 0 2.1 ± 0.05∗ 1.0 ± 0

Horizontal grid Grip strength Body tone Pinna reflex Corneal reflex Toe pinch

2.4 ± 0.1 1.0 ± 0 1.6 ± 0.1 1.4 ± 0.1 2.1 ± 0.2

2.6 ± 0.1 1.0 ± 0 1.1 ± 0.1∗∗ 1.1 ± 0.1∗ 1.3 ± 0.1∗∗

Horizontal wire Wire maneuver

1.1 ± 0.4

1.2 ± 0.3

Supine restraint Skin color Heart rate Limb tone Abdomen tone Lacrimation Salivation Biting

1.0 ± 0 1.0 ± 0 1.4 ± 0.2 1.0 ± 0 0±0 0±0 0±0

1.0 ± 0 1.0 ± 0 1.3 ± 0.1 1.0 ± 0 0±0 0±0 0.1 ± 0.1

Tube Contact righting

0.9 ± 0.1

1.0 ± 0

3.3. Motor coordination

Vertical grid Geotaxis

0.4 ± 0.2

0.4 ± 0.1

Handling Fear Irritability Aggression Vocalization

0.2 ± 0.2 1.0 ± 0 0±0 0±0

0±0 1.0 ± 0 0.1 ± 0.1 0±0

Body measures Weight (g) Length (cm)

20.3 ± 0.6 8.3 ± 0.1

23.2 ± 0.5∗∗ 8.6 ± 0.1∗

Additional signs Paw clasping

0±0

0±0

As seen in Fig. 1, although their latencies before falling were equivalent to those of controls (t(41) = 0.97, p > 0.05), SOD1/wt transgenic mice crossed fewer segments on the stationary beam (t(41) = 3.93, p < 0.001). There was no significant correlation between distance travelled on the stationary beam and either body weight or length (p > 0.05) in the SOD1/wt group. In contrast to the stationary beam test, there was no intergroup difference for any aspect of coat-hanger (Table 2) and rotorod (Fig. 2) tests. Indeed, the only significant difference in the rotorod paradigm was the trial block effect (F(11,429) = 15.39, p < 0.001), as both groups were able to stay longer on the beam with repeated practice, although some fluctuation was observed with respect to the drop in performance on the first trial of test days 2 and 3, presumably a result of incomplete consolidation of motor abilities.

∗ ∗∗

p < 0.05 vs. controls (unpaired t-test). p < 0.01 vs. controls (unpaired t-test).

3.2. Exploratory movements In the open-field, the number of segments crossed by SOD1/wt transgenic mice did not differ from that of nontransgenic controls, either in the periphery (t(41) = 0.94, p > 0.05) or center (t(41) = 0.98, p > 0.05). The mean values were 123.4 ± 4.8 for SOD1/wt versus 131.9 ± 8.4 for controls in the periphery, and 17.3 ± 1.9 versus 20.8 ± 3.0, respectively, in the center. Likewise, no significant difference was revealed in the emergence (p > 0.05) test. On the initial trial of the small enclosure, the mean values were 5.1 ± 0.8 for SOD1/wt versus 7.6 ± 2.5 for controls with the 2-paw criterion and 7.0 ± 1.1 versus 12.8 ± 4.8, respectively, with the 4-paw criterion.

R. Lalonde et al. / Brain Research Bulletin 64 (2004) 251–258

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Table 2 Mean ± S.E.M. movement times (MTs), latencies before falling, and number of falls by SOD1/wt transgenic mice and non-transgenic controls in the coathanger test Tests

Controls

MTs in s (maximal score: 240) Initial 93.9 ± 15.0 End 161.2 ± 13.0 2-paw 200.1 ± 11.5 3-paw 210.4 ± 9.8 4-paw 224.4 ± 7.1 Midway 229.1 ± 5.7 Top 237.4 ± 2.6 Fall latencies

191.4 ± 11.4

SOD1/wt 112.0 ± 10.3 174.2 ± 10.8 209.5 ± 8.3 223.0 ± 7.1 228.6 ± 6.2 231.3 ± 4.8 237.8 ± 1.4 194.6 ± 7.1

are opposite to the diminished body weight reported in ALS mouse models, including one without paresis [30]. In view of the enhanced neuronal survival to various types of toxicity seen after SOD1/wt expression [3,6,22,38,47,48], it is possible that the transgene caused a growth-promoting action in various organs during development. A second possibility is a decrease in litter size of the SOD1/wt progeny, leading to bigger animals. However, we did not find a change in litter size of the SOD1/wt progeny (unpublished observations). A third possibility is that SOD1/wt pups were more competitive or aggressive than their wild-type littermates with respect to access to the dam. In addition to physical differences, SOD1/wt transgenic mice were distinguishable from controls in certain sensorimotor tests. In the placing test, mice held by the tail and slowly lowered toward a horizontal surface extend their limbs in anticipation of touching the ground, either from a certain distance (visual placing) or with vibrissae contact. By con-

Fig. 1. Mean ± S.E.M. number of segments crossed and latencies before falling from the stationary beam by 3-month-old hemizygous SOD1/wt transgenic mice and non-transgenic controls: ∗ p < 0.001 vs. controls.

4. Discussion 4.1. SHIRPA primary screen By contrast to mice expressing mutated SOD1 [23,35], SOD1/wt transgenic mice display no overt sign of neurological dysfunction, such as paresis, tremor, and pathological limb-clasping reflexes. Nevertheless, 3-month-old hemizygous SOD1/wt transgenic mice were distinguishable from non-transgenic controls in regard to body weight and length as well as sensorimotor tests. In particular, the SOD1/wt transgenic mice were heavier and were longer in size. These results

Fig. 2. Mean ± S.E.M. latencies before falling from the rotorod by 3-monthold hemizygous SOD1/wt transgenic mice and non-transgenic controls.

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trast to the DBA strain exhibiting only visual placing [30], the reactivity of the FVB/N strain was more variable, consisting either of visual or tactile placing. This variability is likely due to retinal damage caused by an abnormal allele of the Pdeb gene [14,49]. We found that the visual placing response was even rarer in the SOD1/wt group. A loss in sensorimotor responsiveness in the SOD1/wt group was not limited to the visual modality, but extended as well to three somatosensory tests. In the pinna reflex test, the response of the control group to touching with a probe inside of the ear was variable, consisting either of an active single flick or else of multiple flicks. The same pattern was disclosed with respect to the corneal reflex, as wild-type mice either exhibited a single blink or else multiple ones. Relative to controls, the somatosensory responsiveness of SOD1/wt transgenic mice declined in both tests. In each case, the diminished responsiveness did not reach pathological levels, as defined by values below the active response, but reduced the probability of multiple responses characteristic of the FVB/N strain. In the transgenic group, corneal and pinna scores were linearly correlated, an indication that the subsensitivity is due to a single common factor. A loss in somatosensory function was also disclosed in the SOD1/wt group during toe-pinching to a hand-held forceps. The reduced sensitivity to visual and tactile stimuli observed in SOD1/wt transgenic mice may be akin to the sensory deficits described in Down syndrome for visual [55], tactile [4], and auditory [1,16,33,54] modalities. In addition to brain anomalies, this hyposensitivity may be caused by structural damage, as reported in otitis media-induced hearing loss. Nevertheless, one possible cause of these deficits is the overexpression of SOD1 in brain. It remains to be determined whether the transgene-induced hyposensitivity can be generalized to other transgenic models expressing SOD1. A second possible cause of the hyposensitivity is the insertion of the human as opposed to the endogenous gene.

In addition, lesion studies in rats indicate that stationary beam performance is dependent on the integrity of motor and posterior parietal cortex [25,26]. SOD1/wt transgenic mice crossed fewer segments than controls on the stationary beam. On the basis of normal values observed in open-field and emergence tests, the hypoactivity in the balance beam is unlikely to be due to reduced brain activation or increased anxiety. Instead, these results point toward a deficiency in the sensorimotor control required for moving along the beam, not observable with the larger surface area in the open-field. Nevertheless, this deficit appeared mild, as indicated by normal latencies before falling on stationary beam, coat-hanger, and rotorod tests. The absence of a deleterious effect on the rotorod in these 3-month-old hemizygous SOD1/wt transgenic mice reproduces previous results with a different apparatus in 10-month-old homozygous mice [21]. This test has also been used in the evaluation of transgenic mice with segmental trisomy-16 (Ts65Dn) spanning the SOD1 locus. On one hand, a deficit was found in one rotorod test [9], but not in others, perhaps because of a lower initial starting speed [2,20]. The relative sensitivity of the stationary beam as opposed to the other two tests reflect their different motor demands. Mouse movement on the rotorod is impelled by beam rotation. Mouse movement on the coat-hanger is impelled by their upside-down position, providing motivation to move toward the extremity of the horizontal bar and to right themselves. But no factor impels mouse movement on the stationary beam, as a fall may be avoided simply by remaining still. Thus, the mild hypo-responsiveness revealed in tactile tests of the SHIRPA screen appear insufficient to cause major defects in sensorimotor integration. We will evaluate older hemizygous as well as homozygous mice in the same battery in order to determine whether more severe deficits are discernable with advanced aging.

4.2. Exploratory movements

Acknowledgements

In view of the bulbar-like symptoms seen in ALS [36,45] and the increased impulsivity seen in children with Down syndrome [8,39], one would expect evidence of behavioral disinhibition in an animal model mimicking such disorders. However, SOD1/wt transgenic mice did not differ from nontransgenic controls in terms of exploration of an open-field and emergence from a small toy object, indicating normal levels of cerebral activation and emotional reactivity.

This study was supported by grants from the European Community (BMH14-CT95-3039) and the “Fondation J´erome Lejeune” to JL and from the “Fondation pour la Recherche M´edicale de Lorraine” to CS.

4.3. Motor coordination In view of the loss in balance and equilibrium observed in children with Down syndrome [24], motor coordination was examined in stationary beam, coat-hanger, and rotorod tests. These tests are sensitive to cerebellar degeneration in mice with natural mutations [28] as well as transgenic mice with neurofilament [7,11,29,30] and muscular [46,50] anomalies.

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