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Human Molecular Genetics Advance Access originally published online on July 6, 2005
Human Molecular Genetics 2005 14(16):2369-2385; doi:10.1093/hmg/ddi239
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Profound ataxia in complexin I knockout mice masks a complex phenotype that includes exploratory and habituation deficits

Dervila Glynn1, Cheney J. Drew1, Kerstin Reim2, Nils Brose2 and A. Jennifer Morton1,*

1Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK and 2Max-Planck-Institute for Experimental Medicine, Hermann-Rein-Str. 3, 37075 Göttingen, Germany

* To whom correspondence should be addressed. Tel: +44 1223334057; Fax: +44 1223334040; Email: ajm41{at}cam.ac.uk

Received April 21, 2005; Revised June 8, 2005; Accepted June 29, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Complexins are presynaptic proteins that bind to the SNARE complex where they modulate neurotransmitter release. A number of studies report changes in complexins in psychiatric (schizophrenia and depression) and neurodegenerative disorders (Huntington's disease, Wernicke's encephalopathy and Parkinson's disease). Here, we characterize the behavioural phenotype of Cplx1 knockout (Cplx1–/–) mice. Cplx1–/– mice develop a strong ataxia in the absence of cerebellar degeneration. Although originally reported to die within 2–4 months after birth, when reared using an enhanced feeding regime, these mice survive normally (i.e. >2 years). Cplx1–/– mice show pronounced deficits in motor coordination and locomotion including abnormal gait, inability to run or swim, impaired rotarod performance, reduced neuromuscular strength, dystonia and resting tremor. Although the abnormal motor phenotype dominates their overt symptoms, Cplx1–/– mice also show other behavioural deficits, particularly in complex behaviours. They have deficits in grooming and rearing behaviour and show reduced exploration in several different paradigms. They also show deficits in tasks reflecting emotional reactivity. They fail to habituate to confinement and show a ‘panic’ response when exposed to water. The abnormalities seen in the behaviour of Cplx1–/– mice reflect those predicted from the distribution of complexin I in the brain. Our data show that complexin I is essential not only for normal motor function in mice, but also for normal performance of other complex behaviours. These results support the idea that altered expression of complexins in disease states may contribute to the symptomatology of disorders in which they are dysregulated.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Complexins are small, highly homologous, hydrophilic proteins that bind to the neuronal SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) complex in the brain (1Go,2Go). Current evidence supports the idea that they play an important role in the modulation of neurotransmitter release (3Go–5Go).

There are two major isoforms of complexin in the brain, complexin I and complexin II (1Go), and a third isoform that has a more restricted expression (6Go). Complexin I and complexin II have largely reciprocal patterns of distribution in rodent brain (7Go–10Go), although there are some brain regions in which both isoforms are expressed (9Go). Complexin I is mainly found in axosomatic synapses, whereas complexin II is mainly found in axodendritic synapses (9Go), and it has been suggested that complexin I and complexin II modulate inhibitory and excitatory transmission, respectively (7Go,11Go–13Go). However, this is not the rule in all the regions of the brain, because in the mouse brain, Cplx2 is expressed by inhibitory gamma-aminobutyric acid (GABA) output neurons in the striatum (10Go).

Although the role of complexins in the brain is not clear, they have been implicated in learning (10Go,14Go,15Go) and in disease (11Go–13Go,15Go–23Go). Indeed, a growing number of studies report changes in complexin expression in both psychiatric and neurodegenerative disorders such as schizophrenia (11Go,12Go,15Go–19Go), bipolar and unipolar disorders (16Go,18Go), Huntington's disease (HD) (20Go,21Go), Wernicke's encephalopathy (13Go) and Parkinson's disease (22Go). It has also been suggested that complexin I may play a role in diabetes (23Go).

From our previous expression studies (10Go), we suggested that complexin II is likely to play a major role in cognition, emotional behaviour and control of voluntary movement, whereas complexin I would play a major role in motor learning and sensory processing. In support of this idea, we have shown that Cplx2 knockout (Cplx2–/–) mice (14Go) have abnormalities in exploration and social behaviour as well as subtle progressive deficits in motor coordination, learning and reversal learning, whereas Cplx1–/– mice develop a strong ataxia (3Go).

The phenotype of Cplx1–/– mice is intriguing. Although there are many mouse models of ataxia (reviewed in 24–26), the majority of these exhibit profound cerebellar degeneration (27Go,28Go). In contrast, the ataxia shown by Cplx1–/– mice is seen in the absence of degeneration. Cplx1–/– mice suffer from sporadic seizures, are unable to reproduce and were reported to die within 2–4 months after birth (3Go). The behaviour of these mice has not been characterized in detail, because the early death of Cplx1–/– mice made detailed analysis of their behavioural phenotype difficult. In this study, we modified the husbandry of Cplx1–/– mice to improve their survival. This allowed us to characterize their behavioural phenotype. We find that the deficits in Cplx1–/– mice are not restricted to the cerebellar ataxia initially described, but that they have complex behavioural abnormalities that include not only motor deficits, but also abnormalities in behaviours that reflect ‘emotional’ deficits.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cplx1–/– mice have reduced body weights but normal survival rates
All mice used in the behavioural analysis described here were raised using a modified feeding regime (see Materials and Methods). Figure 1A shows the weights of Cplx1–/– mice, Cplx1 heterozygote (Cplx1–/+) and Cplx1 wild-type (Cplx1+/+) mice bred and reared in the Cambridge facility. Although Cplx1–/– mice thrived, they were consistently lighter than Cplx1+/+ and Cplx1–/+ mice (genotype, F2,55=47.78, P<0.001; age, F11,684=35.26, P<0.001 and agexgenotype, F11,605=1.78, P<0.05). Further, although Cplx1+/+ and Cplx1–/+ mice continued to grow throughout the course of the study, Cplx1–/– mice stopped growing at ~4 months of age. It seems likely that difficulties in accessing food caused the initial reported reduced mortality rates (3Go). This is supported by data from the imported founder mice that had been fed on a standard regime (hard pellets in a suspended hopper). Immediately following the implementation of the new feeding regime (soft moist food on the cage floor), all Cplx1–/– mice gained weight (Fig. 1B) [P<0.01, after 2 weeks, the body weights of Cplx1–/– and wild-type (Cplx1+/+) mice increased by 30 and ~12%, respectively].



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  		Figure 1. Body weights of Cplx1+/+, Cplx1–/+ and Cplx1–/– mice bred ‘in-house’ are shown in (A). Body weights of Cplx1–/– mice were similar to those of Cplx1+/+ mice until 4 months of age. After this time, Cplx1–/– mice stopped growing. The effect of supplementary feeding of the founder Cplx1–/– mice was marked, with a significant increase in body weight seen in both Cplx1+/+ and Cplx1–/– mice in the first 2 weeks after a change in the feeding regime (B, arrow). Symbols show the mean±SEM body weight of each group. Where error bars are not visible, they are obscured by the symbols. Asterisks indicate significant differences between Cplx1+/+ and Cplx1–/– mice (***P<0.001).

 
When given supplementary food in an easily accessible form, Cplx1–/– mice survived to at least 2 years and were no more susceptible to illness than Cplx1+/+ mice. During this study, there was no difference in the number of Cplx1+/+ and Cplx1–/+ mice that died before 2 years of age.

Although seizures were originally described as being part of the phenotype (3Go), seizures have not been observed in mice in the Cambridge colony. Therefore, none of the behavioural tasks described in this paper were compromised by seizure activity. The reason for the lack of seizures in mice in the Cambridge facility is not clear. We have not examined the susceptibility of Cplx1–/–mice to seizures, although this would clearly make an interesting subject for future studies.

Cplx1–/– mice are ataxic and exhibit dystonia and tremor
We characterized the motor behavioural phenotype of Cplx1–/– mice using footprint analysis (Fig. 2) and open field observations (Fig. 5). Cplx1–/– mice suffer from a severe ataxia with dystonia. The ataxia of Cplx1–/– mice is characterized by gait abnormalities (discussed subsequently), which impair their ability to control the direction of their movements. These are complicated by involuntary movement of the extremities and defective coordination between the movements of the limbs. The ataxic gait is apparent by postnatal day 7 (P7) and is pronounced by the time they are weaned at 21 days. Adult Cplx1–/– mice are not capable of coordinated running.



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  		Figure 2. Photographs show typical Cplx1+/+ (A) and Cplx1–/– (B) mice at 6 months of age. The adult Cplx1–/– mouse seen standing on its toes (arrows) is smaller than Cplx1+/+ mice, but still appears healthy with a glossy coat and bright eyes. Dystonia is particularly obvious in the tail suspension test. When a Cplx1+/+ mouse is suspended by its tail, its characteristic response is to try to escape by splaying its hindlimbs away from the trunk of its body (C). In contrast, when a Cplx1–/– mouse is suspended by its tail (D), its hindlimbs are held tonically against its trunk in an abnormal dystonic posture (white arrows). The parameters measured in the footprint analysis are shown in (E). In (F) and (G), purple and orange footprints represent fore- and hindpaws, respectively. Representative walking footprint patterns of 7-month-old Cplx1+/+ (F) or Cplx1–/– (G) mice reveal that Cplx1–/– mice are ataxic and walk with an abnormal wide-based gait. Cplx1–/– mice walk with their heals down (black arrow heads in G), in a ‘leap-frog’ motion where the hind footprint (orange) falls in front of the preceding front footprint (purple), and show a greater proportion of scuffled footprints (black arrows in G and H). Quantitative analysis of the footprints shows significant differences in hind and front base width (I) and stride length (J). Bars represent mean±SEM on each measure. Asterisks indicate significant differences between Cplx1+/+ and Cplx1–/– mice (*P<0.05, **P<0.01 and ***P<0.001).

 


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  		Figure 5. Cplx1–/– mice show deficits in behaviour in the open field when compared with Cplx1–/+ and Cplx1+/+ mice. Naïve adult Cplx1–/– mice took significantly longer time to reach the periphery upon being placed in the centre of the open field (A). Cplx1–/– mice spent more of their time in motion (B), but did not travel a significantly greater distance when compared with the other two groups (C). Cplx1–/– mice entered a similar proportion of central squares when compared with Cplx1–/+ and Cplx1+/+ mice (D). Cplx1–/– mice show deficits in grooming behaviour (E). Cplx1–/– mice do not perform complete grooming cycles and showed significant deficits in face washing and body washing. Although there was a trend for Cplx1–/– mice to exhibit a lower frequency of scratching, this did not reach significance. There were no significant differences in the frequency of supported rearing (F), but Cplx1–/– mice were unable to rear without support in the periphery or in the centre of the open field. Bars indicate the mean±SEM of each group on each measure. Where error bars are not visible, they are obscured by the symbols. Asterisks indicate significant differences between Cplx1+/+, Cplx1–/+ and Cplx1–/– mice (*P<0.05, **P<0.01 and ***P<0.001).

 
In addition to ataxia, Cplx1–/– mice exhibit dystonia as characterized by twisting movements and abnormal postures. Dystonia is never seen in Cplx1+/+ mice (Fig. 2A and C) or in Cplx1–/+ mice (data not shown), but is seen in all Cplx1–/– mice (Fig. 2B and D). Abnormal dystonic postures are commonly observed not only at rest, but also during locomotion. These include futile paddling of the hindlimbs, arched back, extended limbs, flexed limbs held close to the trunk or twisting and elevation of the hindlimbs above the base of the tail. Cplx1–/– mice also exhibit head bobbing and twitching both at rest and in motion. In addition to the dystonia and ataxia, adult Cplx1–/– mice exhibit a pronounced resting tremor (visible by 6–7 weeks) that is not seen in Cplx1+/+ or Cplx1–/+ mice. Nevertheless, despite their pronounced locomotor disabilities, adult Cplx1–/– mice have a normal righting reflex and look healthy with glossy coats and bright eyes.

Cplx1–/– mice do not have diabetes
No Cplx1–/– or Cplx1+/+ mice tested positive for glycosuria during this study (data not shown). Blood glucose levels in Cplx1+/+ and Cplx1–/– mice were tested after fasting and 1 h after a glucose challenge, at 2–3 months and in mice at >1 year of age. A mouse was considered diabetic if it had a concentration of ≥18 mmol glucose per litre. No mouse used in this study had elevated blood glucose levels after 16 h fasting at 2–3 months [6.8±0.3 (Cplx1+/+, n=10); 5.0±1.0 (Cplx1–/–, n=4); mmol/l±SEM] or at >1 year [6.9±0.8 (Cplx1+/+, n=2); 5.4±0.2 (Cplx1–/–, n=3); mmol/l±SEM]. Similarly, there was no evidence of abnormally elevated glucose levels taken 1 h after the glucose challenge at 2–3 months [11.2±0.9 (Cplx1+/+); 9.8±0.4 (Cplx1–/–); mmol/l±SEM] or at >1 year [12.4±0.9 (Cplx1+/+); 8.8±1.1 (Cplx1–/–); mmol/l±SEM].

Cplx1–/– mice show abnormalities in their gait and stride length
Footprints analysis confirmed the marked difference in gait between Cplx1+/+ and Cplx1–/– mice (Fig. 2E–J). Although Cplx1+/+ mice walked in a straight line, with a regular and even alternating stride, Cplx1–/– mice displayed an abnormal wide-based gait of both fore- and hindpaws (Fig. 2I, P<0.01 and P<0.05, respectively). A significantly greater number of Cplx1–/– mice showed ‘scuffled’ footprints when compared with Cplx1+/+ mice [Fig. 2G (arrows) and H, P<0.001]. This ‘scuffling’ is caused by an unsteadiness of the paws of Cplx1–/– mice while in motion and demonstrates an overall loss of control of fine movement. Moreover, during normal locomotion in mice, the centre of the hindlimb footfall falls on top of, or just behind, the centre of the preceding forelimb footfall. In contrast, Cplx1–/– mice showed another unusual feature of gait in that they walked in a ‘leap-frog’ motion, in which the hindlimb footfall was in front of the preceding forelimb footfall. In addition, they often walked on lowered heels (Fig. 2G, arrow heads), and displayed a trend for increased stride length when compared with Cplx1+/+ mice that reached significance for right hind stride and right front stride (Fig. 2J, P<0.01 and P<0.05, respectively).

Cplx1–/– mice cannot swim
The swim tank was used to investigate motor function and coordination of mice at 6–7 months of age. Cplx1+/+ mice swim normally (Fig. 3A). When placed in the water, they swam with their noses and tails elevated above the water line, tucking their forelimbs into their chest and used their hindlimbs to propel themselves. In contrast, Cplx1–/– mice could not swim at all (Fig. 3B). On being placed in the water, they adopted abnormal dystonic postures and kicked in a splayed and uncoordinated manner with both fore- and hindlimbs. They twisted and rotated in the water and were sometimes observed to somersault backwards or forwards, spiral underwater or even plummet to the bottom. They did not appear to be capable of orientating themselves properly with respect to the surface of the water.



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  		Figure 3. Motor function and coordination of Cplx1+/+ and Cplx1–/– mice aged 6–7 months were tested using the swim tank. Representative still images captured from video (recorded at 25 frames/s) are shown for Cplx1+/+ mice (A) and Cplx1–/– mice (B). Cplx1+/+ mice were able to swim normally (A), with their noses elevated above the water line, their forelimbs tucked into their chest and their hindlimbs used to propel themselves. Cplx1–/– mice were unable to swim (B). Upon being placed in the water, this Cplx1–/– mouse dived under the water and was completely submerged, before it rolled over on its back leaving just its nose above the water surface. It then somersaulted backwards until finally emerging holding its trunk vertically out of the water. In addition to the abnormalities illustrated here, Cplx1–/– mice were also observed floating in abnormal dystonic postures, somersaulting, spiralling underwater and nose-diving to the bottom of the swim tank.

 
Cplx1–/– mice have high SHIRPA abnormality indices
Abnormality indices
The mean abnormality index in the SHIRPA test was significantly higher for Cplx1–/– mice than that for Cplx1+/+ and Cplx1–/+ mice at 9 weeks (Fig. 4A, P<0.001). This was expected (given the abnormal overt phenotype exhibited by Cplx1–/– mice) and was mainly attributable to the abnormal gait, lowered tail and pelvic elevation and initial inactivity of Cplx1–/– mice upon cage opening.



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  		Figure 4. Cplx1–/– mice have impaired strength and motor coordination. Cplx1–/– mice have higher abnormality indices during SHIRPA analysis when compared with Cplx1–/+ and Cplx1+/+ mice (A) and show impaired neuromuscular strength and a lack of coordinated motor control as measured using the hang wire test (B) and grip strength meter (C). Cplx1–/– mice perform poorly on the rotarod. Although there were no significant differences in rotarod performance between Cplx1+/+, Cplx1–/+ and Cplx1–/– mice aged 4.5 weeks at 5 r.p.m., Cplx1–/– mice performed significantly worse than Cplx1+/+ and Cplx1–/+ mice at 8 and 15 r.p.m. (D). In contrast, naïve adult Cplx1–/– mice fail to perform on the rotarod at 0 or 5 r.p.m. at various ages (E) when compared with the typical Cplx1+/+ mouse performance indicated by the dashed line. The values of mean±SEM of the latency to fall (maximum trial length=60 s) for four trials at each speed were recorded. Bars indicate the mean±SEM for latency to fall from the rotarod. Where error bars are not visible, they are obscured by the symbols. Asterisks indicate significant differences between Cplx1+/+, Cplx1–/+ and Cplx1–/– mice (*P<0.05, **P<0.01 and ***P<0.001).

 
Hang wire and grip strength
The hang wire test was used to assess neuromuscular strength. Cplx1–/– mice fell off the hang wire earlier when compared with Cplx1+/+ and Cplx1–/+ mice at 9 weeks of age. Cplx1+/+ and Cplx1–/+ mice could hang from the wire for at least 30 s before releasing the wire. In contrast, Cplx1–/– mice were unable to cling to the wire for any extended period of time (Fig. 4B, P<0.001), suggesting either a lack of coordinated motor control or reduced strength. A grip strength meter was used to assess forelimb grip strength. Grip strength of Cplx1–/– mice was significantly weaker than that of Cplx1+/+ and Cplx1–/+ mice (Fig. 4C, P<0.001) at 9 weeks of age.

Cplx1–/+ and Cplx1–/– mice show impaired performance on the rotarod
Cplx1+/+, Cplx1–/+ and Cplx1–/– mice aged 4.5 weeks were trained on the rotarod at 5, 8 and 15 r.p.m. The data from the first day of training for all three groups are shown in Figure 4D. At this age, all three groups performed comparably at 5 r.p.m. However, the latency of Cplx1–/– mice to fall was significantly shorter than either Cplx1+/+ or Cplx1–/+ mice at 8 and 15 r.p.m. A second group of naïve mice was tested for the first time from 8 weeks of age. At this age, Cplx1–/– mice could not stay on a rotating rotarod even at 5 r.p.m. (Fig. 4E). Indeed, their constant uncoordinated movement impaired their ability to balance on the rotarod, even when it was not rotating.

Given the complete failure of adult Cplx1–/– mice on the rotarod task, we wondered whether there was a gene dosage effect of the Cplx1 gene on this task. To test this, adult Cplx1+/+ and Cplx1–/+ mice were trained and then tested on the rotarod at 6 months and again at 9 months (Table 1). Both groups of mice were trained for four trials per day at 24 r.p.m. for three consecutive days and learnt the task in a similar manner (data not shown). On the day following training, both groups were tested at eight different speeds. At 6 months of age, all mice performed equally well at the lowest four speeds. However, at all of the higher speeds, Cplx1–/+ mice performed poorly and fell off the rotarod significantly sooner than Cplx1+/+ mice (genotype, F1,23=8.45, P<0.01 and speed, F7,161=21.19, P<0.001). There was also a significant interaction between speed and genotype, as the performance of the Cplx1+/+ mice remained constant across all speeds, whereas Cplx1–/+ mice performance was worse at all of the higher speeds (speedxgenotype, F7,161=8.83, P<0.001). At 9 months, the performance of Cplx1–/+ mice remained consistently worse than that of Cplx1+/+ mice, although there was no further deterioration in the rotarod performance of Cplx1–/+ mice (genotype, F1,22=5.45, P<0.05; speed, F7,154=15.72, P<0.001 and speedxgenotype, F7,154=3.66, P<0.001).


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  		Table 1. Rotarod performance in Cplx1+/+ and Cplx1–/+ mice aged 6 and 9 months
 
Open field testing revealed locomotor and exploratory deficits in Cplx1–/–mice
Latency to reach the periphery
When naïve mice were placed in the open field, Cplx1+/+ and Cplx1–/+ mice moved quickly to the periphery. However, Cplx1–/– mice did not move immediately and took more than twice as long as Cplx1+/+ and Cplx1–/+ mice to move to the periphery (Fig. 5A) (F2,45=6.001, P<0.01).

Activity levels
Cplx1–/– mice spent significantly more time moving in the open field than Cplx1+/+ and Cplx1–/+ mice (Fig. 5B) (F2,46=12.01, P<0.001). Although Cplx1+/+ and Cplx1–/+ mice displayed the bursts of activity that are typical of mice placed in the open field, Cplx1–/– mice moved almost constantly. However, all groups entered a similar total number of squares [215.9±29.1 (Cplx1+/+ mice); 157.5±19.2 (Cplx1–/+ mice); 189.8±16.6 (Cplx1–/– mice); total number of squares entered±SEM] and covered a similar distance over the 10 min trial period (Fig. 5C). Thus, although Cplx1–/– mice spent more time moving, they did not travel further. Rather, their locomotion was inefficient when compared with Cplx1+/+ and Cplx1–/+ mice.

Cplx1+/+, Cplx1–/+ and Cplx1–/– mice entered a similar percentage of central squares (Fig. 5D), suggesting that there was no difference in anxiety levels as measured by the open field between the three groups (29Go).

Urinations and faecal boli
There were no significant differences between Cplx1+/+, Cplx1+/– and Cplx1–/– mice in the number of faecal boli or urinations deposited in the open field during the 10 min period (data not shown). Together with the central square data, this suggests that there was no difference in anxiety levels between the three groups of mice.

Grooming
A grooming cycle in a normal mouse consists of an ordered sequence of face washing, body washing and scratching of the shoulders, chest and hindquarters (30Go,31Go). Cplx1–/– mice do not groom properly in the open field and were unable to perform complete grooming cycles (Fig. 5E) (chi-square, {chi}2=6.161, P<0.05). Cplx1–/– mice exhibited a lower frequency of face washing and body washing that was significantly lower than that seen in Cplx1–/+ mice (Fig. 5E; P<0.05 and P<0.01, respectively). In addition, Cplx1+/+ and Cplx1–/+ mice washed their face by bringing their forepaws down over their entire face (from ear to mouth), whereas Cplx1–/– mice tended only to wash their mouth and under their chin. Cplx1–/– mice also displayed a trend for a lower frequency of scratching (Fig. 5E), although this did not reach significance when compared with Cplx1+/+ or Cplx1–/+ mice. We normally house our mice in mixed genotype groups, and all mice are well groomed. However, when Cplx1–/– mice are housed without wild-type mice, they remain well groomed and in good condition. This suggests that, although the mice appear not to perform complete cycles in the open field, they perform specific components of the grooming cycle well enough to keep themselves clean.

Exploration and rearing
Cplx1–/– mice exhibited marked differences in rearing activity in the open field (Fig. 5F). The frequency of supported rearing (where forepaws were propped against the wall) was similar in all three groups. However, Cplx1+/+ mice rear with fully extended hindlimbs, their head and body held erect, whereas Cplx1–/– mice did not extend their hindlimbs when rearing, but instead sat back on their haunches. In addition, Cplx1–/– mice frequently lost their balance while rearing, resulting in the mouse falling over onto its back or side. Cplx1–/– mice were rarely able to rear without forelimb support in the periphery (where 38.5% of Cplx1–/– mice reared compared with 70.6% of both Cplx1–/+ and Cplx1+/+ mice, F2,46=4.607, P<0.05). In the centre of the open field, none of the Cplx1–/– mice reared compared with 11.7% of Cplx1–/+ and 35.2% of Cplx1+/+ mice ({chi}2=7.018, P<0.05).

Cplx1–/– mice exhibit reduced exploration of novel stimuli
Rearing activity is usually a good measure of exploratory behaviour, and the rearing deficits observed during open field behaviour suggest a decrease in exploration. However, the difficulties that Cplx1–/– mice have in maintaining normal balance meant that rearing was not a good measure of exploratory behaviour. To study the exploratory deficits in Cplx1–/– mice further, we used tasks that did not require the same level of coordination as rearing in the open field. We used three tests of exploration; a novel environment (clean cage with opened lid), a familiar enclosure (home cage igloo) within a novel environment (open field) and a novel complex environment (a ‘playground’). When placed in a simple novel home cage environment, Cplx1–/– mice show a different pattern of exploratory behaviour from that exhibited by Cplx1+/+ mice. Cplx1+/+ mice actively explored their new surroundings, sniffing the air and bedding, rearing at the cage walls or in the centre of the cage, exploring the wider environment and eventually trying to climb out of the box, whereas Cplx1–/– mice explored the new cage for only a short period (15–30 min) and then went to sleep. This is illustrated in Figure 6A and B, where Cplx1+/+ and Cplx1–/– mice (aged 5 months) were photographed after being placed in a clean open-topped cage. Both groups were active and explored their new surroundings for the first 15 min (Fig. 6A). However after 1 h (Fig. 6B), although Cplx1+/+ mice continued to explore actively, all of the Cplx1–/– mice were huddled together, asleep in a corner.



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  		Figure 6. Cplx1–/– mice exhibit reduced exploration of novel stimuli and environments. This behaviour is illustrated in (A) where Cplx1+/+ and Cplx1–/– mice were photographed 15 min after being placed in a novel environment. Both groups were active and explored their new environments with interest. The same mice were photographed again 45 min later (B). Although Cplx1+/+ mice continued to explore their surroundings, all Cplx1–/– mice were asleep. During the emergence test (C–E), Cplx1–/– mice took significantly longer time to emerge from the igloo house (C), made a significantly greater number of aborted excursions from the igloo (D) and spent a significantly greater proportion of time within the igloo (E). The ‘playground’ used for measuring exploratory behaviour is shown in (F). In each of the three trials, Cplx1–/– mice spent significantly less time in contact with the toys in the playground when compared with Cplx1–/+ and Cplx1+/+ mice (G). Bars indicate the mean±SEM of each group on each measure. Where error bars are not visible, they are obscured by the symbols. Asterisks indicate significant differences between Cplx1+/+, Cplx1–/+ and Cplx1–/– mice (*P<0.05, **P<0.01 and ***P<0.001).

 
Cplx1–/– mice show exploratory deficits in the emergence test
Exploratory behaviour was assessed in Cplx1 mice using an ‘emergence test’ in which mice were allowed to explore the open field with the option of returning to a safe and familiar enclosure (the igloo house from their home cage). When compared with Cplx1+/+ and Cplx1–/+ mice, Cplx1–/– mice took significantly longer time to emerge from the igloo house during the test period (Fig. 6C, P<0.001) and made a significantly greater number of aborted excursions from the igloo (Fig. 6D, P<0.01 and P<0.001 when compared with Cplx1+/+ and Cplx1–/+ mice, respectively). Further, Cplx1–/– mice spent a significantly greater proportion of time within the igloo than Cplx1–/+ or Cplx1+/+ mice (Fig. 6E, P<0.001).

Cplx1–/– mice show reduced exploration in the playground
Mice were assessed in the ‘playground’, a free exploration paradigm (Fig. 6F). Running wheels and wooden climbing apparatus (‘toys’) provided novel objects to explore, and the mice also had the opportunity for social stimulation and interaction. In each of the three trials conducted on three consecutive days, Cplx1–/– mice spent significantly less time on the toys in the playground than Cplx1+/+ mice (Fig. 6G). Interestingly, Cplx1–/+ mice also appeared to exhibit subtle deficits in exploratory behaviour in the playground, spending only ~50% of their time using the different apparatus in the playground (compared with ~70% of time spent by Cplx1+/+ mice). Although the amount of time Cplx1–/+ mice spent exploring was not significantly different from Cplx1+/+ mice in any trial, by the third trial, significant differences between Cplx1–/+ and Cplx1–/– mice had also disappeared.

Mice were scored for being ‘inactive’ when they were observed sleeping or not moving. There was no significant difference in time spent inactive during trial one. However, in trials two and three, Cplx1–/– mice spent >50% of their time inactive (usually sleeping or huddled together in the igloo) compared with 10% (P<0.01) in Cplx1+/+ mice and 30% (P<0.05) in Cplx1–/+ mice. Notably, in contrast to Cplx1–/– mice, Cplx1+/+ and Cplx1–/+ mice did not sleep while in the igloo during their ‘inactive’ time, and often remained outside in the playground sitting still or grooming.

Cplx1–/– mice fail to habituate to confinement
Upon being placed in a small ‘confinement’ chamber, Cplx1+/+ mice quickly acclimatized to their surroundings; sitting still and only occasionally turning around or sniffing the air or walls of the chamber. In contrast, Cplx1–/– mice became very agitated and remained so during the 30 min testing period. This agitation was characterized by frequent movement, scratching at the walls of the box and persistent sniffing of the corners and air holes of the chamber. Cplx1–/– mice also adopted abnormal postures that included placing the plantar surface of their hindpaws on the walls of the chamber. The aberrant response of Cplx1–/– mice to confinement was scored over five consecutive days (Table 2). Cplx1–/– mice exhibited a significantly higher ‘agitation’ score than Cplx1+/+ mice during the observation period (Fig. 7) (genotype, F1,10=18.52, P<0.01). There was also a significant genotypexday interaction (F4,40=2.22, P<0.05), as Cplx1+/+ mice habituated to the chamber, whereas Cplx1–/– mice failed to habituate over the five testing days and remained restless and agitated.


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  		Table 2. Habituation to confinement scoring system
 


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  		Figure 7. Upon being placed in a small ‘confinement’ chamber, Cplx1+/+ mice quickly acclimatized to their surroundings, whereas Cplx1–/– mice failed to habituate to confinement and exhibited a significantly higher anxiety score than Cplx1+/+ mice during the 5-day observation period that reflected their agitation. Symbols indicate the mean±SEM of each group on each measure. Where error bars are not visible, they are obscured by the symbols. Asterisks indicate significant differences between Cplx1+/+ and Cplx1–/– mice (*P<0.05, **P<0.01 and ***P< 0.001).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Cplx1–/– mice were originally reported to suffer from sporadic seizures and die within 2–4 months after birth (3Go). Here, we have shown that with an enhanced feeding regime, in addition to other improvements in husbandry, survival of Cplx1–/– mice was markedly improved (to >2 years) and Cplx1–/– mice do not exhibit seizures. Because Cplx1–/– mice in our study survived to maturity, we were able to characterize their behaviour. Interestingly, Cplx1–/– mice show abnormalities in a number of complex motor behaviours, not just locomotion. Cplx1–/– mice show exploration deficits and abnormalities in tasks reflecting deficits in emotional reactivity. Our findings show that, as previously shown for complexin II (14Go), complexin I is essential for normal motor behaviour in mice. It seems likely that loss of either of the complexin isoforms will contribute deleteriously to neurological symptoms seen in diseases where complexin expression is dysregulated.

The ataxia of Cplx1–/– mice is their most obvious behavioural abnormality. There are many mouse models of ataxia (Table 3); the best described include the leaner (32Go), weaver (28Go), staggerer (33Go), lurcher (27Go), stargazer (34Go), lethargic (35Go) and tottering mutants (36Go). However, it is notable that onset of the ataxic phenotype in Cplx1–/– mice is much earlier than that reported for any of the other models (P7 in Cplx1–/– mice compared with ~P14–P21 in most other mutants). Further, with the exception of the lethargic and stargazer mutants, most other models exhibit profound cerebellar degeneration (35Go,37Go). Cplx1–/– mice exhibit their profound and complex deficits despite showing no obvious brain abnormalities (3Go). Given the normal structural and morphological appearance of the brains of Cplx1–/– mice and the fact that complexins are modulators of neurotransmitter release, it seems most likely that ataxia and other deficits observed in Cplx1–/– mice are not due to degenerative changes, but represent ‘functional’ abnormalities mediated by loss of complexin I at synapses.


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  		Table 3. Characteristics of abnormal motor phenotypes in mice reported to have ataxia
 
The behavioural phenotype of Cplx1–/– mice was consistent with that predicted from the expression pattern of Cplx1 in mouse brain (10Go). Cplx1 expression is found in the cortex (especially layers IV and V), thalamus, the CA3 region of the hippocampus and the deep cerebellar nuclei. Given the high expression of Cplx1 in the deep cerebellar nuclei, it is not surprising that Cplx1–/– mice suffer from ataxia as functional alterations in the cerebellar circuitry and also structural changes of the cerebellar cell types are known to produce neurological defects, which manifest as ataxia (38Go–41Go). The thalamus is also likely to be a key player in the expression of the Cplx1–/– phenotype. Outside the cerebellum, the region expressing the highest levels of Cplx1 in the brain is in the thalamus. The thalamus modulates motor function by transmitting information from the cerebellum and basal ganglia to the motor regions of the frontal lobe (primary motor cortex and higher-order motor area). The basal ganglia and cerebellum receive input from the cerebral cortex and form a projection loop back to the cerebral cortex via the thalamus. Although the cerebellum directly regulates execution of movement, the basal ganglia and the thalamus are involved in higher-order control. This includes cognitive aspects of motor control such as the planning and execution of complex motor strategies (42Go). Lesions of the thalamus have been shown to cause impaired rotarod behaviour in rats (43Go) and dystonia, ataxia and tremor in humans (44Go–47Go). This is interesting given that ataxia, dystonia and tremor dominate the overt phenotype in Cplx1–/– mice. The presence of a resting tremor in Cplx1–/– mice is particularly noteworthy given that this is one of the primary features of Parkinson's disease, and that abnormal complexin I levels have been identified in Parkinson's disease patients' brains (22Go).

In addition to the cerebellum and thalamus, Cplx1 expression is strong in the cortex, hippocampus and medial septal nucleus (10Go). Thus, we predicted that in addition to motor deficits, Cplx1–/– mice would have other behavioural abnormalities. These are likely to include cognitive deficits, as the cortex, hippocampus and medial septal nucleus are all important structures involved in learning and memory (48Go–52Go) and it would be particularly interesting to investigate cognitive function in Cplx1–/– mice. However, the cognitive testing of Cplx1–/– mice is limited by their severe motor impairment, and there are no cognitive tasks available that do not require some level of coordinated movement. We previously used spatial cognitive memory tasks and simple visual discrimination to identify cognitive deficits in Cplx2–/– mice (14Go). However, both of the tasks that we used, the Morris water maze and two-choice swim tank, required the mouse to swim. Because of the inability of Cplx1–/– mice to swim, water maze tasks could not be used as a means of cognitive testing. We have conducted some preliminary experiments, which show that Cplx1–/– mice appeared to be unable to alternate spontaneously in the T-maze task (A.J. Morton and C.J. Drew, unpublished data). However, behavioural deficits again confounded the performance of this task, because when placed in the T-maze, Cplx1–/– mice moved rapidly and impulsively. They also became agitated upon being put in a confined space, such as the start box of the maze, thus making these data difficult to interpret. Until a task is devised that can measure cognitive function without the need for coordinated motor control, the true extent of the cognitive deficit in Cplx1–/– mice will remain untested. However, it seems likely that cognitive deficits will be present in these mice, particularly as it was shown recently that Cplx1 is important in the consolidation phase of memory after spatial learning (53Go).

Despite difficulties in testing cognitive function, we have identified a number of deficits in the behaviour of the Cplx1–/– mice, which suggest a breakdown in ‘higher’ brain function. We found deficits in grooming behaviour and exploratory behaviour in the open field. These are complex behaviours that require not only motor coordination, but also integration of sensory input. Normal sensory function is critically important in social and exploratory behaviour. However, we do not know whether basic sensory function in Cplx1–/– mice is intact. We had planned to test sensory perception using von Frey's filaments, but Cplx1–/– mice failed to habituate to the testing chamber used to restrict their movement, thus making it impossible to measure their sensory responsiveness.

Our swim tank data suggest that sensory gating in Cplx1–/– mice is not normal. Swimming is an innate behaviour in mice. The normal response of a mouse placed in deep water is to swim with its nose and tail above the water line. However, Cplx1–/– mice were unable to orientate themselves in the water and were often observed diving to the bottom of the swim tank. Although Cplx1–/– mice show similarities in their ‘non-swimming’ behaviour to vestibular mutant mice such as the tilted mutant (54Go,55Go), Cplx1–/– mice do not show other symptoms seen in the tilted mutant. However, it seems unlikely that the swimming deficit is solely due to cerebellar dysfunction. Although two other cerebellar mutants, the leaner mouse (32Go) and the tottering mouse (36Go) are unable to swim, many mice with cerebellar deficits, including the weaver and lurcher mice, are capable of swimming (reviewed in 24). One interesting possible explanation for the lack of swimming ability in the Cplx1–/– mice is that the deficits seen are part of a ‘panic’ response to the loss of sensory information about position in space when the mice are put into water. Although naïve Cplx1–/– mice cannot find the surface of the water and would drown if not rescued, with training, Cplx1–/– mice are able to overcome their initial panic when put into water. Indeed, some Cplx1–/– mice have learnt to swim from one end of the tank to the other, although their swimming ability and style remains very poor (A.J. Morton and C.J. Drew, unpublished data).

Cplx1–/– mice show reduced exploratory behaviour in two different tests (novel environment and the emergence test). In addition, they show reduced exploration of novel objects in the ‘playground’. Although the ataxia limits their locomotion, the emergence test and the playground task did not depend on the locomotor ability. The change in exploratory behaviour is particularly interesting because novelty seeking behaviour is thought to be related to brain systems modulated by dopamine (56Go) and is perturbed in many clinical manifestations including Parkinson's disease. Parkinson's disease is characterized by, among other things, a lower-than-average tendency to seek out new experiences (57Go). Studies of patients with Parkinson's disease have shown that the degree of reduced novelty seeking was correlated with specific patterns of impaired dopamine functioning (58Go). Although not all studies found a significant correlation between reduced novelty seeking and dopaminergic activity in Parkinson's disease (59Go,60Go), it has also been shown that the dopamine D4 receptor (D4R) exon III polymorphism is associated with the novelty seeking trait in humans (61Go) Interestingly, D4R knockout mice have been shown to be significantly less behaviourally responsive to novelty than their wild-type littermates (62Go). These mice also showed increased anxiety levels further implicating a role for dopamine in the behavioural phenotype of Cplx1–/– mice. Although we have not investigated the dopamine system in Cplx1–/– mice directly, it would clearly be of interest to do so in the future, particularly given the fact that complexin I is dysregulated in Parkinson's disease (22Go).

Complexin I has been recently shown to be important in pancreatic ß-cells, regulating glucose-induced secretion of insulin (23Go), we did not find any evidence for diabetes or glucose insensitivity (as measured by glycosuria and blood glucose testing) in Cplx1–/– mice. The most likely explanation for this is that another functional isoform of complexin is present in the pancreas.

Heterozygous mice (Cplx1–/+) are normal in appearance, with no ataxia. In contrast to Cplx1–/– mice, they can perform most motor tasks. Nevertheless, there appears to be a gene dosage effect of the Cplx1 gene on some aspects of coordination and motor behaviour, because Cplx1–/+ mice have impaired rotarod performance when compared with Cplx1+/+ mice. However, Cplx1–/+ mice possessed normal locomotor ability in the open field and did not show any abnormalities in hang wire or grip strength at the ages tested in this study. Furthermore, Cplx1–/+ mice do not show any abnormalities in swimming behaviour (A.J. Morton and C.J. Drew, unpublished data). Thus, it may be possible to test the role of complexin I in cognitive function in spatial learning and non-spatial learning using water-based tasks with heterozygous mice that are currently not feasible with Cplx1–/– mice.

The fact that single knockout mice lacking either complexin I or complexin II are viable, whereas complexin I/II double knockouts die at birth, suggesting that the two complexin isoforms have redundant functions in brain regions that are essential for postnatal survival, e.g. in brain stem systems that control breathing, thermoregulation or electrolyte homeostasis. At a synaptic level, this redundancy is seen in cultured glutamatergic hippocampal neurons which coexpress complexins I and II, because single knockout of complexin I or II has no phenotypic consequences, whereas complexin I/II double knockout neurons show a dramatic reduction in transmitter release (3Go). However, the different behavioural and neurological phenotypes of Cplx1–/– and Cplx2–/– mice demonstrate that complexins I and II are not redundant throughout the brain. The differences in behavioural phenotype of Cplx1–/– and Cplx2–/– mice are more likely to be due to different expression levels and subcellular distribution of the two complexins (9Go,10Go), rather than to functional differences between the two proteins.

We have previously shown that some of the cognitive deficits seen in the Cplx2–/– mice are similar to those in the R6/2 mouse of HD (14Go), in which there is a progressive depletion of complexins (21Go,63Go). Interestingly, Cplx1–/– mice also share similarities in their abnormal motor phenotype with late stage R6/2 mice. Dystonia, tremor and a susceptibility to seizures are present in R6/2 mice (64Go,65Go), in addition to abnormalities in swimming and rotarod performance (65Go). Indeed, R6/2 mice become ataxic late in the course of their disease (65Go) at a time that coincides with the significant depletion of Cplx1 in the R6/2 mouse (63Go). However, in spite of some similarities in behaviour, it is clear that R6/2, Cplx1–/– and Cplx2–/– mice all exhibit very different overall phenotypes (see Table 4 for a summary of the behavioural changes in Cplx1–/– mice and Cplx2–/– mice), and neither the Cplx1–/– nor the Cplx2–/– mouse represents a phenocopy of the R6/2 mouse. This is not particularly surprising, given the fact that there are many genes dysregulated in addition to Cplx1 and Cplx2 in the R6/2 mouse (63Go,66Go). Nevertheless, the deficits seen in the Cplx1–/– and Cplx2–/– mice support the idea that changes in complexins will contribute to the deficits seen in the R6/2 mouse. Complexins may provide interesting therapeutic targets not only for HD patients, but also for patients with other psychiatric disorders in which alterations in complexin expression have been shown.


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  		Table 4. Summary of behavioural abnormalities in Cplx1–/–mice (this study) and Cplx2–/– mice (14Go)
 
In summary, our study suggests that complexin I may play an important role not only in the motor system control, but also in the control of emotional reactivity. Thus, decreased expression of complexins is likely to contribute to progressive neurological decline in psychiatric and neurodegenerative disorders where it has been shown to be dysregulated. Together with our data from the Cplx2–/– mice (14Go), a picture emerges whereby changes in either isoform of complexin could cause or contribute to a complex set of neurological symptoms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals
Cplx1 mice were generated by homologous recombination in embryonic stem cells (3Go). All Cplx1 mice used in this study were F1 or F2 mice bred from founders with a mixed genetic background (129Ola/C57Bl6). Cplx1 mice have been backcrossed onto a C57/Bl6 inbred background for five generations, but no change in the overt phenotype of Cplx1–/– mice has been observed (data not shown). Three experimental groups were used (see Table 4 for details of the testing history of each group).

Cplx1 experimental group 1 consisted of 13 Cplx1–/– mice and four Cplx1+/+ controls, which were born in the Max-Planck-Institute for Experimental Medicine, Göttingen, Germany and were imported to Cambridge. Experimental group 2 consisted of 13 Cplx1–/–, 20 Cplx1–/+ and 24 Cplx1+/+ mice. Experimental group 3 consisted of 16 Cplx1–/–, 20 Cplx1–/+ and 20 Cplx1+/+ mice. Group 2 and group 3 were taken from a colony established in the Department of Pharmacology, University of Cambridge. All experimental procedures were licensed and undertaken in accordance with the regulations of the UK Animals (Scientific Procedures) Act 1986.

Mice were housed in hard-bottomed polypropylene experimental cages in groups of nine to 16 mice. The housing facility temperature was maintained at 21–23°C and relative humidity was maintained at 55±10%. Clean cages were provided twice weekly with corncob bedding and fine shredded paper nesting material. Mice from group 3 were housed with a red Perspex igloo and a wooden Aspen tunnel (4x4x10 cm3) (Datesand, Manchester, UK) in their home cage. Lighting was controlled on a 12 h light: 12 h dark cycle. All mice were tested during the light phase. The mice had ad libitum access to water and standard dry chow (Datesand). It was noted on their arrival in the Cambridge facility that Cplx1–/– mice had difficulty in reaching the hard pellets in the food hopper and drinking from the water bottles. R6/2 mice have similar difficulties, which were improved by a change in feeding regime (3Go), where lowered water spouts were provided and access to normal laboratory chow was improved by providing mash on the floor of the cage twice daily at 9 a.m. and 4 p.m. Mash was made by soaking 100 g of chow pellets in 230 ml water for 60 min until the pellets were soft and fully expanded. Despite their motor difficulties, Cplx1–/– mice were able to eat the wet mash from the cage floor and drink from the lowered water spouts without difficulty. This caused a dramatic improvement in body weight, with imported Cplx1–/– mice increasing their body weight by 30% (Fig. 1B, P<0.01) within 2 weeks of the change in feeding regime.

Genotyping
Cplx1 genotyping, using DNA prepared from tail biopsies, to identify mice with a homozygous or heterozygous deletion of the Cplx1 gene was a modification of the method of Reim et al. (3Go). The primers used for the Cplx1+/+ gene were forward 5'-AGT ACT TTT GAA TCC CCT GGT GA-3' and reverse 5'-TAG CTA TCC CTT CTT GTC CTT GTG-3'. Primers used for the Cplx1–/– gene were forward 5'-CGC GGC GGA GTT GTT GAC CTC G-3' and reverse 5'-CTG GCT TGT CCC TGA ATC CTG TCC-3'. PCR was conducted as using 0.3 ng DNA and the cycling conditions used were 3 min at 94°C, 45x (30 s at 94°C, 30 s at 53°C and 1 min at 72°C) and 7 min at 72°C. The PCR products obtained were then run on a 1.5% agarose gel for 1 h at 145 V alongside {phi}X174 DNA/Hae III marker (Promega, WI, USA) and visualized under ultraviolet using a transilluminator.

Body weight
Group 1 mice were weighed upon arrival in the colony and then twice weekly over the course of the experiment. All other mice were weighed once a month between 1 and 12 months.

Modified SHIRPA analysis
Mice were tested using a modified version (14Go) of the primary screen of the SmithKline Beecham Pharmaceuticals; Harwell, MRC Mouse Genome Centre and Mammalian Genetics Unit; Imperial College School of Medicine at St Mary's; Royal London Hospital, St Bartholomew's and the Royal London School of Medicine; Phenotype Assessment (SHIRPA) protocol (68Go,69Go). The data were quantified using a binary scoring system (14Go). A ‘normal’ behaviour received a score of 0. ‘Abnormal’ behaviour received a score of 1. This permitted a global abnormality score to be determined for each mouse, with a higher overall score corresponding to a greater degree of abnormality.

Blood and urinary glucose testing
To assess the possibility of diabetes in all mice, urinary glucose levels were measured using urinary glucose measuring sticks (Diastix, Bayer PLC, UK). Urine samples were placed on the Diastix and excess urine removed. Urine samples were left for 30 s and the results were compared with the reference guide. The amount of glucose present in urine was estimated by comparing the colour of the Diastix with the colours of standard urinary glucose concentrations.

In order to determine the blood glucose readings, mice were fasted for 16 h. Following food deprivation, the tip of each mouse tail was anaesthetized (ethyl chloride, BP) and a short (<2 mm) segment removed. A drop of blood from the tail was dropped onto a OneTouch Ultra test strip (Lifescan). The test strip was placed into a OneTouch Ultra (Lifescan) blood glucose monitoring unit and the prechallenge fasting blood glucose concentrations were taken. Mice were then dosed with a glucose challenge (1.5 g/kg dissolved in distilled water and delivered i.p.) and blood glucose concentrations were measured again 1 h postchallenge.

Behavioural testing
Mice were trained on a battery of behavioural tests. These comprised the open field test (locomotor activity, anxiety, neuromuscular strength and exploration), swim tank (swimming, motivation and coordination), footprints (gait and locomotion), rotarod (coordination, balance and neuromuscular strength), hang wire and grip strength (grip strength), playground, habituation to confinement and emergence test (anxiety, exploration of novel environments and stimuli). Specific groups and ages of the mice tested in the different experiments are given in Table 5.


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  		Table 5. Behavioural testing history and summary of findings in Cplx1 mice used in this study
 
Footprint analysis
The footprint test as described previously (65Go) was used to assess the gait of Cplx1–/– and Cplx1+/+ mice.

Swim tank
To monitor swimming ability and efficiency, mice were trained to swim from one end of a water-filled glass tank to a visible escape platform at the opposite end (14Go,65Go,70Go). The glass tank (90x30x16 cm3) was filled to a depth of 20 cm with water maintained at a temperature of 23°C. The escape platform was made from black Perspex (6 cm2 and 20.5 cm high) with the top surface protruding 0.5 cm above the water level. To monitor their swimming ability, Cplx1–/– and Cplx1+/+ mice were placed at one end of the water-filled glass tank with a visible escape platform at the opposite end for a maximum of 120 s. Mice that were unable to swim were guided by hand to the escape platform, placed on the platform and allowed to remain there for 15 s, before being removed and returned to the home cage. The swimming behaviour of Cplx1 mice in the swim tank was video-recorded. Owing to the severity of their impairment in the swim tank, Cplx1–/– mice were tested only once.

Hang wire
Individual mice were placed on a wire cage lid and the lid was gently moved back and forth so as to enable the mouse to grip the wire. The lid was then turned upside down, ~6 in. above the surface of the bedding material. Latency to fall onto the bedding was recorded, with a 60 s cut-off time.

Grip strength
The grip strength meter (Ugo Basile, Biological Research Apparatus, Varese, Italy) was used to assess the forelimb grip strength. Each mouse was given four successive trials and a mean grip strength reading was calculated.

Rotarod
The rotarod apparatus (Accelerating model, Ugo Basile) was used to assess motor coordination, strength and balance (14Go,65Go). Three different protocols were employed. Juvenile Cplx1+/+, Cplx1–/+ and Cplx1–/– mice (aged 4.5 weeks) received four trials (maximum 60 s each) per day, at 5, 8 and 15 r.p.m. for three consecutive days. Adult Cplx1–/– mice received four trials per day, at 0 and 5 r.p.m. for three consecutive days. During the training period, adult Cplx1+/+ and Cplx1–/+ mice (at 6 and 9 months) received four trials per day, at 24 r.p.m. for three consecutive days. The latency to fall off the rotarod within this time period was recorded. On the fourth day, mice underwent testing. During testing, each animal received two trials (maximum 60 s each) at eight increasing speeds (5, 8, 15, 20, 24, 31, 33 and 44 r.p.m.). The mean latency to fall off the rotarod (for each experimental protocol) was recorded and used in the subsequent analysis.

Open field testing
The open field as described previously (14Go,71Go) was used to assess the locomotor activity and exploratory behaviour in Cplx1+/+, Cplx1–/+ and Cplx1–/– mice. Movement of each mouse within the open field was tracked using the Field 2020 system (HVS Image, Buckingham, UK).

Emergence test
The emergence test was used to assess the exploratory behaviour from a ‘safe’ enclosure in the open field. Twenty Cplx1+/+, 19 Cplx1–/+ and 16 Cplx1–/– mice aged 4 months were used for this task. A red igloo house was positioned in a corner of the open field arena. During each testing session, individual mice were initially placed inside the igloo house and observed for a 10 min period. Mice were habituated to the red Perspex igloo house in their home cage for at least 48 h prior to testing. Parameters measured during the 10 min trial included latency to leave the house (defined as all four paws leaving house), total time spent in house, and number of aborted excursions (defined as head and at least one paw exiting before returning inside the house).

Exploration in the playground
The playground was developed for this study as a passive form of environmental stimulation and exploration. The playground is composed of an open-topped clear Perspex box (60x45x30 cm3), which had a cylinder (14.5 cm long with a radius of 6.5 cm) protruding outwards in the bottom right corner and a three-step staircase (15x8 cm2 with 1.5 cm increments between each stair) leading up to the cylinder. The floor of the box was covered in fresh corncob bedding and contained a variety of interactive and inanimate objects as shown in Figure 6F. These included a yellow upright running wheel, a red igloo house with a running wheel attached at a 45° angle, (Fast track, Datesand), a wooden cross with a ladder, a pink Perspex tunnel, a wooden seesaw, an elevated wooden house and a wooden hexagonal maze and were placed in the box. Groups of mice (five mice per group of the same genotype) were placed into the playground and their activity was monitored for 60 min. The exact activity of each mouse was scored once every minute (i.e. sleeping, exploring or using a particular toy). For the purpose of analysis, the different activities were pooled according to the type of activity observed. Each point at which a mouse was recorded sitting quietly or sleeping was deemed ‘time spent inactive’. All points when mice were directly sniffing a toy or in physical contact with any apparatus was considered ‘time spent using toys’. After 60 min, mice were removed from the playground and returned to their home cage. All playground items were removed and washed between trials. Between each test group, the observation box was emptied and cleaned using 1% acetic acid. Each group was tested on three consecutive days. The arrangement of the individual playground items in the box was altered on each testing day. The behavioural response of five Cplx1+/+, five Cplx1–/+ and five Cplx1–/– mice aged 5.5 months was evaluated in detail.

Habituation to confinement
Reaction and habituation to a confined space were examined in six adult Cplx1+/+ and six Cplx1–/– mice aged 8.5 months. The confinement chamber consisted of an opaque, Perspex box measuring 9x4.5x5 cm3 with a hinged lid and a wire grid floor surface. Individual mice were placed in the confinement chamber for a period of 30 min and their activity was monitored every 2 min (for scoring system see Table 2). The confinement chamber was cleaned thoroughly with water after each test session. Male and female mice were tested in separate sessions. The habituation test was repeated on five consecutive days.

Statistical analysis
For all experiments presented, unless stated otherwise, data were subjected to analysis of variance (ANOVA) repeated measures, with one or two between-subject factors (genotype and or gender) and one within-subject factor (age/day/block of trials) as appropriate to the particular test. In cases of a significant interaction (genotypexage/day/block of trials), Newman–Keuls test (for one between subject factor) or Sidak's test (for two between-subject factors) was used for multiple independent post hoc pairwise comparisons between different genotypes at each relevant age, day or block of trials (72Go). For data that were not normally distributed, the Kruskal–Wallace test with Dunnet's post hoc test was used. An unpaired two-tailed t-test (or a Mann–Whitney two-tailed test in the case of non-parametric data) was applied to test the significance of differences between mean values where factorial ANOVA was not required. Proportions were compared using contingency tables and P-values were calculated using the {chi}2 test. Although the data from males and females were separated in all analyses, data have been pooled for clarity of presentation of the results. With the exception of body weight, no sex differences existed between male and female mice of the different genotypes on any of the tests described. Statistical analyses were performed using GraphPad Prism (Version 3.0, San Diego, CA, USA) and Genstat (Release 4.1, NAG Ltd, Oxford, UK). A critical value for significance of P<0.05 was used throughout the study.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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