THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE The Effect of Sex on Motor Coordination in Juvenile Mice

Understanding sex differences in motor abilities is important for understanding motor development in children as well as many disease phenotypes. However, whether there are biologically based differences in motor abilities of both human children and mice is still in debate. There are sex differences in the cerebellum of juvenile mice which suggests that sex differences in motor coordination are likely. Based on the past findings that females have higher calbindin mRNA expression in the juvenile mouse cerebellum, and that mice with reduced calbindin show severe motor deficits, it was hypothesized that females would perform better on strenuous tests of motor coordination. In order to address this hypothesis, juvenile mice were tested in an open field test and an accelerating rotarod test. The results demonstrated that there is not a significant sex difference in juvenile mice in either test. The effect of sex on motor coordination is not well classified, but these results indicate that there is not a large sex difference in pre-pubescent mice. THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE 4 The Effect of Sex on Motor Coordination in Juvenile Mice It is generally believed that in humans there is a small sex difference, favoring males, in motor coordination in childhood and that difference increases once boys reach puberty. A metaanalysis of motor performance studies found many tasks that follow this pattern of a slight difference that exists throughout childhood and increases at puberty, at least in part due to increases in size and strength of boys during puberty. With the exceptions of throwing velocity and distance, the sex differences seen prior to puberty were small and could be attributed to social differences in treatment of the children by parents, coaches, and teachers (Thomas & French, 1985). In a study of Portuguese elementary school children, boys were shown to have significantly better motor speed and coordination than girls (Martins et al., 2005), yet evidence was not given to determine if this effect was biologically or socially based. In support of a biological basis of sex difference, a study done in infants indicated that, while males and females are very similar in their motor development, females develop fine motor skills slightly sooner and males develop gross motor skills sooner (Touwen, 1976). It has also been shown in infants that females have greater synchrony between the joints of their arms than males (Piek, Gasson, Barrett, & Case, 2002). Together these studies indicate that in humans there are some innate biological differences in motor coordination, but these differences can be exaggerated by social environmental factors. The cerebellum is a brain structure that, among other tasks, is essential for motor coordination. There are some sex differences in the cerebellum of different species. The male human cerebellum is larger than that of the female. The female rat cerebellum has more extensive dendritic branching (Nguon, Ladd, Baxter, & Sajdel-Sulkowska, 2005). These THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE 5 differences may have implications for motor function in these species and may be important differences to investigate in other species, such as the mouse used in this study. The cerebellum is unique from other brain regions in that its development occurs largely after birth and in parallel with motor development (Swinny, van der Want, & Gramsbergen, 2005). In rodents, cerebellar Purkinje cells, the only cells that project out from the cerebellum, have their final mitosis from embryonic day 11-13 (E11-13). On E14-E17 in rats, or after birth in mice, these cells mature and migrate to their final positions as a single cellular layer. However, it is not until postnatal weeks 2 and 3 that the greatest synapse formation occurs between Purkinje cells and parallel fibers, the excitatory main input to Purkinje cells. The mouse cerebellum is not fully mature until postnatal week 5 (Iacopino, Rhoten, & Christakos, 1990). NMDA receptor signaling between Purkinje cells and parallel fibers during post natal day 15-16 (PN15-PN16) is important for the pruning of synapses between Purkinje cells and climbing fibers, another important input to Purkinje cells, and the failure to reduce these connections has been associated with reduced motor coordination (Swinny et al., 2005). In rats, Purkinje cells begin to show expression of a calcium binding protein, Calbindin D-28k (calbindin), during late prenatal cerebellar development (Swinny et al., 2005). In male C57BL/6J mice, calbindin protein and mRNA levels peak at postnatal week 2 and then drop 47% to a steady level by weeks 4 and 8. By 120 weeks calbindin levels had reduced to near that at the day of birth (Iacopino et al., 1990). Calbindin is an EF-hand protein meaning that the fifth and sixth α-helix form a metal binding region, and it is a fast, high-affinity calcium buffer involved in calcium homeostasis and regulation of Ca signals (Farre-Castany et al., 2007; Schwaller, Meyer, & Schiffmann, 2002). In the cerebellum, calbindin is expressed exclusively in Purkinje THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE 6 cells (Schwaller et al., 2002). The level of calbindin expression can have a significant impact on synaptic transmission (Farre-Castany et al., 2007). Interestingly, calbindin knockout mice develop relatively normally, showing typical growth, lifespan and fertility. They do not show significant compensatory up-regulation of the other calcium binding proteins calretinin, parvalbumin, and calmodulin in either the cerebellum or the whole brain. Behaviorally, these mice show no major differences in locomotion in the home cage (Airaksinen et al., 1997), but when presented with more difficult tasks, such as an elevated runway test where mice walk along a 2cm wide runway, they showed significantly more slips and falls than wild type animals (Airaksinen et al., 1997; Farre-Castany et al., 2007). Mice heterozygous for the calbindin gene mutation had significantly reduced calbindin expression in the cerebellum and showed significantly impaired motor coordination on an elevated runway task as compared to wild type mice. However, the deficits of these heterozygous mice were not as severe as those observed in the full calbindin knockout animals that are homozygous for the mutation (Airaksinen et al., 1997), indicating that the level of calbindin expression is related to the level of motor performance. In addition, double mutants that are knockouts of both calbindin and parvalbumin, another calcium binding protein, do not show any greater deficit in motor coordination than calbindin single knockouts, indicating that the presence of calbindin specifically, is essential for normal motor coordination. Similar conclusions were also reached through assessment of mice of the same genotypes on a rotarod assay (Farre-Castany et al., 2007). Calbindin protein levels, as well as levels of several other proteins, were significantly lower in the cerebellum of 129X1/SvJ mice as compared to C57BL/6J mice. These mice also showed significantly reduced motor performance on a rotarod assay as compared to C57BL/J6 THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE 7 mice. This indicates that the reduced protein levels in the cerebellum of 129X1/SvJ mice may be involved in the impairment of motor coordination (Pollak et al., 2005). An accelerating rotarod study of eight strains of inbred adult mice revealed that of all of the strains surveyed C57BL/6J mice showed the best rotarod performance. Running time has also been shown to be significantly affected by weight and body length, with smaller mice running longer (McFadyen, Kusek, Bolivar, & Flaherty, 2003). Conditional knockout mice with calbindin knocked out in Purkinje cells only, were utilized to test how cell type specific calbindin expression affects motor behavior (Barski et al., 2003). These knockout mice did not show significant differences from wild type littermates in an open field test, but they did show significant deficits in an elevated runway task (Barski et al., 2003) similar to complete calbindin knockout mice. It was demonstrated that repeated training on this task lead to some improvement in motor performance in the Purkinje cell specific calbindin knockout mice, but they were not able to attain levels of motor performance comparable to wild type animals regardless of amount of training. The Purkinje cell specific calbindin knockout mice show reduced motor capacity again in a horizontal bar task where they were able to hold onto the bar for a significantly shorter period of time than wild type mice. Purkinje cells, which provide the only output from the cerebellum, clearly require calbindin for their essential function in motor coordination (Barski et al., 2003). Calbindin knockout mice do not show increased cell death or neuronal degeneration (Schwaller et al., 2002). Neuron number in cerebellum can be related to behavior; those mice with higher cell numbers are able to perform better on measures of motor coordination (Goldowitz, Moran, & Wetts, 1992). One extreme example of the importance of cell numbers in motor coordination is the Lurcher mouse. These mice have an autosomal dominant mutation that THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE 8 causes loss of 100% of cerebellar Purkinje cells, 90% of granule cells and 75% of neurons in the inferior olive between PN9 and PN30 (Goldowitz et al., 1992). Lurcher mice have significant motor deficits including ataxia, balance difficulties, and intention tremor (Goldowitz et al., 1992). Interestingly, Lurcher chimeras, which have an intermediate number of cerebellar Purkinje and granule cells between lurchers and wild type mice, do not show these motor difficulties on simple tasks, but do have reduced motor abilities in more strenuous motor tests as compared to wild type mice. They may have increased numbers of synapses on their Purkinje cells to compensate for reduction in total cell number (Goldowitz et al., 1992). Cerebellar involvement in some sexually dimorphic disorders suggests that sex differences in the cerebellum may have implications for human motor coordination, as well as cognitive difficulties. While the cerebellum is vital in motor coordination, balance, and posture, it is also involved in emotion and cognition. Human patients with cerebellar lesions have been shown to have symptoms including anxiety, anhedonia, aggression, and ruminativeness. In addition many neuropsychiatric disorders such as autism, schizophrenia, mood and anxiety disorders, and Attention Deficit Hyperactivity Disorder (ADHD) have been associated with cerebellar abnormalities (Hoppenbrouwers, Schutter, Fitzgerald, Chen, & Daskalakis, 2008). The expression of these disorders is sexually dimorphic with autism, schizophrenia, ADHD, and Parkinson’s being more prevalent in males and mood disorders being more prevalent in females (Nguon et al., 2005; Parker & Brotchie, 2010; Rucklidge, 2010). In autistic patients there is often a reduction in Purkinje cell number and this may be caused be increased apoptosis (Nguon et al., 2005). The cerebellar involvement in these disorders, together with their sexually dimorphic expression indicates that any sex differences present in the cerebellum can have major THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE 9 implications for motor coordination as well as higher cognitive function and neurological disorders. Sex differences exist not only in the prevalence of disorders that involve the cerebellum, but also in the amounts of calbindin produced in the brain. Calbindin gene expression is sexually dimorphic in the hypothalamus, but has more recently been shown to be sexually dimorphic in the fontal cortex and the cerebellum. Female weanling-aged (PN21-25) mice have significantly higher levels of calbindin mRNA in both the frontal cortex and the cerebellum (Abel, Witt, & Rissman, 2011). Interestingly, expression of parvalbumin, a calcium binding protein also found in Purkinje cells (Schwaller et al., 2002), was measured and shown not to have a significant sex difference in the cerebellum (Abel et al., 2011). This demonstrates that there was not a significant increase in another important calcium binding protein to compensate for the lower levels of calbindin expression in males. In addition to sex differences seen in the brain, and specifically in the cerebellum, sex differences have been studied in behaviors such as motor coordination. In an accelerating rotarod task C57BL/6J female mice were able to run for significantly longer than C57BL/6J male mice (McFadyen et al., 2003). Yet, this sex difference in motor performance has not consistently been shown in other studies. Another study in this lab showed no effect of gonadal sex on performance on an accelerated rotarod task gong to a maximum speed of 40rpm (McPhieLalmansingh, Tejada, Weaver, & Rissman, 2008). A third study using a lower speed accelerating rotarod protocol had mice running on a portion of the rod that was not textured and showed no significant sex difference in motor performance (Jyotika, McCutcheon, Laroche, Blaustein, & Forger, 2007). Variations in the rotarod apparatus and protocols may have lead to differences in the effects seen in the different studies (McFadyen et al., 2003). There is clearly THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE 10 not a consensus on the effect of sex on motor coordination in adult mice. By focusing on adult mice, these studies ignored the effect of sex on motor coordination in prepubertal animals, thus ignoring a time period during which there are significant sex differences in the cerebellum and its development. This study sought to compare the motor coordination of male and female mice at weaning using an open field test and an accelerating rotarod test. Given the impact of calbindin levels on motor coordination in mice (Airaksinen et al., 1997; Barski et al., 2003; Farre-Castany et al., 2007; Pollak et al., 2005), and the sex difference seen in calbindin gene expression in the cerebellum of juvenile mice (Abel et al., 2011), it was hypothesized that female mice would show greater motor performance. Since no difference in home cage behavior was observed even in calbindin knockout mice (Airaksinen et al., 1997; Barski et al., 2003), a sex difference was not expected for the open field task. With the added challenge it was hypothesized that a sex difference would be apparent in the accelerating rotarod task. Female mice were expected to run for longer on the rotarod before falling than male mice, given their higher levels of calbindin expression in the cerebellum. While other research has studied sex differences in the motor coordination of adults (Jyotika et al., 2007; McFadyen et al., 2003; McPhie-Lalmansingh et al., 2008), this study is novel in that it studies the effect of sex on motor coordination in juveniles. Method Animals For these experiments wild type C57BL/6 mice were used, which were housed in the University of Virginia School of Medicine, Jordan Hall Animal Facility. Animals were maintained on a 12:12h light: dark cycle with food and water provided ad libitum. Animals were weaned at post natal day 21 and group housed with same sex littermates unless no same sex THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE 11 littermates were present, in which case animals were single housed. All mice were transported to the testing room 15 minutes prior to the commencement of testing each day, to allow them to habituate to the room. All animal procedures were approved by the University of Virginia Animal Care and Use Committee. Open Field In the first cohort, all mice were given an open field test during the light phase of their light:dark cycle. Ten male and 9 female mice were tested between PN21 and PN25. Each mouse was placed in an empty Plexiglas box and allowed to move freely for twenty minutes. During this time a Mouse-E-Motion sensor in the cage lid detected the occurrence of movement in each one second interval and recorded the total number of movements over the twenty minute test period. The box was cleaned with ethyl alcohol between tests. Rotarod A separate cohort of 12 male and 11 female mice was tested on the rotarod near the end of the light phase of their light:dark cycle. Mice were tested on a modified version of the accelerating rotarod protocol described by Pollak et al. (2005). Each mouse was tested once a day for four days on an accelerating rotarod protocol (PN21-PN24), using a Med Associates ENV-576M rotarod. On the first day of testing each mouse was placed on the rotarod and allowed to run at a constant speed of 4rpm for 180 seconds to habituate to the rod. Any mice that fell from the rotarod during the habituation phase were placed back on the rod and allowed to complete the 180 seconds of habituation. The mouse was then placed in a clean holding cage for 150 seconds to rest. The mouse was then placed back on the rotarod and it was accelerated from 4rpm to 40rpm over 300 seconds. After 300 seconds the rod maintained a constant speed of 40rpm and mice were allowed to run for a maximum of 360 seconds per trial. The latency to THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE 12 cling to the rod and passively rotate around, as well as the latency to fall, were recorded. The latency to fall was measured by a timer that stopped automatically when a laser beam at the base of the apparatus was broken. The latency to passively rotate around the rod was measured manually using a stopwatch. Throughout the protocol mice were placed onto the rod backward to force them to turn around while the rod was spinning and thus increase the difficulty of the task. The apparatus was cleaned with ethyl alcohol following the testing of each mouse. Statistical Analysis A t-test was computed to determine if there was an effect of sex on number of movements in the open field task. A repeated measures ANOVA was calculated to determine if sex had an effect on rotarod performance. Results Open field Male and female mice had comparable levels of activity in the open field test. As hypothesized there was no significant effect of sex on number of movements (t(17)= -0.421; p=0.679) (see Figure 1). Rotarod Contrary to the hypothesis, sex was not found to have a significant effect on latency to fall from the rotarod (F(1,90)=0.09; p=0.77) or time until the mouse passively rotated around the rod (F(1,90)<0.005; p=0.98) (See Figure 2). There was not a significant correlation between body weight and latency to fall (r=.006; p=.956), but there was a significant correlation between body weight and time until the mouse passively rotated around the rod (r=.210; p=.046). There was a significant effect of trial (F(3,90)=12.44; p=0.000002), with the fourth trial showing THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE 13 significantly longer latency to fall than the first or second trial (See Figure 2), indicating that the mice were learning and improving at the task. Discussion In this study it was hypothesized that female mice would show greater motor coordination as compared to male mice, due to differences in levels of calbindin expression in the juvenile mouse cerebellum (Abel et al., 2011), and the importance of calbindin in normal motor functioning (Airaksinen et al., 1997; Farre-Castany et al., 2007). It was predicted that there would be no significant effect of sex on number of movements in an open field task, but that females would run longer than males on an accelerating rotarod protocol, as this was a much more rigorous test of motor ability. Male and female mice had similar numbers of movements in the open field task which is consistent with the hypothesis. Male and female mice also showed similar latencies to fall from the rotarod as well as similar times until passively rotating around the rod. Thus, this study found that there was not a significant effect of sex on motor coordination at PN21. This indicates that any sex difference present is too small to be detected using an accelerating rotarod protocol. The results of this study can be extended to ideas of sex differences in motor coordination in human children, and lend support to the idea that the biologically based sex differences are minimal and much of the sex difference can be explained socially. The results of the open field test are consistent with expectations based on previous studies. It has been demonstrated that calbindin knockout mice show significant motor deficits when strenuously tested on an elevated runway task, but show no impairments in the home cage (Airaksinen et al., 1997). In addition Purkinje cell specific calbindin knockout mice were shown not to have any significant differences from wild type mice in an open field task (Barski et al., THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE 14 2003). Given that mice with complete elimination of calbindin do not show impairment in this task it would not be expected for the normal level of variation in calbindin expression between the sexes to lead differences in behavior in the open field task. Previous reports on sex differences in rotarod tasks have been inconsistent, thus it is not possible to state whether the current results fit with previous data. The results of this study are consistent with a subset of previous research, which has reported no sex difference in the motor coordination of adult mice. One study demonstrated an effect of sex on rotarod performance similar to that hypothesized in the current study, with females running significantly longer than males (McFadyen et al., 2003). Yet other studies have shown no sex difference in rotarod performance, consistent with the current results (Jyotika et al., 2007; McPhie-Lalmansingh et al., 2008). Thus the effect of sex on motor coordination remains uncertain, but any effect is likely small and requires a more rigorous test to fully classify. The effect of repeated trials on rotarod performance is much more clearly established. The improvement seen in the current study is consistent with other studies that found improvement in rotarod performance over repeated trials (Gobeske et al., 2009; Jyotika et al., 2007; McFadyen et al., 2003). Wild type mice are obviously able to learn this motor task and improve their performance with practice. In this study it did not appear that the known sex differences in calbindin expression in the cerebellum lead to differences in motor coordination. Given that calbindin mRNA was twice as abundant in the female cerebellum as in the male (Abel et al., 2011), and that mice with reduced calbindin have significant motor deficits (Airaksinen et al., 1997; Barski et al., 2003; Farre-Castany et al., 2007; Pollak et al., 2005), it was predicted that female mice would show greater motor coordination. There are several possible explanations for the lack of an effect. In THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE 15 a study of calbindin knockout mice it was hypothesized that while there was not a significant increase in parvalbumin in calbindin knockout mice, the high levels normally present in Purkinje cells may be sufficient to partially compensate for the loss of calbindin (Airaksinen et al., 1997). It has been shown that there is not a significant sex difference in expression of parvalbumin in the cerebellum (Abel et al., 2011), but its normal level of expression may be sufficient to even out any effects caused by the difference in calbindin expression. It is also possible that other untested calcium binding proteins show sexually dimorphic expression in the cerebellum, compensating for differences in calbindin expression. In addition the normal variation between males and females in calbindin expression may fall within a normal range that has insufficient variation to cause differences in motor coordination. Finally, the developmentally high levels of calbindin at PN21 may be enough that both sexes gain the maximum benefit in motor performance and the higher level in females is not able to provide any additional improvement in motor coordination. A new theory by De Vries (2004), surrounding the importance of sexual dimorphism in the brain could provide an alternative explanation for why sex differences were not seen in motor coordination. Sexual dimorphisms in the brain may not directly cause sex differences in behavior, they may also compensate for other physiological differences, such as gonadal hormone levels, in order to eliminate sex differences in behavior and other functions (De Vries, 2004). Based on this idea, it is possible that the higher levels of calbindin expression seen in female mice as compared to males may help offset another physiological difference that would lead to a sexual dimorphism in motor coordination. Given the inconsistency in results of studies of sex differences in motor coordination it is possible that there is a small effect of sex that was not detected in this study. The normal THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE 16 variation in calbindin levels may lead to differences in motor coordination that require more taxing protocols to detect. While no significant effect of sex was found on overall rotarod performance, either measured by latency to fall or time until the mouse passively rotated around the rod, it is clear that variations in the testing protocol as well as the apparatus can have an impact on results (McFadyen et al., 2003). Most rotarod protocols are designed for adult mice and rats and thus the protocol for this study was repeatedly modified to test juvenile mice. It has been shown that mice with lower body weights and shorter body lengths were able to run for longer (McFadyen et al., 2003), and this may contribute to the juveniles’ ability to run substantially longer than adult mice in other protocols (Jyotika et al., 2007; McPhie-Lalmansingh et al., 2008). The reduced size of juveniles may have allowed mice to keep their body weight closer to the rod and to maintain a more normal stride length and pattern, making the task easier. The differences in juvenile ability and exact protocol may have masked any sex differences that exist. It is also possible that any sex differences that exist in adult rotarod ability develop after the age tested in this study, perhaps as an effect of puberty. The current study was limited by its inclusion of only open field and rotarod tasks. It could have been improved by including other tests that may assess different aspects of motor coordination such as a horizontal rod test or an elevated runway test. In addition, the variation between rotarod protocols used in various studies, and the lack of established protocols for the testing of juveniles, limit the ability to replicate and validate any results obtained. Further research is necessary to fully understand motor coordination and its development. To further investigate if there is a sex difference in juvenile mouse motor coordination additional assessments of motor coordination, such as an elevated runway task, should be run on weanling THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE 17 mice. To understand the development of motor coordination and any sex differences present tests should be run at several ages in order to determine when any differences between male and female mice noted in the literature appear. Calbindin knockout mice should also be tested using an accelerating rotarod protocol to determine if they show a sex difference, and if the sex difference in calbindin expression is compensating for another sexual dimorphism. The development of motor coordination in mice is clearly a complex process with many contributing factors. While small sex differences may exist in the motor coordination of mice prior to puberty, there are no drastic differences in basic abilities. As further research is done with more rigorous tests of motor performance, a clearer picture of how sex effects motor coordination can be developed. The current findings lend support to theories that there is little biological basis for sex differences in motor coordination in human children. By showing no major differences in motor abilities in mice it can be suggested that social influence may be important for the development of sex differences in motor coordination of children prior to puberty. THE EFFECT OF SEX ON MOTOR COORDINATION IN JUVENILE MICE 18