The current experimental results reveal that both male and female CGG KI mice are impaired in performance of a skilled ladder rung walking task compared to wildtype littermates. Specifically, CGG KI mice showed a greater number of foot slips with increasing CGG repeat length (). These findings suggest that the length of an expanded CGG trinucleotide repeat on the Fmr1 gene is related to impaired locomotor performance in CGG KI mice as observed in the ladder rung task. The present data also provide the first demonstration of motor deficits in CGG KI mice under 12 months of age. Interestingly, mice as young as two months of age appeared to show motor deficits similar to the mice over 12 months of age. Contrary to our initial hypotheses, age did not contribute to task performance, nor were there differences between sexes for skilled ladder rung performance. Male and female mice showed similar decrements in performance with increasing CGG repeat length (). These data suggest that the ladder rung task is likely revealing an early motor deficit as opposed to directly modeling the late onset cerebellar gait ataxia reported in human cases of FXTAS.
The early appearance of motor deficits was not entirely unexpected, considering recent reports that embryonic cortical development is abnormal in CGG KI mice [22
] and that dendritic complexity is reduced and synaptic structure is altered in cultured hippocampal neurons [16
]. Furthermore, it has been found that CGG repeat length and age modulate performance on a spatial processing task in 23–43 year old human female fragile X premutation carriers [30
The lack of differential performance between sexes was unexpected as it has been reported that fragile X syndrome and FXTAS are more prevalent in males than females, presumably due to a protective influence of a second non mutated FMR1
gene on the second X chromosome [7
]. Despite the reduced prevalence of FXTAS in female carriers of the fragile X premutation relative to males, females with FXTAS do not show reduced FXTAS symptoms once diagnosed [7
]. Furthermore, it is possible that the ladder rung task is sensitive enough to probe the underlying motor networks that may be similarly disrupted in male and female CGG KI mice.
Although not quantified, CGG KI mice also showed a hunched posture all ages and a discernible shaking while walking along the ladder rung apparatus. Similar behaviors were not observed in wildtype mice. This tremoring during the performance of a motor task is of interest because no gross motor abnormalities or tremoring are apparent when CGG KI mice are observed in an open field. These results suggest that motor abnormalities in CGG KI mice may not be apparent until the mice are challenged by a difficult motor task, such as the ladder rung task, which may unmask a previously unidentified motor tremor. These postural tremor-like behaviors need to be further investigated and carefully described in CGG KI mice.
These results also indicate that the ladder rung task is a sensitive and robust assay that allows for a high throughput analysis of motor function in CGG KI mice. As each mouse was only exposed to the apparatus for approximately 2 min in the present experiment and there was no adaptation period preceding data collection, performance of the task served as a rapid assay of motor function without the potential confounds of motor learning that may mask between group effects, as suggested in earlier studies [24
]. This point is important as the rotarod task used to test motor function in mice requires the mouse be placed on a rotating drum and the time to fall is typically used as the outcome measure. For the rotarod, there are often early training trials given to mice on the rotarod apparatus that may potentially mask any differences that are present in baseline motor function as mice are trained to set performance criteria before administration of accelerating rotarod testing [58
The present experiment did not explicitly employ the subtle gait measurements described by Whishaw and colleagues [8
]. This is because the sides of the apparatus used in this study were opaque and prevented the requisite recording the mouse’s foot placement from the side for more precise analysis of limb movements. Such measurements could provide additional evidence for subtle motor deficits, as well as the precise nature of the observed missteps. For example, more sophisticated analyses of gait would reveal whether animals make predictable errors such as consistently under or overestimating the location of subsequent beams. Such errors could indicate a possible dysfunction in frontal-parietal network-dependent vector calculations underlying action in peripersonal space [23
]. Alternately, if animals showed a trend toward a general clumsiness or lack of precision in motor performance that could indicate a more purely motor deficit [4
]. The first possibility is intriguing in for the CGG KI mice as frontal-parietal network dysfunction is hypothesized to underlie spatiotemporal, arithmetic, and attentional deficits in FXS as well as the fragile X premutation [3
], as well as being involved in skilled walking behaviors [2
]. The latter possibility, based more directly on motor function is also important for the extension of the CGG KI mouse as a murine model of motor deficits present in FXTAS [62
]. Such follow-up studies are currently underway to explore these possibilities in CGG KI mice as well as to correlate any performance deficits to neuropathological features, which are present throughout the neocortex and cerebellum of CGG KI mice [37
The primary benefit of this modification of the ladder rung task is that extensive pre-training is not required and that testing times and trials are significantly shortened compared to the versions of the task reported previously [24
]. Furthermore, spontaneous exploration is encouraged, and the effect of this behavior on potential error production can be considered. This results in a high throughput screen that is sufficiently sensitive to detect subtle motor impairments in transgenic mouse models.
In summary, the present experiment identified age-independent motor deficits in CGG KI mice. The detection of motor deficits in young CGG KI mice is important as performance on the ladder rung task may be used as outcome measures for behavioral or pharmacological therapeutic intervention in this mouse model as it pertains to FXTAS as well as other late onset neurodegenerative disorders. This modification might make the test more attractive to other groups.. Because mice as young as 2 months of age show deficits, the need to limit testing to mice greater than 12 months of age to identify potential endpoints and outcome measures is mitigated.