The present results show that CGG KI mice with trinucleotide repeat expansions between 150-200 CGG repeats did not preferentially explore the earlier presented object in a sequence of objects, indicating either a deficit in processing sequential relationships among remembered stimuli, or a deficit in judging the relative memory strength among the stimuli [26
]. While the deficit could reflect impaired learning or impaired recall of the order of exposure to the objects, it is not possible to make this distinction based on the present data . This is because the experimental design of the temporal order for visual objects task was not explicitly designed to test for strength of memory for sequences, and thus it is not possible to discriminate between the use of sequential processing per se and judgments of relative memory strengths among stimuli [3
]. We assume that the present task could be solved either by explicit sequential processing of the visual object information or by weighting the relative strengths of the memory traces of experienced visual objects [28
]. Since the present task was designed to evaluate temporal ordering in CGG KI mice, not to elucidate the component processes underlying the temporal ordering in CGG KI mice, we shall refer to both processes collectively as “temporal ordering” (cf. [28
Interestingly, CGG KI mice with CGG trinucleotide repeat expansions >150 repeats (High CGG repeat) showed temporal ordering deficits compared to wildtype mice, whereas those with CGG trinucleotide repeat expansions <100 repeats (Low CGG repeat) showed no evidence for temporal ordering deficits and performed similarly to wildtype mice. Both groups of female CGG KI mice were able to identify and selectively explore a novel visual object, suggesting that the temporal ordering deficits reflected impaired temporal processing/judging of relative memory strength and not global memory deficits or altered visual perception [25
]. This is the first report of impaired temporal ordering in the female CGG KI mouse as previous studies have been focused on the spatial domain [30
The temporal ordering or visual objects task presented to CGG KI mice has been described as an episodic-like memory task, as episodic memory relies on the separation of experienced behavioral episodes in time in order to recall specific episodes, either by explicitly recalling time-stamped memories or discriminating the differential strengths of memory traces [12
]. In this way, the temporal ordering deficits in CGG KI mice with >150 CGG trinucleotide repeats supports previous studies that have suggested abnormal hippocampal activity during episodic recall in fragile X premutation carriers [41
]. The present experiment also extends previous work by Van Dam et al. [65
] and Hunsaker et al. [30
] who were able to demonstrate spatial processing deficits in male CGG KI mice. The Hunsaker et al. [30
] report suggested that male CGG KI mice showed deficits for fine spatial processing that contribute to the memory deficits observed by Van Dam et al. [65
] using the Morris water maze test of spatial learning and memory. A deficit in temporal ordering as revealed in the present study suggests that deficits in the fragile X premutation may involve larger networks of structures that interact in temporal and/or episodic memory [13
]. Furthermore, the present data in combination with those reported by Hunsaker et al. [30
] support the hypothesis that a spatiotemporal hypergranularity may underlie some cognitive deficits seen in carriers of the fragile X premutation.
In rat lesion studies, diverse brain regions such as the anterior thalamus [69
], rostral infralimbic and prelimbic cortices (referred to as medial prefrontal cortex by [24
]), perirhinal cortex [23
], and the hippocampus [28
] have been shown to be involved in temporal ordering. Lesions of any one of these structures lead to qualitatively similar deficits for temporal ordering. In humans, the parietal cortex has further been shown to be involved in processing temporal relationships [9
]. Due to the apparently distributed nature of the processes underlying temporal ordering within the brain [13
], it is difficult to assign abnormalities within any specific anatomical loci as underlying the observed deficits in temporal ordering in CGG KI mice.
Brain anomalies in fragile X premutation carriers have been reported, and include grey matter volume decreases in the hippocampus [32
], the left thalamus [47
], the insula, inferior temporal cortex, pre and post central gyrus, and the inferior parietal cortex, as well as white matter abnormalities in the cingulum and frontal-temporal white matter tracts [47
]. All of these areas have been implicated in temporal and/or episodic memory [13
]. Unfortunately, studies evaluating similar neuropathological features have not been performed in the CGG KI mouse [4
], so it is not yet possible to make comparisons between the human and mouse. Longitudinal studies of large groups of CGG KI mice using noninvasive imaging technologies (e.g., high field MRI) that bypass fixation and histological artifacts are underway to characterize regional volumetric differences in the CGG KI mouse well enough to make a direct comparison between the CGG KI mouse and the fragile X premutation carrier.
Willemsen et al. [68
] showed the presence and relative abundance of intranuclear inclusions in male CGG KI mice (on a mixed FVB/N × C57BL/6J background). These inclusions appeared similar to those reported in FXTAS cases. They found that at 48-52 weeks of age, CGG KI mice showed intranuclear inclusions in the thalamus, hypothalamus, cingulate cortex (anterior cingulate), periamygdaloid cortex, rostral cortex, and in the hippocampus at slightly lower levels—all regions implicated in temporal ordering. Hunsaker et al. [30
] and Wenzel et al. [67
] undertook limited analyses of intranuclear inclusions in male CGG KI mice (on a congenic C57BL/6J background) and found a similar pattern of inclusions, though quantitative analyses were not undertaken. What remains absent from the literature is the distribution and relative abundance of intranuclear inclusions in heterozygous female CGG KI mice. Wenzel et al. [67
] evaluated a pair of aged female CGG KI mice that would be in the High CGG repeat group in the present study, and found a similar distribution of inclusions as in the male CGG KI mice, but analyses of female mice in the Low CGG repeat group have yet to be undertaken, although these female mice do have intranuclear inclusions (unpublished observations).
Because levels of Fmr1
mRNA and Fmrp levels have not yet been quantified in female CGG KI mice it is not yet possible to relate these molecular measure to the present temporal order deficits. To date, these measurements have only been carried out in male mice [6
], and unequal X inactivation in females renders any Fmr1
and Fmrp measurements in males and females impossible to directly compare (cf. [57
]). Studies are currently underway to quantify Fmr1
and Fmrp levels in female CGG KI mice.
The present data demonstrate impaired temporal ordering in the CGG KI mouse. Furthermore, this study provided insights into potential differences between CGG KI mice with repeat lengths below 100 CGG trinucleotide repeats and those with repeat lengths above 150 CGG trinucleotide repeats that bear further study using both the murine model as well as with fragile X premutation carriers. These data also suggest some additional parallels in the cognitive deficits seen in fragile X premutation carriers in the CGG KI mouse model of the fragile X premutation, although additional studies will be necessary in order to determine how strong such parallels might be. The present results also point to the need to develop behavioral and neurocognitive tasks for CGG KI mice that more closely model the neurocognitive phenotype of fragile X premutation carriers so that the underlying neural circuitry that is impaired in fragile X premutation carriers (both with and without FXTAS) can be more directly explored. Behavioral tests in CGG KI mice that more closely map onto the neurocognitive deficits reported in fragile X premutation carriers would also make such mice valuable tools for the development and evaluation of treatment for the disorder [33
The present data show that female CGG KI mice developed to model the fragile X premutation show deficits in temporal ordering, but only when the repeat expansions approach the upper end of the premutation range. What remains to be studied are the implications of this finding for human fragile X premutation carriers and whether such deficits can be attributed to spatiotemporal hypergranularities as has been suggested [58