Research reportEarly detection of cognitive deficits in the 3xTg-AD mouse model of Alzheimer's disease
Introduction
The 3xTg-AD mouse model of familial Alzheimer's disease (AD) was created by inserting the Swedish amyloid precursor protein (APPswe) and tau (TauP301L) genes into the embryo of a PS1M146V transgenic mouse [1]. The APPswe gene is a human gene with a mutation associated with familial AD and the PS1M146V gene is a mouse presenilin 1 (PS1) gene which has a human mutation associated with familial AD inserted. The TauP301L mutation is associated with human tau pathology. With these transgenes the 3xTg-AD mouse develops both amyloid and tau pathology. The first detectable pathology is the development of intracellular amyloid beta at 3 months of age followed by the development of extracellular plaques in the neocortex and hippocampus at 6 months of age [2], [3]. The development of tau pathology begins at 6 months of age, when phosphorylated tau is detectable in the hippocampus. The phosphorylated tau develops into neurofibrillary tangles between 18 and 26 months of age [3].
There have been several studies delineating the cognitive behavior phenotype of the 3xTg-AD mouse and using behavioral bio-assays to evaluate novel drug treatments for AD. However the results have not been consistent; differing according to the age and sex of the mice and the test procedures used. In the Y-Maze test of spontaneous alternation, a commonly used measure of short term memory, some studies reported that female 3xTg-AD mice have no deficit at 6 months of age, some reported a deficit at 6 months of age, and others found no deficits until 7 months of age [4], [5], [6]. In the novel object recognition task, some studies found deficits in 6-month-old female 3xTg-AD mice compared to B6129SF2 mice in a 15 min delay task [7], [8]. However Clinton et al. [9] did not find a deficit in novel object recognition in either male or female 3xTg-AD mice until 9 months of age in either the 1.5 h or 24 h delay task, likewise Martinez-Coria et al. [10] found a deficit in both 1.5 h and 24 h retention in the novel object recognition task in 9-month-old male and female 3xTg-AD mice compared to age-matched B6129SF2 wildtypes. In the Barnes maze, 6-month-old 3xTg-AD mice of both sexes had significantly longer escape latencies and made more errors than control mice [11]. In the radial arm maze the 3xTg-AD mice were impaired in both working and reference memory relative to B6129SF2 control mice starting at 2 months of age [12]. Some studies have reported deficits in contextual fear conditioning by 6 months of age in 3xTg-AD mice of both sexes [2], [13]. Others found no difference between 6-month-old 3xTg-AD and B6129SF2 wildtype mice in contextual fear conditioning with a 24 h delay, but the 3xTg-AD were impaired in longer term memory [14]. On the other hand Chu et al. [15] reported no difference between 3xTg-AD and B6129SF2 mice of either sex in cued or contextual fear conditioning at 13–14 months of age.
The 3xTg-AD mice are commonly used to assess potential therapies for the treatment of AD [16], [17], [18], [19], [20]. In order to determine the effectiveness of novel therapeutic agents on cognition in the 3xTg-AD mice, one or more of many behavioral tasks, which differ in sensitivity to detect cognitive impairments in this strain, have been used. Although there is variability in the ages of the 3xTg-AD mice to test potential AD treatments, cognitive deficits have been reported by 6.5 months of age. However, the effect size for the cognitive deficits are seldom reported in these studies. If the effect sizes for the cognitive deficits is very small it may not be replicable and the effect of any drug treatments will be non-significant; if there are no deficits then cognition cannot be improved with any drug treatment. The purpose of the present experiment, therefore, was to test male and female 3xTg-AD mice on a number of commonly used behavioral tests of cognitive function, chosen to assess several cognitive domains. The tests were: spontaneous alternation in the Y-Maze, a measure of exploratory and spatial memory; novel object recognition (NORT), a measure of recognition memory; the Barnes maze, a measure of spatial learning and memory; and cued and contextual fear conditioning tasks, measures of classically conditioned fear memory. We evaluated these tests in order to determine which was the most sensitive to the cognitive deficits of these mice at this age. The sensitivity of each test was evaluated by comparing the effect sizes for the genotype differences detected in that test. Knowing which test is the most sensitive will allow researchers to more efficiently screen new therapies by decreasing the number of animals required to detect a difference and increasing the likelihood of detecting any differences. Because sex differences in memory have been found in transgenic mice [21], including the 3xTg-AD mice [22], we also examined the effect sizes of sex differences in these cognitive tasks.
Section snippets
Animals
Eighty-five mice, 42 3xTg-AD (21 female and 21 male, Stock #004807), and 43 B6129SF2 (22 male and 21 female, Stock #101045), were bred in our lab from parents purchased from the Jackson Laboratory (Bar Harbor, Maine). The mice were weaned at 21 days of age and tested at 6.5 months of age in three cohorts of approximately 28 mice. The mice were housed in groups of 2–4 same sex littermates in plastic cages (18.75 cm × 28 cm × 12.5 cm), with a PVC tube (4 cm diameter × 7 cm length) for enrichment, wood chip
Y-Maze test of spontaneous alternation
Three measures of alternation behavior were calculated: the percentage of spontaneous alternation behaviors (SAB), the percentage of alternate arm returns (AAR), and the percentage of same arm returns (SAR). For the percentage of SABs the linear regression model with the lowest AICc was the model with only genotype (AICc = 714.038, weight = 0.429, Supplemental Table 1.1, Fig. 1A), which differed significantly from the null model (F(1,83) = 6.273, p = 0.014). Confidence intervals indicated that the
Discussion
In this study, the Barnes maze was the most sensitive test for detecting cognitive deficits in 3xTg-AD mice. We found evidence for a deficit in spatial learning and memory in the Barnes maze in both male and female 3xTg-AD mice at 6.5 months of age, as 3xTg-AD mice made more errors during the acquisition learning phase than B6129SF2 wildtype mice (Fig. 3D), and spent less time in the correct zone during the memory probe trial (Fig. 4A). The 3xTg-AD mice moved faster than B6129SF2 wildtype mice
Acknowledgments
This research was funded by an NSERC grant to REB. The authors would like to thank Dr. Rachel Dingle and Dr. Aimee Wong for their assistance in this project.
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