Behavioural Brain Research 273 (2014) 116–122

Contents lists available at ScienceDirect

Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr

Research report

Learning strategy preference of 5XFAD transgenic mice depends on the sequence of place/spatial and cued training in the water maze task Woo-Hyun Cho a , Jung-Cheol Park a , ChiHye Chung a , Won Kyung Jeon b , Jung-Soo Han a,∗ a b

Department of Biological Sciences, Konkuk University, Seoul 143-701, Republic of Korea Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon 305-811, Republic of Korea

h i g h l i g h t s • • • •

5XFAD mice with serial cued- and place training preferred a cued strategy. 5XFAD mice with serial place- and cued training showed impairment in place learning. The highest levels of A␤42 were observed in the hippocampus of 5XFAD mice. Differential functional recruitment of learning-related brain structures in AD.

a r t i c l e

i n f o

Article history: Received 18 February 2014 Received in revised form 20 June 2014 Accepted 21 July 2014 Available online 28 July 2014 Keywords: Alzheimer’s disease Amyloid beta Cued training Place/spatial training Learning strategy

a b s t r a c t Learning strategy preference was assessed in 5XFAD mice, which carry 5 familial Alzheimer’s disease (AD) mutations. Mice were sequentially trained in cued and place/spatial versions of the water maze task. After training, a strategy preference test was conducted in which mice were required to choose between the spatial location where the platform had previously been during the place/spatial training, and a visible platform in a new location. 5XFAD and non-transgenic control mice showed equivalent escape performance in both training tasks. However, in the strategy preference test, 5XFAD mice preferred a cued strategy relative to control mice. When the training sequence was presented in the reverse order (i.e., place/spatial training before cued training), 5XFAD mice showed impairments in place/spatial training, but no differences in cued training or in the strategy preference test comparing to control. Analysis of regional A␤42 deposition in brains of 5XFAD mice showed that the hippocampus, which is involved in the place/spatial learning strategy, had the highest levels of A␤42 and the dorsal striatum, which is involved in cued learning strategy, showed a small increase in A␤42 levels. The effect of training protocol order on performance, and regional differences in A␤42 deposition observed in 5XFAD mice, suggest differential functional recruitment of brain structures related to learning in healthy and AD individuals. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Studies in humans and rodents indicate that different neural systems are engaged in performing navigational tasks, depending on the learning strategies being employed [1–3]. Specifically, in the Morris water maze task, a place/spatial strategy is an allocentric navigation strategy requiring spatial information, and depends on the hippocampal function. In contrast, a cued learning strategy is an egocentric navigation strategy that requires instrumental

∗ Corresponding author at: Department of Biological Sciences, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea. Tel.: +82 2 450 3293; fax: +82 2 3436 5432. E-mail address: [email protected] (J.-S. Han). http://dx.doi.org/10.1016/j.bbr.2014.07.033 0166-4328/© 2014 Elsevier B.V. All rights reserved.

learning, and depends on the striatal function. In patients with AD, and in transgenic AD mice, hippocampal dependent memory is impaired, whereas hippocampal-independent memory is intact [4–7]. Animal models of Alzheimer’s disease (AD) contribute to our understanding of the molecular and pathophysiological mechanisms underlying cognitive impairment in the disease [8,9] as well as may provide research platform for studying differences between healthy and AD individuals in functional recruitment of brain structures involved in multiple memory system. Transgenic AD animal models with mutant forms of human amyloid precursor protein (APP), presenilin (PS), and/or tau genes exhibit pathological features of AD, including cognitive deficits [10]. In particular, hippocampal-dependent memory has been intensively examined in AD transgenic studies, using well-established behavioral tests that rely on intact hippocampal functioning.

W.-H. Cho et al. / Behavioural Brain Research 273 (2014) 116–122

Examples of such tests include the Morris water maze to measure spatial memory, and Pavlovian conditioning to measure context fear memory [11–13]. Additional behavioral tasks, such as the Yor T-maze, have been used to measure spatial memory or spatial working memory in transgenic AD mice [13]. The 5XFAD APP/PS1 transgenic mouse, which possesses 5 familial AD mutations (Swedish, Florida, and London human APP mutations; M146L and L286V PS1 mutations), is one of the most severe amyloid mouse models of early-onset AD pathology [14]. The 5XFAD mice develop neuropathological characteristics of AD, including amyloid plaques and gliosis, at 2 months of age [14]. Cognitive deficits are observed at 5–6 months of age [14–16]. Hongpaisan et al. [15] used the Morris water maze task, which is typically conducted to assess hippocampal function, and reported that spatial reference memory was impaired in 5XFAD mice. Spatial working memory assessed with spontaneous alternation Y-maze tasks [14], and fear memory assessed using context fear conditioning tasks, were also found to be impaired in 5XFAD mice [17]. Evidence for multiple memory systems is observed in studies demonstrating behavioral strategy difference in two inbred strains of mice, C57B/6 and DBA/2, using a redundant place/cued version of the water maze task adapted from McDonald and White [1,18]. Sung et al., (2008) showed that mice received training to a stationary platform across 12 consecutive days with the platform visible on some days and submerged on others and a preference test on the 13th day. Both C57B/6 and DBA/2 mice showed similar escape performance in cued and place/spatial training, but C57B/6 mice preferred a place/spatial strategy more often than DBA/2 mice on the preference test. Effects of age on strategy preference were examined in C57BL/6 × SJL F1 mice, a common background strain for AD transgenic models [19], using a modified behavioral protocol of a redundant place/cue version of the water maze task [1]. Aged mice (23 months) and adult mice (12 months) were sequentially trained in cued and place/spatial versions of the Morris water maze task. Mice then underwent a strategy preference test. No differences in performances between aged and adult mice were reported in cued and place/spatial training, but the aged mice were biased towards a cued strategy on the preference test. Based on the findings reported above, we examined the learning strategy preferences of 5XFAD mice using this same training order (i.e., cued training followed by place/spatial training), and vice versa (i.e., place/spatial training followed by cued training), to examine the effect of training order on learning strategy. 2. Materials and methods 2.1. Subjects Transgenic mice (5XFAD) were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA). Six-month-old hemizygous 5XFAD mice (33 male, 21 female) and non-transgenic control mice (29 male, 22 female) were used at the beginning of the experiments. Mice were housed in groups of 4 per cage, in a temperature- and humidity-controlled room, on a 12-h light/dark cycle (lights on 07:00-19:00). Food and water were available ad libitum. All testing was performed during the light cycle. The Institutional Animal Care and Use Committee of Konkuk University approved all protocols described in this study. 2.2. Apparatus The water maze consisted of a circular tank (1.83-m diameter and 0.58-m height) with an escape platform (20-cm diameter) centered in one of the four maze quadrants. Water (27 ◦ C) was made opaque with nontoxic white paint. The visible escape platform was

117

raised 2 cm above the water surface for cued training. The hidden platform was located 0.5 cm beneath the surface for place training. The maze was surrounded by white curtains, on which black felt patterns were affixed to provide distal visual (spatial) cues. Data were recorded using an HVS Image tracking system (Hampton, UK). 2.3. Behavioral training procedure Cued training: Mice received 4 trials/day (10-min intertrial interval, maximum trial duration of 60 s, with 20 s on the platform at the end of each trial) in which a visible platform was moved to different locations in the pool between trials. Blank white curtains were drawn around the pool during cue training to occlude extramaze cues. Place/spatial training: Mice received 4 trials/day (10-min intertrial interval, maximum trial duration of 60 s, with 20 s on the platform at the end of each trial), with each trial beginning at one of four equidistantly located positions at the perimeter of the maze. The location of the platform remained constant across all training trials. Mice were placed into the water facing the wall and were allowed to swim for a maximum of 60 s. The trial ended when a mouse climbed onto the available platform, or after the 60-s interval had elapsed. If a mouse did not locate the platform during a trial, it was placed on the platform by the experimenter. Mice were left on the platform for 20 s, and then moved to a holding cage for a 10-min intertrial interval. Test for preference of learning strategy: On day 9, a competition test was given in which the visible platform was positioned in the SW quadrant (opposite to its placement on place training days). Two trials were given with start points equidistant from the two platform locations (NE and SW). Video recordings were analyzed to determine if mice swam to the previous hidden platform location prior to escaping to the visible platform. Training protocol: The first group of 5XFAD mice (n = 25) and control mice (n = 25) first received cued training for 4 days, followed by place/spatial training for 4 days. On day 9, a competition test was conducted. The second group of 5XFAD (n = 29) mice and control mice (n = 26) received the same training, but in the reverse order (i.e., place/spatial training before cued training), and a competition test was conducted on day 9. The spatial bias of mice that first received place/spatial training was assessed with one probe trial prior to the start of cued training. During the probe trial, mice swam for 30 s with the platform retracted to the bottom of the pool. 2.4. Histology and ELISA All mice were sacrificed 7 days following the competition test. One hemi-brain of each mouse was stored in 4% paraformaldehyde, while the hippocampus, dorsal striatum, prefrontal cortex, cortex, and cerebellum were rapidly dissected from the remaining hemibrain and frozen at −80 ◦ C until further processing. Human A␤42 levels were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (ImmunoBiological Laboratories Co., Gunma, Japan). The dissected tissues were sonicated in radioimmunoprecipitation assay (RIPA) buffer (1% NP-40, 150 mM NaCl, 50 mM Tris–Cl, [pH 8.0], 0.1% sodium dodecyl sulfate [SDS], and 0.5% deoxycholic acid) that contained 50 mM NaF, 1 mM Na3 VO4 , and a 1× protease inhibitor cocktail. Protein extracts (800 ␮g/␮l) were centrifuged at 100,000 × g for 1 h at 4 ◦ C. Supernatants were transferred to a new tube and used to obtain the soluble A␤42 sample. Pellets were supplemented with 400 ␮l of 70% formic acid, and sonicated. Samples were then centrifuged at 100,000 × g for 1 h at 4 ◦ C. Formic acid extracts (50 ␮l) were evaporated for 30 min at 40 ◦ C. The resulting insoluble A␤42 samples were suspended in 1 mL RIPA buffer, and further diluted in RIPA buffer in the same to achieve A␤42 concentrations within

118

W.-H. Cho et al. / Behavioural Brain Research 273 (2014) 116–122

the standard curve. All ELISA experimental procedures were performed in accordance with the manufacturer’s instructions, and the absorbance was read at 450 nm using a plate reader. Data were expressed as ng A␤42/mg total protein. One hemi-brain from each mouse was stored in 4% paraformaldehyde for at least 5 days, and then kept in 30% sucrose until sinking to the bottom of the container. Brains were sectioned sagittally on a freezing microtome at 40 ␮m. For A␤42 immunoreactivity, endogenous peroxidase activity of free floating sections was quenched by a 30-min incubation in 3% H2 O2 /10% MeOH in PBS. The sections were then incubated for 1 h at room temperature in PBS with 0.3% Triton-X 100 and 10% serum (PBS-T-S). The sections were then incubated with A␤42 antibody (1:500, Invitrogen) for 16 h at 4 ◦ C and 2 h at room temperature in PBS-T-S solution. The sections were then incubated for 1 h in goat anti-rabbit antibody (1:500, Vector Labs) and then for 1 h in ExtrAvidin peroxidase conjugate (1:1000, Sigma–Aldrich). Finally, the sections were reacted with a Vector SG substrate kit (Vector Labs) for peroxidase and mounted onto resin coated slides, after which they were dried for up to 5 days. Slides were then coverslipped with permount reagent 2.5. Behavioral and statistical analyses Escape latencies during cued and place training were separately analyzed using a repeated measures two-way analysis of variance (ANOVA; group × trial session [day]) to evaluate acquisition. The average proximity of an animal’s position with respect to the hidden platform position was used to measure spatial bias in the probe trial conducted after the place/spatial training that first received, and the first trial of place/spatial training that was followed by cued learning. Swimming search pattern was classified as spatial search strategy or nonspatial search strategy for each mouse on every trial in the place/spatial training followed by cued training according to the criterion adopted by Janus [20]. Student’s t-test or Mann–Whitney U test was used to evaluate differences between 5XFAD and control mice. For the competition test, mice were given two trials. Fig. 1D shows representative swim paths for mice using two different strategies. Mice designated as using a “place/spatial strategy” visited the location where the platform had been in the previous training days, before escaping to the newly located visible platform. In contrast, mice using the “cued strategy” swam directly to the visible platform in its new location. Using the criterion established by McDonald and White [1], mice were classified as using a place strategy if they visited the previous platform location during either of the two trials. All other mice were classified as using a cued strategy. A 2 analysis was employed to evaluate differences in the frequency of strategies during the competition test. A two-way ANOVA (group × region [hippocampus, dorsal striatum, prefrontal cortex, cortex, and cerebellum]) was conducted to examine insoluble and soluble A␤42 levels. Post hoc Fisher’s Least Significant Differences test was conducted to reveal regional differences in brains of 5XFAD mice. p values less than 0.05 were considered significant. All data are expressed as mean ± S.E.M. 3. Results Performances for the group in which visible platform training (cued) preceded hidden platform training (place/spatial) are shown in Fig. 1. Groups of 5XFAD and control mice perform equally during visible platform trials. Repeated measures two-way ANOVA indicated that the performance of both groups improved over the course of training (F(3,144) = 82.91, p < 0.001, main effect of session), and that there was no significant main effect of group (F(1,48) = 1.25, p = 0.27) or significant group × session interaction (F(3,144) = 0.61,

p = 0.61). Thus, the rate of learning was the same in both groups. In the subsequent hidden platform trials (place/spatial training), there was also no difference in performance between 5XFAD and control mice. Repeated measures two-way ANOVA indicated that performance improved over the course of training (F(3,144) = 6.10, p < 0.001, main effect of session). As in the visible platform trials, there was no significant main effect of group (F(1,48) = 0.04, p = 0.84) or significant group × session interaction (F(3,144) = 1.33, p = 0.27), indicating that the rate of learning rate was the same in both groups (Fig. 1A). Furthermore, no differences in average proximity to the platform for the first training trial of the place/spatial training was observed between 5XFAD and control mice (t(48) = −0.260, p = 0.80; Fig. 1B). When the cued training was conducted first, mice might acquire some procedural knowledge of the task, which could affect performances in the subsequent place/spatial training. Therefore, we divided the place/spatial training into two sessions (the first 8 trials vs. the second 8 trials) and performed an analysis of search strategies adopted by mouse during navigation [20]. We compared the used frequencies of spatial search strategy in the second sessions with those in the first session using Mann–Whitney U test. Control mice developed spatial search strategy, but 5XFADmice did not (U = 216.00, p = 0.05; Fig. 1C). In the competition test, 15 of 25 control mice used a place strategy, whereas only 8 of 25 5XFAD mice did (Fig. 1D and E). The observed difference between the control and 5XFAD mice in terms of strategy selection was significant as determined by the 2 analysis (2 = 3.950, p = 0.04). Performances for the group in which place/spatial training preceded cued training are shown in Fig. 2. 5XFAD mice performed worse than control mice during place/spatial learning. Repeated measures two-way ANOVA indicated that the performance of both groups improved over the course of training (F(3,159) = 17.801, p < 0.001, main effect of session). However, there was a significant main effect of group (F(1,53) = 6.05, p = 0.02) and a significant group × session interaction (F(3,159) = 3.05, p = 0.03), indicating different rates of learning for control and 5XFAD mice. In order to confirm that there are differences between 5XFAD and control mice in place/spatial learning conducted without prior training, a probe trial was given 24 h later after the place/spatial learning. Differences in average proximity in the probe trial were observed between 5XFAD and control mice (t(52) = −2.02, p = 0.05; Fig. 2B). In the subsequent cued training, there were no differences in performance between 5XFAD and control mice. Repeated measures two-way ANOVA indicated that the performance of both groups improved over the course of training (F(3,159) = 71.50, p < 0.001, main effect of session). However, there was no significant main effect of group (F(1,53) = 0.04, p = 0.84) and no significant group × session interaction effects (F(3,159) = 0.23, p = 0.87), indicating that the rate of learning was the same for both groups (Fig. 2A). As shown in Fig. 2C, in the competition test, 12 of 26 control mice, and 15 of 29 5XFAD mice, used a place strategy. No difference between the control and 5XFAD mice in terms of strategy selection was observed, as determined by the 2 analyses (2 = 0.17, p = 0.79). Fig. 3A shows the expression of A␤42 in the hippocampus including cortex, prefrontal cortex, dorsal striatum, and cerebellum in 5XFAD mice. A␤42-positive signals were massively observed in the hippocampus and cortex, but, in dorsal striatum, a few A␤42positive signals in 2 of 9 mice were observed and no A␤42-positive signals in rest of them (Fig. 3A). To determine whether 5XFAD mice exhibit A␤42 deposition, we measured insoluble and soluble A␤42 levels in the hippocampus, prefrontal cortex, dorsal striatum, and cerebellum of 5XFAD ( n= 8) and control mice (n = 8), using A␤42-specific sandwich ELISA. Statistical analysis showed that the effects of group and region were significant (F(1,70) ≥ 47.60, p < 0.001; F(4,70) ≥ 12.43, p < 0.001) and that the interaction of group and region was significant (F(4,70) ≥ 12.17, p < 0.001). By post hoc

W.-H. Cho et al. / Behavioural Brain Research 273 (2014) 116–122

119

Fig. 1. (A) Performances of 5XFAD and non-transgenic control mice for cued training and subsequent place/spatial training, and for selection of learning strategy. 5XFAD (n = 25) and control mice (n = 25) received cued training for 4 days and subsequently place/spatial training for 4 days. No differences between 5XFAD and control mice were observed during cued training or place/spatial learning (A). No differences between 5XFAD and control mice were observed in terms of proximity to the platform in the first trial of the place/spatial training (B). 5XFAD mice used spatial search strategy less than control mice in the second half of the place/spatial training (C). (D) Representative swim paths are shown for a mouse choosing a “place/spatial strategy” (right) and a mouse using a “cued strategy” (left) in the competition test (day 9). Note that in the right panel, the mouse crossed the location where the escape platform had been during the previous 4 days of training whereas in the left panel, the mouse swam directly to the visible platform in its new location. 5XFAD mice used a cued strategy rather than a place/spatial strategy (E). The differences between 5XFAD and control mice in terms of strategy selection were statistically significant. Non-Tg: non-transgenic control mice.

analyses, among the examined regions in 5XFAD mice, insoluble A␤42 levels were highest in the hippocampus, followed by the cortex and prefrontal cortex (p < 0.05). Insoluble A␤42 levels in dorsal striatum were lower than those of brain regions except the cerebellum (Fig. 3B). Like insoluble A␤42 levels, among the examined regions in 5XFAD mice, the highest levels in soluble A␤42 were observed in the hippocampus, followed by prefrontal cortex and cortex. Soluble A␤42 levels of dorsal striatum were not statistically different from those of prefrontal cortex and cortex (Fig. 3C). No notable insoluble and soluble A␤42 level was measured in cerebellum of 5XFAD mice.

4. Discussion The present study was conducted to assess the learning strategy preference of 5XFAD mice as compared with non-transgenic control mice. To accomplish this, one group of control and 5XFAD mice sequentially received cued and place/spatial training in a traditional water maze, while a second group received the same training, albeit in the opposite order (place/spatial followed by cued). After training, the learning strategy preference of control and 5XFAD mice in both groups was evaluated by presenting a choice between the spatial location of the platform during place training

120

W.-H. Cho et al. / Behavioural Brain Research 273 (2014) 116–122

Fig. 2. Performances of 5XFAD and non-transgenic control mice for place/spatial training and subsequent cued training, and for selection of learning strategy. 5XFAD (n = 29) and control mice (n = 26) received place/spatial training for 4 days, followed by a probe test 24 h later, followed by the cued training over the next 4 days. Significant differences between 5XFAD and control mice were observed during place/spatial training (A, left panel) and the probe test (B). No differences between 5XFAD and control mice were observed during the cued training (A, right panel). No differences between 5XFAD mice and control mice were found in terms of the selection of learning strategy (C). Non-Tg: non-transgenic control mice.

(a place/spatial strategy) and a visible platform in a new location (a cued strategy). In the first experiment, 5XFAD and control mice underwent cued training for different platform locations, and then place/spatial training for a single platform location in a water maze. These strategies were then placed in competition, and mice were required to choose between the spatial location of the platform in the place training trials, and a visible platform located in a new location. Performances, as assessed by latency, in the cued and place/spatial tasks did not differ between 5XFAD and control mice. However, control mice used spatial search strategy more frequently than 5XFAD mice in the second half of the place/spatial training. And, on the strategy preference test, 60% of control mice visited the place location before escaping to the visible platform, whereas only ∼30% of 5XFAD mice did. These results demonstrate a strong preference of 5XFAD mice for using a cued learning strategy in a task where both place/spatial and cued strategies are available. By contrast, unlike the first group, in which cued training preceded place training, and 5XFAD performed comparably to control mice in terms of spatial learning, in the second experiment, 5XFAD mice took longer to find the hidden platform when place training preceded cued training. 5XFAD mice also exhibited less spatial bias in the probe trial conducted after the initial place/spatial training, compared with control mice. However, no differences in performance between 5XFAD and control mice were found in the cued

training that followed. On the preference test, there was no difference between 5XFAD and control mice in terms of preferred learning strategy. Thus, even though cognitive deficits in 6-monthold 5XFAD mice were evident in both experiments, impairments in cognitive ability of 5XFAD mice compared with control mice depended on the training procedure, type of probe test, and even more importantly, presence/absence of prior training. Overall, these findings are consistent with previous study indicating that 5XFAD mice are impaired in hippocampusdependent learning [15], while providing new evidence that striatal-dependent learning is spared in 5XFAD mice. But, the deficit of 5XFAD mice was disappeared on a measure of latency at place/spatial training task compared with control mice, when the place/spatial training was followed by the cued training. However, analysis of search strategy adopted by mice during the place/spatial training revealed that control mice developed place/spatial search strategy in the later session of place/spatial training, but 5XFAD mice did not. These results may be interpreted as follows: when trained in the cued learning first, the control mice acquired some procedural knowledge of the cued learning task, which could interfered the development of spatial search strategy in the early training when place/spatial training was then given. 5XFAD mice rapidly develop several features of AD amyloid pathology and exhibit intraneuronal A␤42-indiced neurodegeneration [14]. Here our A␤42 analysis of 5XFAD mice showed that A␤42

W.-H. Cho et al. / Behavioural Brain Research 273 (2014) 116–122

121

Fig. 3. A␤42 deposition in hippocampus, prefrontal cortex, cortex, dorsal striatum, and cerebellum of 5XFAD and non-transgenic control mice. The representative photomicrograph illustrates the presence of A␤42 in the brains of 5XFAD and control mice (A). A␤42-specific sandwich ELISA analyses showed regional differences in insoluble and soluble A␤42 levels in 5XFAD mice (B, C). Among brain structures examined, the hippocampus (##) of 5XFAD mice had the highest level of insoluble and soluble A␤42, followed by the prefrontal cortex and cortex (#). Dorsal striatal A␤42 levels were significantly lower compared with those of hippocampus and prefrontal cortex.

levels were highly elevated and massively aggregated in the hippocampus and prefrontal cortex, whereas dorsal striatum showed low levels of A␤42 and exhibited very little A␤42 aggregation. Relative to control mice, 5XFAD mice showed deficits in performing hippocampus-dependent task (i.e. spatial learning), while comparable performance in striatum-dependent task (i.e. cued learning). However, these behavioral differences between 5XFAD and control mice were observed in different behavioral tasks depending on training protocol. Taken together, the dependence of training protocol order on learning strategy preference, and regional variability in A␤42 deposition observed in 5XFAD mice, suggest that, in AD, different brain structures are recruited based on the nature of a given learning task, and the absence or presence of prior learning experiences. In a previous study of 5XFAD mice, plaques appeared at 2 months in the layer V of the cortex and in the subiculum, and were observed in the all areas of cortex and hippocampus at older ages. We found that, in 5XFAD mice, the A␤42 levels were high in the hippocampus and prefrontal cortex; A␤42 levels in the dorsal striatum were lower than in these two regions. These results support the findings that 5XFAD mice preferred a cued learning strategy rather than a place/spatial learning strategy, and showed impairments in the place/spatial training when presented first, as well as in the spatial probe trial that followed. This is consistent with evidence supporting a multiple memory system, that is,

damage or inactivation of the hippocampus impairs place learning and enhances cued learning, whereas damage or functional inactivation of the dorsal striatum impairs cued learning and facilitates place learning [1,2]. But, because preference of learning strategy also depends on natural fluctuations in ovarian steroids in female rodent [21], an explanation of the findings would be strengthened by further study regarding sex differences of 5XFAD mice or with estrous cycle-linked behavioral testing. Recently, behavioral tasks examining hippocampusindependent functions have been conducted to study the brain function of 5XFAD mice. These tasks include the conditioned taste aversion task, in which memory formation of animal involves the amygdala and insular cortex [22]. 5XFAD mice showed deficits in this memory task, and had A␤ deposition in the amygdala and insular cortex [23]. Another task for studying cognitive functions involving the frontal cortex [24], the olfactory H-maze, was devised by Girard et al. [25]. 5XFAD mice showed impaired performances in this task, and greater A␤ deposition was observed in the frontal cortex compared with control mice [25]. It is interesting that significant A␤42 deposition was observed in the prefrontal cortex of 5XFAD mice in our study, given that the prefrontal cortex is reportedly involved in performing the tasks that we used [26–28]. In conclusion, our results demonstrate that 5XFAD mice receiving sequential cued and place/spatial training prefer a cued learning strategy, but only when cued training precedes place learning. We

122

W.-H. Cho et al. / Behavioural Brain Research 273 (2014) 116–122

expect that, at 6 months of age, the dorsal striatum might be functionally intact in 5XFAD mice, but functions of the hippocampus and prefrontal cortex are impaired by A␤ deposition. Our experimental paradigm will be particularly useful in future studies investigating cognitive function in animal models that have abundant intraneuronal A␤ accumulation or other AD features, as well as in the evaluation of possible AD interventions. Acknowledgments This work was supported by a Korea Research Foundation Grant funded by the Korean Government (KRF-2010-0023880) and a grant (kiom-2010-2) from the Inter-Institutional Collaboration Research Program provided by the Korea Research Council of Fundamental Science & Technology (KRCF). References [1] McDonald RJ, White NM. Parallel information processing in the water maze: evidence for independent memory systems involving dorsal striatum and hippocampus. Behavioral and Neural Biology 1994;61:260–70. [2] Packard MG, McGaugh JL. Inactivation of hippocampus or caudate nucleus with lidocaine differentially affects expression of place and response learning. Neurobiology of Learning and Memory 1996;65:65–72. [3] Hartley T, Maguire EA, Spiers HJ, Burgess N. The well-worn route and the path less traveled: distinct neural bases of route following and wayfinding in humans. Neuron 2003;37:877–88. [4] Ammassari-Teule M, Middei S, Passino E, Restivo L. Enhanced procedural learning following beta-amyloid protein (1–42) infusion in the rat. Neuroreport 2002;13:1679–82. [5] Beaunieux H, Eustache F, Busson P, de la Sayette V, Viader F, Desgranges B. Cognitive procedural learning in early Alzheimer’s disease: impaired processes and compensatory mechanisms. Journal of Neuropsychology 2012;6:31–42. [6] Hirono N, Mori E, Ikejiri Y, Imamura T, Shimomura T, Ikeda M, et al. Procedural memory in patients with mild Alzheimer’s disease. Dementia and Geriatric Cognitive Disorders 1997;8:210–6. [7] Middei S, Geracitano R, Caprioli A, Mercuri N, Ammassari-Teule M. Preserved fronto-striatal plasticity and enhanced procedural learning in a transgenic mouse model of Alzheimer’s disease overexpressing mutant hAPPswe. Learning & Memory 2004;11:447–52. [8] Ashe KH. Learning and memory in transgenic mice modeling Alzheimer’s disease. Learning & Memory 2001;8:301–8. [9] Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, et al. Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 1996;274:99–102. [10] Hall AM, Roberson ED. Mouse models of Alzheimer’s disease. Brain Research Bulletin 2012;88:3–12.

[11] Anagnostaras SG, Gale GD, Fanselow MS. Hippocampus and contextual fear conditioning: recent controversies and advances. Hippocampus 2001;11: 8–17. [12] Morris RG, Garrud P, Rawlins JN, O’Keefe J. Place navigation impaired in rats with hippocampal lesions. Nature 1982;297:681–3. [13] Yuede CM, Dong H, Csernansky JG. Anti-dementia drugs and hippocampaldependent memory in rodents. Behavioural Pharmacology 2007;18: 347–63. [14] Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J, et al. Intraneuronal betaamyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: potential factors in amyloid plaque formation. The Journal of Neuroscience 2006;26:10129–40. [15] Hongpaisan J, Sun MK, Alkon DLPKC. epsilon activation prevents synaptic loss, Abeta elevation, and cognitive deficits in Alzheimer’s disease transgenic mice. The Journal of Neuroscience 2011;31:630–43. [16] Ohno M. Failures to reconsolidate memory in a mouse model of Alzheimer’s disease. Neurobiology of Learning and Memory 2009;92:455–9. [17] Kimura R, Ohno M. Impairments in remote memory stabilization precede hippocampal synaptic and cognitive failures in 5XFAD Alzheimer mouse model. Neurobiology of Disease 2009;33:229–35. [18] Sung JY, Goo JS, Lee DE, Jin DQ, Bizon JL, Gallagher M, et al. Learning strategy selection in the water maze and hippocampal CREB phosphorylation differ in two inbred strains of mice. Learning & Memory 2008;15:183–8. [19] Nicolle MM, Prescott S, Bizon JL. Emergence of a cue strategy preference on the water maze task in aged C57B6 × SJL F1 hybrid mice. Learning & Memory 2003;10:520–4. [20] Janus C. Search strategies used by APP transgenic mice during navigation in the Morris water maze. Learning & Memory 2004;11:337–46. [21] Korol DL, Malin EL, Borden KA, Busby RA, Couper-Leo J. Shifts in preferred learning strategy across the estrous cycle in female rats. Hormones and Behavior 2004;45:330–8. [22] Yamamoto T, Fujimoto Y. Brain mechanisms of taste aversion learning in the rat. Brain Research Bulletin 1991;27:403–6. [23] Devi L, Ohno M. Genetic reductions of beta-site amyloid precursor proteincleaving enzyme 1 and amyloid-beta ameliorate impairment of conditioned taste aversion memory in 5XFAD Alzheimer’s disease model mice. The European Journal of Neuroscience 2010;31:110–8. [24] Del’Guidice T, Nivet E, Escoffier G, Baril N, Caverni JP, Roman FS. Perseveration related to frontal lesion in mice using the olfactory H-maze. Behavioural Brain Research 2009;205:226–33. [25] Girard SD, Baranger K, Gauthier C, Jacquet M, Bernard A, Escoffier G, et al. Evidence for early cognitive impairment related to frontal cortex in the 5XFAD mouse model of Alzheimer’s disease. Journal of Alzheimer’s Disease 2013;33:781–96. [26] Kim C, Nam DW, Park SY, Song H, Hong HS, Boo JH, et al. O-linked beta-Nacetylglucosaminidase inhibitor attenuates beta-amyloid plaque and rescues memory impairment. Neurobiology of Aging 2013;34:275–85. [27] Rich EL, Shapiro M. Rat prefrontal cortical neurons selectively code strategy switches. The Journal of Neuroscience 2009;29:7208–19. [28] Rich EL, Shapiro ML. Prelimbic/infralimbic inactivation impairs memory for multiple task switches, but not flexible selection of familiar tasks. The Journal of Neuroscience 2007;27:4747–55.