A condensed summary of the outcomes of the behavioral experiments and the brain structures believed to be involved in each task is shown in .
Summary of behavioral experiments, statistical data, interpretation, and involved brain regions
Exploratory behavior in a novel environment and general locomotor activity were assessed in automated activity chambers for 10 min (). Median tracks of Thy1-hAPPLond/Swe+ and control littermates are shown in . A minute-to-minute analysis revealed that Thy1-hAPPLond/Swe+ mice consistently moved a longer distance than their control littermates (; effect of genotype, F1, 21 = 17.54, P = 0.0004; genotype × time interaction, F9, 189 = 0.93, P = 0.50). Accordingly, the total (cumulative) distance moved in the novel environment was significantly higher in Thy1-hAPPLond/Swe+ than in control mice (; P = 0.0016). Both groups of mice showed higher activity in the perhipheral zone than in the central zone both in terms of the distance moved (; effect of zone, F1, 21 = 59.25, P < 0.0001) and the time spent in the two zones (; effect of zone, F1, 21 = 140.3, P < 0.0001). Thy1-hAPPLond/Swe+ mice tended to show more activity in the peripheral zone than the control mice; however, the genotype × zone interaction did not achieve statistical significance for either distance moved (genotype × zone interaction, F1, 21 = 2.33, P = 0.14) or time spent in zones (genotype × zone interaction: F1, 21 = 2.82, P = 0.11). Thy1-hAPPLond/Swe+ mice engaged in significantly more rearing behavior than their control littermates (, 1g; effect of genotype, F1, 21 = 4.68, P = 0.042).
Figure 1 Activity chamber. (a) Activity was monitored for 10 min in the activity chamber (upper panel). Display of tracks of median Thy1-hAPPLond/Swe+ and control mouse (lower panels). (b) Thy1-hAPPLond/Swe+ traveled a longer distance than control mice (P = 0.0004, (more ...)
The open-field test was used for assessment of gross locomotor activity and exploration behavior in a relatively large novel environment as compared to the activity chamber (). Thy1-hAPPLond/Swe+ mice moved a longer distance in the open field compared with control animals ( and 2c; effect of genotype, F1, 21 = 9.10, P = 0.007; genotype × time interaction, F9, 189 = 0.80, P = 0.61) and showed a significantly increased velocity (control: 9.26 ± 0.24 cm/s; mutant: 11.03 ± 0.35 cm/s; P = 0.006). Both genotypes moved a longer distance in the periphery zone than the center zone (; effect of zone, F1, 21 = 934.6, P < 0.0001), but the effect of zone was more pronounced in the Thy1-hAPPLond/Swe+ mice (genotype × zone interaction, F1, 21 = 10.62, P = 0.004). Mice spent more time in the center zone (; effect of zone, F1, 21 = 3064.92, P < 0.0001) but this effect was not different between genotypes (genotype × zone interaction, F1, 21 = 1.21, P = 0.28).
Figure 2 Open field. (a) Activity levels were measured in a 10-min open field test (upper panel). Tracks of median Thy1-hAPPLond/Swe+ and control mice are displayed in the lower panels. (b) Thy1-hAPPLond/Swe+ mice traveled a significantly longer distance than (more ...)
Social behavior was assessed with the three-chamber and six-trial social memory tests (). In the three-chamber test, a subject mouse was first habituated to the test environment in a habituation session, then tested for sociability in a sociability session, and finally tested for preference for social novelty in a social novelty session (). No side preference was detected during the habituation session (data not shown). During the sociability test (), both Thy1-hAPPLond/Swe+ and control mice preferred to sniff at a cage containing a stranger mouse versus sniffing at an empty cage (; effect of object, F1, 16 = 34.64, P < 0.0001), and this preference did not differ by genotype (genotype × object interaction, F1, 16 = 0.31, P = 0.58). Calculating a preference index (ratio of time sniffing stranger 1 vs. empty cage) showed no difference between genotypes (P = 0.1). During the subsequent social novelty test, control mice seemed to spend more time sniffing the novel stranger's cage than the now-familiar mouse's cage whereas Thy1-hAPPLond/Swe+ mice did not demonstrate such a preference (). A two-way ANOVA showed a trend close to significance for the object effect (F1, 18 = 4.01, P = 0.06) and genotype × object interaction (F1, 18 = 4.20, P = 0.055). However, the preference index (ratio of time sniffing stranger 2 vs. stranger 1) revealed a significantly decreased preference of mutant mice for the novel stranger's cage (; P = 0.031). Significance level was also reached when two outliers (33 for control mice and 3.5 for mutant mice) were excluded (P = 0.009). In the six-trial social memory test, we found a significant habituation to the SAME intruder (; trial 1–4: effect of object, F3, 75 = 5.69, P = 0.0014) and this effect did not differ by genotype (genotype × object interaction, F3, 75 = 0.33, P = 0.81). Furthermore, we found a significant dishabituation with the presentation of a NOVEL intruder (trial 4–5: effect of object, F1, 25 = 49.73, P < 0.0001, genotype × object interaction, F1, 25 = 0.09, P = 0.77) and a significant effect of an additional presentation of the SAME intruder in trial 6 (trial 5–6: effect of object, F1, 25 = 71.75, P < 0.0001, genotype × object interaction, F1, 25 = 1.22, P = 0.28). No significant differences in genotype × object interactions were detected.
Figure 3 Social behavior. (a) Three-chamber test. After a 10-min habituation to a three-chambered box, an empty cup and a cup containing stranger 1 were introduced in the side chambers for a 10-min sociability session. Thereafter, stranger 2 was added under the (more ...)
The T-maze and Y-maze were used to assess spontaneous alternation and spatial working memory. Thy1-hAPPLond/Swe+ mice showed a deficit in spontaneous alternation both in the T-maze (; P = 0.026) and the Y-maze (; P = 0.04). However, no difference was revealed between genotypes in the number of entries made in the Y-maze (; P = 0.95).
Figure 4 T-maze and Y-maze. (a) T-maze: Thy1-hAPPLond/Swe+ showed significantly (P < 0.05, t-test) less spontaneous alterations than control mice. n(each genotype) = 12. (b) Y-maze: Thy1-hAPPLond/Swe+ showed significantly (P < 0.05, t-test) less (more ...)
Morris water maze
During the training phase of the MWM, both Thy1-hAPPLond/Swe+ and control mice acquired the location of the hidden platform equally well, as indicated by a significant effect of training day on escape latency ( and b; effect of day, F3, 63 = 44.92, P < 0.0001; genotype × day interaction, F3, 63 = 1.21, P = 0.32) and distance moved to find the hidden platform (; effect of day, F3, 63 = 26.62, P < 0.0001; genotype × day interaction, F3, 63 = 0.85, P = 0.47) and a lack of a genotype × day interaction in both cases. For velocity, we did not find a genotype difference (; effect of genotype, P = 0.34). During the probe trial, Thy1-hAPPLond/Swe+ mice and control littermates spent significantly more time in the target quadrant, indicating normal memory retrieval (; effect of genotype, F1, 21 = 0.56, P = 0.462; effect of quadrant, F3, 63 = 19.05, P < 0.0001; genotype × quadrant interaction, F3, 63 = 0.19, P = 0.90). No genotype difference was revealed in the visible platform test (; effect of genotype, F1, 21 = 1.99, P = 0.173; effect of trial, F3, 63 = 5.65, P = 0.0017; genotype × trial interaction, F3, 63 = 0.71, P = 0.55).
Figure 5 Morris water maze. (a) Display of median tracks of Thy1-hAPPLond/Swe+ and control mice during trial 16. Acquisition of the hidden platform location did not differ between genotypes as shown by escape latency (b), and distance moved (c). (d) For velocity, (more ...)
DMP dry maze
Since Thy1-hAPPLond/Swe+ mice did not display a deficit in the water maze tests, we developed a new DMP task using a dry maze (modified Barnes maze) in an attempt to enhance detection of deficits by eliminating the water and swimming factor (). Before testing Thy1-hAPPLond/Swe+ and their control littermates, a validation experiment was conducted using C57BL/6J mice and scopolamine to induce experimental memory impairment. Scopolamine-injected mice exhibited decreased learning as indicated by a significant trial effect for escape latency in combination with a significant treatment × trial interaction (; effect of treatment, F1, 18 = 51.58, P < 0.0001; effect of trial, F15, 270 = 10.00, P < 0.0001; treatment × trial interaction, F15, 270 = 3.15, P < 0.0001). Calculation of the averages of escape latencies per trial confirmed this finding (; effect of treatment, F1, 18 = 51.58, P < 0.0001; effect of trial, F3, 54 = 31.30, P < 0.0001; treatment × trial interaction, F3, 45 = 9.9, P < 0.0001). Both the trial effect and treatment × trial interaction of the escape distance were significant (data not shown). The savings between the first and second trials (T1-T2 savings) and the first and fourth trials (T1-T4 savings) were significantly lower in scopolamine-treated versus vehicle-treated mice (; P = 0.0019 for T1-T2 savings and P = 0.0003 for T1-T4 savings).
Figure 6 Delayed-matching-to-place dry maze. (a) On a circular platform, mice were given four trials over four to five days to find an escape box along three rings of escape holes. (b) Scopolamine-treated mice showed a significantly altered escape latency to find (more ...)
Thy1-hAPPLond/Swe+ mice exhibited a deficit in acquisition of the DMP dry maze task compared to control mice as supported by a significant trial effect on escape latency in combination with significant genotype × trial interaction (; effect of genotype, F1, 18 = 15.72, P = 0.0009; effect of trial, F19, 342 = 14.08, P < 0.0001; genotype × trial interaction, F19, 342 = 2.49, P = 0.0006). Calculation of the trial average of escape latencies revealed the same overall effect (; effect of genotype, F1, 18 = 14.57, P = 0.0013; effect of trial, F3, 54 = 34.06, P < 0.0001; genotype × trial interaction, F3, 45 = 3.93, P = 0.01). A similar trend (but not statistically significant) was detected in both the trial effect and genotype × trial interaction of the escape distances (data not shown). The T1-T2 savings was significantly lower in the Thy1-hAPPLond/Swe+ mice than in their control littermates (; P = 0.037). A trend in the same direction was found for the T1-T4 savings (; P = 0.053).
Tone-cued and contextual FC was used for evaluation of conditional learning and memory. Both genotypes acquired the task equally well as shown by a significant time effect on freezing and a lack of a genotype × time interaction (; effect of genotype, F1, 21 = 3.73, P = 0.067; effect of time F5, 105 = 54.76, P < 0.0001; genotype × time is interaction, F5, 105 = 1.00, P = 0.42). For tone freezing, we found a significant time effect but no significant genotype × time effect (; effect of genotype, F1, 21 = 4.92, P = 0.038; effect of ITIs F4, 84 = 28.13, P < 0.0001; genotype × ITIs is interaction, F4, 84 = 1.64, P = 0.17). Still, a significant overall genotype effect has to be accounted for. In the tone-cued FC test in a novel context, no differences were revealed between genotypes (; P = 0.735). Importantly, freezing during the tone presentation on day 2 was not lower in mutant mice than control mice (data not shown). However, Thy1-hAPPLond/Swe+ mice showed a significant deficit in the contextual memory retrieval test as shown by a significantly decreased freezing behavior (; P = 0.006).
Figure 7 Fear conditioning (FC). (a) Both genotypes acquired the FC task without significant statistical difference in freezing. (b) Mutant mice, however, froze less in response to the tone alone. (c) No freezing differences between genotypes were apparent for (more ...)
Hot plate test
Sensitivity to a painful stimulus (nociception) was assessed using the hot plate test. No difference in latency of reaction to the hot surface was found between Thy1-hAPPLond/Swe+ and control littermates, suggesting no difference in responsiveness to aversive stimuli between the transgenic and control animals (P = 0.068, data not shown).