Body weights
Analysis at 6 months
On two-way ANOVA of mice at 6 months of age, there was an overall difference between groups (F = 14.20, P < 0.0001) but no difference over time (F = 2.07, P > 0.05), and there was no interaction of the two factors (F = 0.07, P > 0.05). Then, the body weight of WT mice injected with saline was compared with that of WT mice injected with D-Ala2GIP. On two-way ANOVA, there was no difference between groups (F = 2.77, P > 0.05), no effect over time (F = 0.91, P > 0.05) and no interaction of the two variables (F = 0.02, P > 0.05). The same analysis was performed comparing the body weight of APP/PS1 mice injected with saline and the body weight of APP/PS1 mice injected with D-Ala2GIP. On two-way ANOVA there was no difference between groups (F = 1.27, P > 0.05), no effect over time (F = 0.0004, P > 0.05) and no interaction of the two variables (F = 0.03, P > 0.05). However, there was a difference in body weight between WT and APP/PS1 mice that were both injected with saline (F = 12.47, P < 0.0001), as well as between WT and APP/PS1 mice that were both injected with D-Ala2GIP (F = 30.40, P < 0.0001). No difference was found over time in the saline groups (F = 0.82, P > 0.05) and D-Ala2GIP groups (F = 1.32, P > 0.05). No effect of interaction of both group and time was shown between the saline groups (F = 0.03, P > 0.05), or between the D-Ala2GIP groups (F = 0.19, P > 0.05). These statistics show that APP/PS1 mice had a higher body weight compared to WT control mice, which was not influenced by the saline or D-Ala2GIP injection (Figure 2A).
Analysis at 12 months
On two-way ANOVA of mice at 12 months of age there was an overall difference between groups (F = 16.85, P < 0.0001) and over time (F = 7.11, P < 0.0001), but no interaction of the two factors (F = 0.10, P > 0.05). Then, the body weight of WT mice injected with saline was compared with the body weight of WT mice injected with D-Ala2GIP. On two-way ANOVA there was a slight difference between groups (F = 6.25, P < 0.05), with an effect over time (F = 5.60, P < 0.0001), but there was no interaction of the two variables (F = 0.09, P > 0.05). The same analysis was performed comparing the body weight of APP/PS1 mice injected with saline and the body weight of APP/PS1 mice injected with D-Ala2GIP. There was no difference between groups (F = 1.08, P > 0.05), and no effect of interaction of the two variables (F = 0.06, P > 0.05), but a slight effect of time was found (F = 2.70, P < 0.05). On two-way ANOVA there was a difference in body weight between WT and APP/PS1 mice that were both mice injected with saline (F = 26.79, P < 0.0001) over time (F = 4.79, P < 0.0001), as well as between WT and APP/PS1 mice that were both injected with D-Ala2GIP (F = 21.08, P < 0.0001) with a slight effect of time (F = 2.88, P < 0.05). No effect of interaction of groups and time was shown between the saline groups (F = 0.27, P > 0.05) or between the D-Ala2GIP groups (F = 0.05, P > 0.05). These statistics show that APP/PS1 mice had a higher body weight compared to WT control mice, and the D-Ala2GIP peptide slightly decreased the body weight when injected in WT mice. Moreover a decrease in body weight was observed over time for all groups (Figure 2B).
Blood glucose and plasma insulin levels
Analysis at 6 months
On two-way repeated measures ANOVA in mice at 6 months of age, there was a difference between groups in glucose concentrations (F = 4.08, P < 0.01), and over time (F = 7.23, P < 0.0001), and an influence of interaction between the two factors (F = 3.6, P < 0.0001) (Figure 3A). Bonferroni post hoc analysis revealed a significant increase in glucose concentrations in APP/PS1 mice injected with saline compared to WT mice injected with saline (P < 0.01) on day 34. Moreover, injection of D-Ala2GIP for 34 days increased blood glucose levels of APP/PS1 mice compared to WT mice (P < 0.0001). However, blood glucose levels stayed in a normal range (between 4 and 8 mM/l) for all groups over time. On two-way repeated measures ANOVA there was no difference between groups in plasma insulin levels (F = 1.75, P > 0.05), no difference over time (F = 1.91, P > 0.05), and no effect of interaction between the two factors (F = 0.96, P > 0.05) (Figure 3B).
Analysis at 12 months
On two way repeated measures ANOVA of mice at 12 months of age there was a slight difference between groups in glucose concentrations (F = 3.84, P < 0.05), and a difference over time (F = 7.14, P < 0.0001), which was not influenced by interaction between the two factors (F = 1.39, P > 0.05) (Figure 3C). Bonferroni post hoc analyses revealed a significant decrease of glucose concentrations in APP/PS1 mice injected with saline compared to WT mice injected with saline (P < 0.05) on day 21 and 36. However, blood glucose levels stayed in the normal range (between 4 and 8 mM/l) for all groups over time. On two-way repeated measures ANOVA there was no difference between groups in plasma insulin levels (F = 1.56, P > 0.05), no difference over time (F = 0.74, P > 0.05) and no effect of interaction for the two factors (F = 0.54, P p > 0.05) (Figure 3D).
Effect of D-Ala2GIP treatment on spontaneous behaviour of APP/PS1 mice and WT littermates
Analysis at 6 months
In the open-field task at 6 months of age, the spontaneous behaviour of WT and APP/PS1 mice injected with saline or D-Ala2GIP was similar. On one-way ANOVA there was no difference between groups in path length (F = 1.99, P > 0.05), number of lines crossed (F = 1.59, P > 0.05), speed (F = 1.99, P > 0.05), exploration levels (F = 0.29, P > 0.05), grooming events (F = 0.85, P > 0.05), or the ratio of time spent in the centre of the arena to the periphery (F = 1.29, P > 0.05) (data not shown).
Analysis at 12 months
In the open-field task at 12 months of age, the spontaneous behaviour of WT and APP/PS1 mice injected with saline or D-Ala2GIP was similar. On one-way ANOVA there was no difference between groups in path length (F = 1.14, P > 0.05), number of lines crossed (F = 0.94, P > 0.05), speed (F = 1.14, P > 0.05), exploration levels (F = 0.56, P > 0.05, grooming events (F = 0.21, P > 0.05), and the ratio of time spend in the centre of the arena to the periphery (F = 0.07, P > 0.05) (data not shown).
Effect of D-Ala2GIP treatment on object recognition memory of APP/PS1 mice and WT littermates
Analysis at 6 months
In the test trial, there was a difference (Student's paired t-test) in the recognition index (RI) of novel vs familiar objects for WT mice injected with saline (t = 2.28, P < 0.05) and with D-Ala2GIP (25 nmol/kg) (t = 2.37, P < 0.05), as well as for APP/PS1 mice injected with saline (t = 3.71, P < 0.01) and with D-Ala2GIP (t = 2.28, P < 0.05). On one-way ANOVA there was no difference between groups in the difference in score between the time spent exploring the novel and the familiar object (F = 0.27, P > 0.05). These results indicate that all groups had an intact object recognition memory at 6 months of age (Figure 4).
Analysis at 12 months
In the test trial, there was a difference (Student's paired t-test) in the RI of novel vs familiar objects for 12-month-old WT mice injected with saline (t = 2.07, P < 0.05) and with D-Ala2GIP (25 nmol/kg) (t = 2.51, P < 0.05), showing an intact object recognition memory in these groups. APP/PS1 mice injected with D-Ala2GIP spent significantly more time exploring the novel object than the familiar one (t = 2.80, P < 0.01), while APP/PS1 control mice injected with saline did not discriminate between the familiar and the novel task, as no difference was found in the time spent between them (t = 1.33, P > 0.05), reflecting an impairment in recognition memory for this group. On one-way ANOVA, there was no difference between groups in the difference in score between the time exploring the novel and the familiar object (F = 1.15, P > 0.05); even the APP/PS1 group injected with saline solution showed a trend towards reduction of the difference in score in comparison to the other groups but this difference was not significant (Figure 4).
Effect of D-Ala2GIP treatment on spatial learning and memory of APP/PS1 mice and WT littermates
Analysis at 6 months
During the acquisition task, all mice learned to locate the hidden escape platform. On two-way repeated measures ANOVA there was a decrease in escape latency across trials of training in the acquisition trial (trials: F = 11.47, P < 0.0001), but there was no difference between groups (F = 0.64, P > 0.05) and no interaction of training trials and groups (F = 0.86, P > 0.05). A decrease in path length was also found across training trials (F = 13.75, P < 0.0001), although no differences were detected between groups (F = 0.75, P > 0.05). There was no interaction of training trials and groups (F = 0.97, P > 0.05). On two-way repeated measures ANOVA there was a difference in swim speed over time (trials: F = 7.01, P < 0.0001) but not between groups (F = 1.96, P > 0.05). No effect was found for the interaction of both factors (F = 1.19, P > 0.05) (data not shown).
In the probe trial, on one-way ANOVA there was an effect of quadrant preference for WT mice injected with saline (F = 9.70, P < 0.0001), and D-Ala2GIP (F = 7.60, P < 0.0001), and the APP/PS1 mice injected with saline (F = 11.83, P < 0.0001) and D-Ala2GIP (F = 6.47, P < 0.01), indicating that all groups remembered the location of the escape platform. In the WT saline-injected group, Bonferroni post hoc analysis showed increased time of stay in the target quadrant (south-west, S-W) compared to the south-east (S-E) (P < 0.001), north-east (N-E) (P < 0.001) and north-west (N-W) (P < 0.01) quadrants. In the WT D-Ala2GIP-injected group, Bonferroni post hoc analysis showed increased time of stay in the target quadrant (S-W) compared to the S-E (P < 0.01), N-E (P < 0.01) and N-W (P < 0.001) quadrants. In the APP/PS1 saline-injected group, Bonferroni post hoc analyses showed increased time of stay in the target quadrant (S-W) compared to the S-E (P < 0.05), N-E (P < 0.001) and N-W (P < 0.05) quadrants. APP/PS1 mice injected with D-Ala2GIP spent more time in the target S-W quadrant compared to the other (P < 0.01) (data not shown).
The probe trial was further analysed using one-way ANOVA, to analyse the difference in time spent in the target quadrant between all groups. No significant difference was found between groups for this parameter (F = 0.37, P > 0.05). Moreover, there was no difference between groups in the time spent crossing the exact previous location of the platform (F = 0.50, P > 0.05).
During the reversal task, all mice learned the new location of the platform. On two-way repeated measures ANOVA there was a significant decrease in escape latency across trials of training in the acquisition trial (trials: F = 5.76, P < 0.0001). A difference in escape latency was also found between groups (F = 4.97, P < 0.01), which was not influenced by interaction of training trials and groups (F = 0.83, P > 0.05). Then, on two-way repeated measures ANOVA to compare the escape latency between WT injected with saline and those injected with D-Ala2GIP there was a difference over time (trials: F = 3.17, P < 0.0001), but not between groups (F = 2.44, P > 0.05), and there was no effect of interaction between both factors (F = 0.54, P > 0.05). The same analysis comparing the APP/PS1 group injected with saline and that injected with D-Ala2GIP showed a significant difference between groups in escape latency (F = 4.36, P < 0.05) and over time (F = 4.00, P < 0.0001), which was not influenced by interaction between factors (F = 0.82, P > 0.05). On two-way ANOVA there was a difference in escape latency between WT and APP/PS1 mice that were both injected with saline (F = 4.85, P < 0.05), as well as between WT and APP/PS1 mice that were both injected with D-Ala2GIP (F = 3.91, P < 0.05). A difference was also found over time for the saline (F = 2.73, P < 0.01) and D-Ala2GIP groups (F = 3.66, P < 0.0001). No effect of interaction of both group and time was shown between the saline groups (F = 1.18, P > 0.05), or between the D-Ala2GIP groups (F = 0.76, P > 0.05).
On two-way repeated measures ANOVA there was a significant decrease in path length across trials of training in the acquisition trial (trials: F = 4.28, P < 0.0001). A difference in escape latency was also found between groups (F = 6.54, P < 0.0001), which was not influenced by interaction of training trials and groups (F = 0.64, P > 0.05). Then, on two-way repeated measures ANOVA to compare the escape latency between WT injected with saline and D-Ala2GIP, there was a difference over time (trials: F = 16.06, P < 0.0001), but not between groups (F = 2.58, P > 0.05) and no effect of interaction between the two factors (F = 3.47, P > 0.05). The same analysis was conducted to compare the APP/PS1 group injected with saline and that injected with D-Ala2GIP, and this showed a significant difference in the escape latency training trials (F = 1.86, P < 0.05), but not between groups (F = 0.63, P > 0.05) and no effect of interaction between factors (F = 0.43, P > 0.05). On two-way ANOVA there was no difference in escape latency between WT and APP/PS1 mice that were both injected with saline (F = 9.7, P > 0.05), or between WT and APP/PS1 mice that were both injected with D-Ala2GIP (F = 8.74, P > 0.05). No difference was found over time for the saline groups (F = 1.93, P > 0.05) and the D-Ala2GIP groups (F = 2.80, P > 0.05). No effect of interaction of group and time was shown between the saline groups (F = 0.95, P > 0.05), or between the D-Ala2GIP groups (F = 0.65, P > 0.05). On two-way repeated measures ANOVA there was no difference between groups in swim speed over time, or interaction between the two factors (trials: F = 1.41, P > 0.05; groups: F = 0.61, P > 0.05; interaction: F = 1.07, P > 0.05).
In the reversal probe trial, on one-way ANOVA there was an effect of quadrant preference for WT mice injected with saline (F = 7.67, P < 0.0001), and D-Ala2GIP (F = 5.13, P < 0.01), and the APP/PS1 mice injected with saline (F = 4.76, P < 0.01) and D-Ala2GIP (F = 7.45, P < 0.0001), indicating that all groups remembered the location of the escape platform. In the WT saline-injected group, Bonferroni post hoc analysis showed increased time of stay in the target quadrant (N-W) compared to the N-E (P < 0.05) and S-E (P < 0.001) quadrants. In the WT D-Ala2GIP-injected group, Bonferroni post hoc analyses showed increased time of stay in the target quadrant (N-W) compared to the N-E (P < 0.01) and S-E (P < 0.05) quadrants. In the APP/PS1 saline-injected group, Bonferroni post hoc analysis showed increased time of stay in the target quadrant (N-W) compared to the others (P < 0.05). APP/PS1 mice injected with D-Ala2GIP spent more time in the target S-W quadrant than in the N-E (P < 0.05) and S-E (P < 0.001) and the S-W (P < 0.05) quadrants.
The reversal probe trial was further analysed using one-way ANOVA, to analyse the difference between all groups in the time spent in the target quadrant. No significant difference was found between groups for this parameter (F = 0.48, P > 0.05). Additionally, one-way ANOVA did not show any difference in the spent time crossing the exact previous location of the platform between groups (F = 0.83, P > 0.05).
Analysis at 12 months
During the acquisition task, on two-way repeated measures ANOVA there was a decrease in escape latency across trials of training in the acquisition trial (trials: F = 10.20, P < 0.0001), with a difference between groups (F = 5.92, P < 0.0001), which was not influenced by interaction of training trials and groups (F = 0.67, P > 0.05). Then, on two-way repeated measures ANOVA to compare the escape latency between WT injected with saline D-Ala2GIP we found a difference over time (trials: F = 4.20, P < 0.0001), and between groups (F = 9.29, P > 0.005), but no effect of interaction between the two factors (F = 0.79, P > 0.05). The same analysis between the APP/PS1 group injected with saline or with D-Ala2GIP and showed a significant difference in escape latency between groups (F = 6.15, P < 0.05) and over time (F = 6.86, P < 0.0001), which was not influenced by interaction between factors (F = 0.0.48, P > 0.05). On two-way ANOVA, there was no difference in escape latency between WT and APP/PS1 mice that were both injected with saline (F = 0.42, P > 0.05), as well as between WT and APP/PS1 mice both injected with D-Ala2GIP (F = 1.77, P > 0.05). However, a difference was found over time for the saline groups (F = 3.35, P < 0.0001) and D-Ala2GIP groups (F = 8.14, P < 0.0001). No effect of interaction of group and time was shown between the saline groups (F = 0.69, P > 0.05), or between the D-Ala2GIP groups (F = 0.45, P > 0.05).
A decrease in path length was also found across training trials (F = 17.60, P < 0.0001), and differences were detected between groups (F = 6.043, P < 0.0001). There was no interaction of training trials and groups (F = 0.73, P > 0.05). Then, on two-way repeated measures ANOVA to compare the path length between WT injected with saline or D-Ala2GIP there was a difference over time (trials: F = 8.48, P < 0.0001), but not between groups (F = 0.01, P < 0.05), and no effect of interaction between the two factors was detected (F = 0.96, P > 0.05). The same analysis between the APP/PS1 group injected with saline or D-Ala2GIP showed a significant difference in escape latency over trials (F = 9.60, P < 0.0001), but not between groups (F = 61.36, P > 0.05), and there was no effect of interaction between factors (F = 0.47, P > 0.05). On two-way ANOVA there was a difference in escape latency between WT and APP/PS1 mice that were both mice injected with saline (F = 4.99, P < 0.05), as well as between WT and APP/PS1 mice both injected with D-Ala2GIP (F = 1.77, P > 0.05). A difference was found over time for the saline groups (F = 9.12, P < 0.0001) and D-Ala2GIP groups (F = 9.28, P < 0.0001). No effect of interaction of group and time was shown between the saline groups (F = 0.88, P > 0.05), or between the D-Ala2GIP groups (F = 0.60, P > 0.05). On two-way repeated measures ANOVA there was a difference in swim speed over time (trials: F = 7.49, P < 0.0001) but not between groups (F = 2.30, P > 0.05). No effect was found for the interaction of the two factors (F = 0.55, P > 0.05) (data not shown).
In the probe trial, one-way ANOVA showed an effect of quadrant preference for WT mice injected with saline (F = 3.02, P < 0.05) or D-Ala2GIP (F = 12.06, P < 0.0001), and the APP/PS1 mice injected with D-Ala2GIP (F = 7.47, P < 0.0001). However, APP/PS1 mice injected with saline spent an equal amount of time in each quadrant of the pool (F = 0.16, P > 0.05), indicating that this group did not remember the location of the escape platform. In the WT saline-injected group, Bonferroni post hoc analysis showed increased time of stay in the target quadrant (S-W) compared to the N-W (P < 0.05) quadrant. In the WT D-Ala2GIP-injected group, Bonferroni post hoc analyses showed increased time of stay in the target quadrant (S-W) compared to the other quadrants (P < 0.001). In the APP/PS1 D-Ala2GIP-injected group, Bonferroni post hoc analyses showed increased time of stay in the target quadrant (S-W) compared to the S-E (P < 0.05), N-E (P < 0.001) and N-W (P < 0.01) quadrants. See Figure 5.
The probe trial was further analysed using one-way ANOVA of the difference between all groups in the time spent in the target quadrant (Figure 5). A significant difference between groups was found for this parameter (F = 3.31, P < 0.05). Bonferroni post hoc analysis showed a decrease in time spent in the target quadrant for the APP/PS1 group injected with saline compared to the WT group injected with D-Ala2GIP (P < 0.05). Moreover, on one-way ANOVA there was no difference in the time spent crossing the exact previous location of the platform between groups (F = 0.87, P > 0.05). However, the APP/PS1 control group showed a trend towards a reduction in the time spent in the platform location compared to the other groups.
During the reversal task, on two-way repeated measures ANOVA there was a significant decrease in escape latency across trials of training in the acquisition trial (trials: F = 4.39, P < 0.0001). A difference in escape latency was also found between groups (F = 7.69, P < 0.0001), which was not influenced by interaction of training trials and groups (F = 0.72, P > 0.05). Then, on two-way repeated measures ANOVA to compare the escape latency between WT injected with saline or D-Ala2GIP, there was a difference over time (trials: F = 3.00, P < 0.0001), and between groups (F = 19.83, P < 0.0001), which was not influenced by interaction between factors (F = 0.45, P > 0.05). The same analysis was conducted between the APP/PS1 group injected with saline or D-Ala2GIP and showed a significant difference in escape latency between trials (F = 1.19, P < 0.05), but not between groups (F = 3.21, P > 0.05), and no effect of interaction between both factors was detected (F = 1.28, P > 0.05). On two-way ANOVA there was no difference in escape latency between WT and APP/PS1 mice that were both injected with saline (F = 1.45, P < 0.05), or over time (F = 1.55, P > 0.05). Moreover, on two-way ANOVA there was no difference in escape latency between WT and APP/PS1 mice that were both injected with D-Ala2GIP (F = 1.56, P > 0.05), but a difference was found over time (F = 4.87, P < 0.0001). No effect of interaction of group and time was shown between the saline groups (F = 0.40, P > 0.05), or between the D-Ala2GIP groups (F = 0.45, P > 0.05).
On two-way repeated measures ANOVA there was a significant decrease in path length across trials of training in the acquisition trial (trials: F = 2.79, P < 0.01), but not between groups (F = 0.99, P > 0.05), and no effect of interaction of both factors was detected (F = 1.13, P > 0.05). On two-way repeated measures ANOVA there was no difference between groups in swim speed over time, or interaction between the two factors (trials: F = 0.46, P > 0.05; groups: F = 0.84, P > 0.05; interaction: F = 0.91, P > 0.05) (data not shown).
In the reversal probe trial, one-way ANOVA showed an effect of quadrant preference for WT mice injected with saline (F = 3.80, P < 0.05), or D-Ala2GIP (F = 5.41, P < 0.01), and the APP/PS1 mice injected with D-Ala2GIP (F = 7.39, P < 0.0001). However, APP/PS1 mice injected with saline spent an equal amount of time in each quadrant of the pool (F = 02.40, P > 0.05), indicating that this group did not remember the location of the escape platform. In the WT saline-injected group, Bonferroni post hoc analyses showed increased time of stay in the S-W quadrant compared to the N-E (P < 0.05) quadrant, indicating that this group did not remember the location of the escape platform. In the WT D-Ala2GIP-injected group, Bonferroni post hoc analyses showed increased time of stay in the target quadrant (N-W) compared to the S-E (P < 0.05) quadrant. The APP/PS1 D-Ala2GIP-injected with D-Ala2GIP spent more time in the target N-W quadrant compared to the N-E (P < 0.05) and S-E (P < 0.001) quadrants. See Figure 5b.
The reversal probe trial was further analysed using a one-way ANOVA of the difference between all groups in the time spent in the target quadrant (Figure 6). A significant difference between groups was found for this parameter (F = 3.27, P < 0.05). Moreover, one-way ANOVA showed a difference in the time spent crossing the exact previous location of the platform between groups (F = 3.42, P < 0.05). Both the WTand APP/PS1group injected with saline showed a trend towards a reduction in the time spent in the platform location compared to the other groups, but the difference was not significant. In the visible platform task, one-way ANOVA did not show any difference in escape latency between groups in visual acuity (F = 0.89, P > 0.05) (Figure 5b).
In summary, the drug treatment did not have any effects in mice that were 6 months old, at an age when the amyloid plaque load and the associated learning impairments had not yet developed. However, D-Ala2GIP improved memory in WT mice and rescued the cognitive decline of 12 month-old APP/PS1 mice in two different memory tasks.
Effect of D-Ala2GIP treatment on synaptic plasticity of APP/PS1 mice and WT littermates
Analysis at 6 months
The pre-HFS baselines of WT mice at 6 months of age were analysed by two-way repeated measures ANOVA (Figure 6a). No difference was found between the saline group and D-Ala2GIP group (F = 1.24, P > 0.05), and no effect of time (F = 0.74, P > 0.05) or interactive effect of group and time was determined (F = 0.61, P > 0.05), indicating that pre-HFF baselines were stable and similar between groups over time. On two-way repeated measures ANOVA there was a clear difference in post-HFS baselines between the WT group injected with saline and the WT group injected with D-Ala2GIP (groups: F = 289.3, P < 0.0001), but not over time (F = 0.63, P > 0.05) and no interactive effect of group and time was found (interaction: F = 0.93, P > 0.05). Altogether, these results indicate that HFS stimulation induced robust LTP in the WT D-Ala2GIP-injected mice compared to the control group. On two-way repeated measures ANOVA there was no difference between groups in paired-pulse facilitation (PPF) (F = 0.26, P > 0.05). Inter-stimulus delay (F = 0.57, P > 0.05) and interaction between both factors (F = 1.11, P > 0.05) were not significant.
The pre-HFS baselines of APP/PS1 mice at 6 months of age were analysed by two-way repeated measures ANOVA. No difference was found between the saline and D-Ala2GIP group (F = 0.00007, P > 0.05), and no effect of time (F = 0.94, P > 0.05) and no interactive effect of group and time was determined (F = 0.60, P > 0.05), indicating that pre-HFF baselines were stable and similar between groups over time. On two-way repeated measures ANOVA there was a clear difference of post-HFS baselines between the APP/PS1 group injected with saline and the APP/PS1 group injected with D-Ala2GIP (groups: F = 309.2, P < 0.0001), but not over time (F = 0.94, P > 0.05) and no interactive effect of group and time was found (interaction: F = 0.60, P > 0.05). Altogether, these results indicate that HFS stimulation induced robust LTP in the APP/PS1 D-Ala2GIP-injected mice compared to the control group. On two-way repeated measures ANOVA there was a difference between groups in PPF (F = 4.07, P < 0.05). Inter-stimulus delay (F = 0.69, P > 0.05) and interaction between the two factors (F = 0.58, P > 0.05) were not significant. See Figure 6a.
Analysis at 12 months
The pre-HFS baselines of WT mice at 12 months of age were analysed by two-way repeated measures ANOVA (Figure 6b). No difference was found between the saline and D-Ala2GIP group (F = 1.18, P > 0.05), but an effect of time was determined (F = 2.00, P < 0.01), which was not influenced by interaction of group and time (F = 0.57, P < 0.01), indicating that pre-HFF baselines were similar between groups. On two-way repeated measures ANOVA there was a clear difference of post-HFS baselines between the WT group injected with saline and the WT group injected with D-Ala2GIP (groups: F = 427.0, P < 0.0001), but not over time (F = 1.19, P > 0.05) and no interactive effect of group and time was found (interaction: F = 0.46, P > 0.05). Altogether these results indicate that HFS stimulation induced robust LTP in the WT D-Ala2GIP-injected mice compared to the control group. On two-way repeated measures ANOVA there was no difference between groups on PPF (F = 0.28, P > 0.05). Inter-stimulus delay (F = 1.41, P > 0.05) and interaction between both factors (F = 0.58, P > 0.05) were not significant.
The pre-HFS baselines of APP/PS1 mice at 12 months of age were analysed by two-way repeated measures ANOVA. No difference was found between the saline and the D-Ala2GIP group (F = 0.00001, P > 0.05), and no effect of time (F = 1.18, P > 0.05) and no interactive effect of group and time was determined (F = 1.18, P > 0.05), indicating that pre-HFF baselines were stable and similar between groups over time. On two-way repeated measures ANOVA there was a clear difference of post-HFS baselines between the APP/PS1 group injected with saline and the APP/PS1 group injected with D-Ala2GIP (groups: F = 234.1.2, P < 0.0001), but not over time (F = 0.54, P > 0.05) and no interactive effect of group and time was found (interaction: F = 0.26, P > 0.05). Altogether these results indicate that HFS stimulation induced robust LTP in the APP/PS1 D-Ala2GIP-injected mice compared to the control group. On two-way repeated measures ANOVA there was no difference between groups on PPF (F = 0.33, P > 0.05). Inter-stimulus delay (F = 0.39, P > 0.05) and interaction between the two factors (F = 0.16, P > 0.05) were not significant. See Figure 6b.
In conclusion, D-Ala2GIP facilitated synaptic plasticity in APP/PS1 and WT mice at all ages tested.
Effect of D-Ala2GIP treatment on cell proliferation and neurogenesis of APP/PS1 mice and WT littermates
Analysis at 6 months
On one-way ANOVA there was a difference between groups in BrdU-positive cells in the dentate gyrus (DG) (F = 12.90, P < 0.0001, Figure 7a). The Bonferroni post hoc test showed that the DG of WT mice injected with D-Ala2GIP contained significantly more BrdU-positive cells than the other groups (P < 0.001). However, on one-way ANOVA there was no difference between groups in the number of double-cortin-positive young neurons (F = 1.49, P > 0.05) (Figure 7b).
Analysis at 12 months
On one-way ANOVA there was no difference between groups in BrdU-positive cells in the DG at 12 months of age (F = 0.07, P > 0.05, Figure 8a). Moreover, there was no difference between groups in the number of double-cortin-positive young neurons (F = 2.11, P > 0.05) (Figure 8b).
Effect of D-Ala2GIP treatment on synaptic density of APP/PS1 mice and WT littermates
Analysis at 6 months
On one-way ANOVA there was a difference between groups in levels of expression of synaptophysin in the exterior cortex (layers 4 to 6) (F = 6.62, P < 0.0001) (Figure 9a) and interior cortex (layers 1 to 3) (F = 5.72, P < 0.01). The Bonferonni post hoc test showed that D-Ala2GIP treatment increased levels of synaptophysin in the interior cortex in APP/PS1 mice compared with the saline-treated APP/PS1 (P < 0.01), and in the exterior cortex compared with both saline-treated APP/PS1 and WT mice (P < 0.01).
On one-way ANOVA there was a difference between groups in levels of expression of synaptophysin in the polymorphic layer (hilus) of the DG (F = 4.95, P < 0.01), the molecular layer of the DG (F = 3.55, P < 0.05) and the stratum pyramidale (CA1) (F = 9.14, P < 0.0001). A decrease in synaptophysin levels was found in APP/PS1 mice compared to the WT control group in the polymorphic layer (hilus) (P < 0.05), and a decrease was found in the stratum pyramidale in the saline group (P < 0.001) and in the D-Ala2GIP group (P < 0.05). D-Ala2GIP treatment increased levels of synaptophysin in APP/PS1 mice compared with the saline-treated APP/PS1 mice in the polymorphic layer of the DG (P < 0.01), as well as in the molecular layer of the DG (P < 0.05). However, D-Ala2GIP treatment did not change synaptophysin expression in WT and APP/PS1 mice in the granular cell layer of the DG (F = 2.65, P > 0.05), in the stratum radiatum (F = 2.15, P > 0.05), or the stratum oriens (F = 1.24, P > 0.05). See Figure 9a.
Analysis at 12 months
On one-way ANOVA there was a difference between groups in levels of expression of synaptophysin in the exterior cortex (layers 406) (F = 10.52, P < 0.0001) and interior cortex (layers 1 to 3) (F = 6.84, P < 0.0001). The Bonferonni post hoc test showed a decrease of synaptophysin expression in the 12 month-old APP/PS1 mice compared to their respective WT control in the exterior cortex (P < 0.001 and P < 0.05). In the interior cortex, APP/PS1 mice treated with saline exhibited lower levels of synaptophysin expression than the WT group (P < 0.001 compared to the saline group, and P < 0.01 compared to the D-Ala2GIP group), while D-Ala2GIP treatment increased levels of synaptophysin expression in APP/PS1 mice compared with the saline-treated APP/PS1 (P < 0.05).
On one-way ANOVA there was a difference between groups in levels of expression of synaptophysin in the molecular layer of the DG (F = 8.71, P < 0.0001), the stratum radiatum (F = 9.39, P < 0.0001), the stratum pyramidale (F = 15.45, P < 0.0001), and the stratum oriens (F = 5.88, P < 0.01) in area CA1. A decrease in synaptophysin levels was found in saline-treated APP/PS1 mice compared to the saline-treated WT control group in the molecular layer and the stratum radiatum (P < 0.01) and the stratum pyramidal (P < 0.001). Moreover, a decrease of synaptophysin levels was also found in D-Ala2GIP-treated APP/PS1 mice compared to the D-Ala2GIP-treated WT control group in the stratum pyramidal (P < 0.01). However, D-Ala2GIP treatment increased levels of synaptophysin in APP/PS1 mice in the molecular layer of the DG (P < 0.05), in the stratum radiatum and stratum pyramidal (P < 0.01), as well as in the stratum oriens (P < 0.001). No change was detected in the polymorphic layer (hilus) (F = 0.76, P > 0.05) and in the granular cell layer of the DG (F = 1.97 P > 0.05) of WT and APP/PS1 mice. See Figure 9b.
Effect of D-Ala2GIP treatment on plaque formation and inflammation in the cortex of APP/PS1 mice
Analysis at 6 months
There was no difference between APP/PS1 mice injected with saline or D-Ala2GIP in the number of plaques in the cortex (t = 0.21, P > 0.05, Student's unpaired t- test). Additionally, D-Ala2GIP peptides showed no effect on the amount of stained area for Congo red (t = 1.30, P > 0.05). However, a slight decrease in activated microglia was found in the cortex of APP/PS1 mice treated with D-Ala2GIP compared to the saline group (t = 2.00, P < 0.05) (Figure 10a).
Analysis at 12 months
There was a significant decrease in the number of plaques (t = 2.41, P < 0.01) and a lower amount of Congo red stained area (t = 1.96, P > 0.05, Student's unpaired t- test) in the cortex of 12 month-old APP/PS1 mice that were chronically treated with D-Ala2GIP. Moreover, a marked decreased in the inflammation response shown in activated microglia was found in the cortex of D-Ala2GIP-treated mice (t = 3.89, P < 0.001) (Figure 10b).