神经损伤与修复
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Figure 1｜ (D-Ser2) Oxm improves working memory and influences theta rhythm in 9-month-old 3xTg mice.
To avoid the influence of stress on the following experiments, the Y-maze spontaneous alternation task was performed before the fear conditioning test. Mice have a propensity to enter a new arm to explore the relatively new environment in the maze (Kraeuter et al., 2019). Two-way ANOVA showed that the genotype and treatment had significant main effects (genotype: F(1, 36) = 46.83, P < 0.001; treatment: F(1, 36) = 9.018, P < 0.01) and an interaction effect (F(1, 36) = 21.42, P < 0.001). Tukey’s post hoc test showed that the correct alternation percentage was significantly decreased from 81.30 ± 2.37% in the WT + saline group to 58.00 ± 2.04% in the 3×Tg + saline group (P < 0.01; Figure 1A). (D-Ser2) Oxm treatment restored it to 73.50 ± 2.00% (P < 0.001, vs. 3×Tg + saline group), nearly equal to the level of the WT + saline group. The results suggested a neuroprotective effect of (D-Ser2) Oxm on short-term spatial memory. There was no significant difference between the groups in total arm entries (F(1, 36) = 0.3231, P > 0.05; Figure 1B). These data suggest that the effects of the APP/PS1/tau gene mutation and (D-Ser2) Oxm treatment were not due to a change in locomotor ability.
During the Y-maze task, we recorded changes in the theta rhythm in the four groups (in the central area before entering the right arm). The trend of the theta rhythms in mice entering the correct arm within 5 seconds in the Y-maze was shown in Figure 1C. Two-way ANOVA showed that the genotype and treatment had significant main effects (genotype: F(1, 36) = 20.16, P < 0.001; treatment: F(1, 36) = 3.435, P = 0.0721) and an interaction effect (F(1, 36) = 9.549, P < 0.05). Tukey’s post hoc test showed that the peak power of the theta rhythm was decreased in 3×Tg + saline mice compared with that in WT + saline mice (P < 0.001). However, the theta rhythm was improved after treatment with (D-Ser2) Oxm (P < 0.01, vs. 3×Tg + saline group; Figure 1C and D). The results showed that (D-Ser2) Oxm improved the short-term working memory and the associated abnormal theta rhythm of the AD model mice.Figure 2｜ (D-Ser2) Oxm relieves fear memory impairment in 9-month-old 3xTg mice.
After the Y-maze experiment, the fear conditioning test was performed. On the first day of fear learning training, as shown in Figure 2A and B, the freezing ratio increased gradually with the increase of conditioning in all four groups. There were no significant differences in the freezing ratios between the four groups at the beginning or the end of the training (P > 0.05), indicating that the APP/PS1/tau gene mutation and (D-Ser2) Oxm had no effect on the fear learning process. During the testing sessions, the freezing ratio induced by the CS in 3×Tg + saline mice was significantly lower than that in WT + saline mice (P < 0.001; Figure 2C and D). The freezing ratio was improved after (D-Ser2) Oxm treatment (P < 0.001, vs. 3×Tg + saline group). The above experiments showed that the fear memory of 3×Tg mice was significantly impaired, and was significantly improved after treatment with (D-Ser2) Oxm.
Figure 3｜(D-Ser2) Oxm increases the release of the theta waveform, pressure perception curve, and corresponding time-frequency heat map of 9-month-old 3xTg mice between the pre-CS and CS stages.
Figure 3 shows the typical original theta rhythm waveforms, pressure perception curves, and corresponding time-frequency thermograms of the four groups during the testing phase. According to the pressure trace curve, the WT mice were more active in the pre-CS stage, and their activity significantly decreased after CS. The 3×Tg mice showed decreased activity in the CS stage in comparison to the pre-CS stage, and their freezing behavior was decreased compared with that of WT mice after CS. The hippocampal theta rhythm curve and time-frequency heat map also showed corresponding differences in 3×Tg mice. The freezing state and the power of theta rhythms were improved after treatment with (D-Ser2) Oxm.
Figure 4｜(D-Ser2) Oxm increases the theta power in the hippocampal CA1 of 9-month-old 3xTg mice.
Figure 4A shows the typical peak frequency of the four groups during the testing phase. Figure 4B and C show the peak frequency and peak power of theta rhythms in the hippocampal CA1 region of mice in each group before and after conditioned stimulation during the fear conditioning test, respectively. The frequency of the theta rhythm in WT + saline mice in the CS stage was higher than that in the pre-CS stage, whereas the theta power in 3×Tg + saline mice was lower than that in WT+ saline mice in the CS phase (P < 0.001), which indicated that the fear memory impairment of 3×Tg mice in the CS stage was accompanied by decreased theta power in the hippocampal CA1 region. After (D-Ser2) Oxm treatment, the peak value and frequency of 3×Tg mice in the CS phase were recovered (both P < 0.001, vs. 3×Tg + saline group). These findings suggested that the fear memory impairment of 3×Tg mice in the CS stage, along with the decreased theta emission power in the hippocampal CA1 region, were improved by (D-Ser2) Oxm treatment.
Figure 5｜(D-Ser2) Oxm increases the density of dendritic spines and the level of synaptic proteins in the hippocampal CA1 region of 9-month-old 3xTg mice.
The synaptic plasticity of the hippocampal CA1 area plays a crucial role in AD cognitive function (Auffret et al., 2009). The above findings demonstrated that (D-Ser2) Oxm improves the working memory and fear memory of 3×Tg mice and the accompanying abnormalities of the theta rhythm. Next, we used Golgi staining to determine whether (D-Ser2) Oxm improves the density of dendritic spines in 3×Tg mice. Two-way ANOVA showed that genotype and treatment had significant main effects on spine density (genotype: F(1, 20) = 128, P < 0.001; treatment: F(1, 20) = 55.28, P < 0.001; genotype × treatment: F(1, 20) = 67.52, P < 0.001). Dendritic spine density in the hippocampal CA1 region of 3×Tg mice was significantly reduced compared with that in the control group (P < 0.001), and dendritic spine density was increased in the 3×Tg + Oxm group compared with that in the 3×Tg + saline group (P < 0.001; Figure 5A and B).
PSD-95 and SYP are involved in the regulation of synaptic activity and plasticity (Liu et al., 2018). Two-way ANOVA showed that genotype and treatment had significant main effects on the level of both PSD-95 (genotype: F(1, 20) = 128, P < 0.001; treatment: F(1, 20) = 55.28, P < 0.001; genotype × treatment: F(1, 20) = 67.52, P < 0.001) and SYP (genotype: F(1, 20) = 62.84, P < 0.001; treatment: F(1, 20) = 30.24, P < 0.001; genotype × treatment: F(1, 20) = 13.34, P < 0.05) in the hippocampal CA1 region. As shown in Figure 5C–E, the protein expression levels of PSD-95 and SYP in the hippocampal CA1 region of mice in the 3×Tg + saline group were significantly decreased compared with those in the WT + saline group. Treatment with (D-Ser2) Oxm increased the level of hippocampal synaptic proteins in 3×Tg mice (P < 0.001 for each comparison, vs. 3×Tg + saline group).