Treadmill Exercise Prevents Decline in Spatial Learning And Memory in 3×Tg-AD Mice Through Enhancement Of Structural Synaptic Plasticity Of The Hippocampus And Prefrontal Cortex Part 3

Synaptic curvature and the thickness of postsynaptic density are closely correlated with postsynaptic information integration and neurotransmitter transmission efficiency [39]. 

Synaptic curvature refers to the degree of curvature of the synaptic area connecting neurons, which is closely related to human memory.

On the one hand, synaptic curvature is one of the important factors in the connection between neurons. When the curvature of the synapse is appropriate, it can enhance the connection between different neurons and promote the information transmission of neurons, thereby improving human cognition and memory.

On the other hand, studies have shown that frequent thinking, learning new knowledge, and maintaining social activities can increase synaptic curvature, thereby promoting the connection and information transmission between neurons. These activities can promote the formation of more connections between neurons and fix these connections, thereby helping people maintain better memory.

Therefore, there is a close relationship between human memory and synaptic curvature. By being proactive, learning new knowledge, and participating in various social activities, synaptic curvature can be increased, the connection between neurons can be strengthened, and thus personal memory can be improved. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because it has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche can also promote the growth and repair of nerve cells, thereby enhancing the connectivity and function of neural networks. These effects can help improve memory, learning ability, and thinking speed, and can also prevent the occurrence of cognitive dysfunction and neurodegenerative diseases.

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Two-way ANOVA showed that genotype and treadmill exercise had significant effects on the synaptic curvature both in the hippocampus (genotype: F1,56 = 17.4, p < 0.001; treadmill exercise: F1,56 = 5.0, p = 0.030; genotype × treadmill exercise interaction: F1,56 = 6.8, p = 0.012; Figure 3F) and prefrontal cortex (genotype: F1,53 = 5.1, p = 0.029; treadmill exercise: F1,53 = 5.3, p = 0.026; genotype × treadmill exercise interaction: F1,53 = 7.0, p = 0.011; Figure 3G). Tukey's post hoc tests indicated that the synaptic curvature of the hippocampus (p < 0.001; Figure 3F) and prefrontal cortex (p = 0.001; Figure 3G) was significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group. 

Treadmill exercise pretreatment increased the synaptic curvature both in the hippocampus (p = 0.001; Figure 3F) and prefrontal cortex (p < 0.001; Figure 3G) in 3×Tg-AD mice. 

Meanwhile, two-way ANOVA indicated that genotype and treadmill exercise had significant effects on the thickness of postsynaptic density, both in the hippocampus (genotype: F1,56 = 26.3, p < 0.001; treadmill exercise: F1,56 = 8.0, p = 0.007; genotype × treadmill exercise interaction: F1,56 = 4.1, p = 0.047; Figure 3H) and prefrontal cortex (genotype: F1,55 = 34.2, p < 0.001; treadmill exercise: F1,55 = 11.1, p = 0.002; genotype × treadmill exercise interaction: F1,55 = 4.9, p = 0.032; Figure 3I). 

Tukey's post hoc tests indicated that the thickness of postsynaptic density of the hippocampus and prefrontal cortex was significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group (p < 0.001; Figure 3F, I). 

Treadmill exercise pretreatment increased the thickness of postsynaptic density both in the hippocampus (p = 0.001; Figure 3F) and prefrontal cortex (p < 0.001; Figure 3I) in 3×Tg-AD mice.

3.3. Effects of Treadmill Exercise on the Expression of Synaptophysin (Syn) and PSD95 of the Hippocampus and Prefrontal Cortex in 3×Tg-AD Mice

Syn is a specific presynaptic marker and PSD-95 is a specific postsynaptic marker for excitatory synapses [53,54]. 

We further investigated the expression of Syn and PSD95 in the hippocampus and prefrontal cortex in these four groups of mice (Figure 4A). For the hippocampus, two-way ANOVA indicated that genotype and treadmill exercise had significant main effects on the expression of Syn (genotype: F1,23 = 5.5, p = 0.029; treadmill exercise: F1,23 = 14.9, p < 0.001; Figure 4B), but there was not a significant interaction between genotype and treadmill exercise on the expression of Syn (genotype × treadmill exercise interaction: F1,23 = 1.2, p = 0.292; Figure 4D). 

For the prefrontal cortex, two-way ANOVA indicated that genotype had no significant main effect on the expression of Syn (genotype: F1,23 = 1.2, p = 0.278), but there was a significant interaction between genotype and treadmill exercise on the expression of Syn (treadmill exercise: F1,23 = 11.9, p = 0.002; genotype × treadmill exercise interaction: F1,23 = 11.2, p = 0.003; Figure 4C). 

Tukey's post hoc tests indicated that the expression of Syn of the hippocampus (p = 0.025; Figure 4B) and prefrontal cortex (p = 0.005; Figure 4C) was significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group. 

Treadmill exercise pretreatment increased the expression of Syn both in the hippocampus (p = 0.002; Figure 4B) and prefrontal cortex (p < 0.001; Figure 4C) in 3×Tg-AD mice. 

For PSD95 expression, two-way ANOVA revealed that genotype and treadmill exercise had no significant effects on the expression of PSD95 both in the hippocampus (genotype: F1,23 = 3.3, p = 0.082; treadmill exercise: F1,23 = 7.5, p = 0.013; genotype × treadmill exercise interaction: F1,23 = 0.9, p = 0.344; Figure 4D) and prefrontal cortex (genotype: F1,23 = 0.4, p = 0.514; treadmill exercise: F1,23 = 15.2, p < 0.001; genotype × treadmill exercise interaction: F1,23 = 3.3, p = 0.083; Figure 4E). The original Western blots are provided in Supplementary Figure S1.

Figure 4. Treadmill exercise facilitates the expression of Syn and PSD95 of the hippocampus and prefrontal cortex in 3×Tg-AD mice. (A) Representative western blots for Syn, PSD95, and GAPDH of the hippocampus and prefrontal cortex homogenates were prepared from these four groups of mice. (B, C) 

Summarized data showed that Syn of the hippocampus (B; * p < 0.05, n = 6) and prefrontal cortex (C; ** p < 0.01, n = 6) were significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group and this decrease was blocked by treadmill exercise pretreatment both in the hippocampus (B) and prefrontal cortex (C) (** p < 0.01, n = 6). (D, E) 

There were no significant effects on the PSD95 between non-Tg control, non-Tg exercise, 3×Tg-AD control, and 3×Tg-AD exercise mice in the hippocampus (D; p > 0.05, n = 6) and prefrontal cortex (E; p > 0.05, n = 6). 

Immunoreactivity was normalized to GAPDH and presented as the percentage of the non-Tg control group. Each data set was obtained from 3 mice.

3.4. Treadmill Exercise Enhanced the Axon Length and Dendritic Complexity of the Hippocampus and Prefrontal Cortex in 3×Tg-AD Mice

Axons and dendrites represent the structural basis of synaptic plasticity. We next assessed the axon length and dendritic complexity of the hippocampus and prefrontal cortex in 3×Tg-AD mice by Golgi staining (Figure 5A). 

Two-way ANOVA revealed that genotype and treadmill exercise had significant effects on the axon length of the hippocampus (genotype: F1,45 = 43.6, p < 0.001; treadmill exercise: F1,45 = 25.7, p < 0.001; genotype × treadmill exercise interaction: F1,45 = 4.1, p = 0.049; Figure 5B), the prefrontal cortex (genotype: F1,43 = 41.6, p < 0.001; treadmill exercise: F1,43 = 28.0, p < 0.001; genotype × treadmill exercise interaction: F1,43 = 5.4, p = 0.025; Figure 5C), and the dendritic complexity of the hippocampus (genotype: F1,36 = 166.8, p < 0.001; treadmill exercise: F1,36 = 57.5, p < 0.001; genotype × treadmill exercise interaction: F1,36 = 5.2, p = 0.029; Figure 5D) and the prefrontal cortex (genotype: F1,42 = 52.3, p < 0.001; treadmill exercise: F1,42 = 62.6, p < 0.001; genotype × treadmill exercise interaction: F1,42 = 5.7, p = 0.022; Figure 5E). 

Tukey's post hoc tests indicated that the axon length and dendritic complexity of the hippocampus and prefrontal cortex were significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group (p < 0.001; Figure 5B–E). 

Treadmill exercise increased the axon length and dendritic complexity in the hippocampus and prefrontal cortex in 3×Tg-AD mice (p < 0.001; Figure 5B–E). Meanwhile, treadmill exercise increased the axon length and dendritic complexity in the hippocampus and prefrontal cortex in non-Tg mice (p < 0.05; Figure 5B–E).

Figure 5. Treadmill exercise enhances the axon length and dendritic complexity of the Hippocampus and prefrontal cortex in 3×Tg-AD mice. (A) Representative Golgi staining images of the hippocampus and prefrontal cortex in non-Tg control, non-Tg exercise, 3×Tg-AD control, and 3×Tg-AD exercise mice. (B, C) Axon length of the hippocampus (B) and prefrontal cortex (C) were significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group (** p < 0.01, n = 9–13 image sections, 3–5 cells/image section) and this decrease was blocked by treadmill exercise pretreatment both in the hippocampus (B) and prefrontal cortex (C) (** p < 0.01, n = 9–13). 

Treadmill exercise increased the axon length in the hippocampus (B) and prefrontal cortex (C) in non-Tg mice (* p < 0.05, n = 9–13 image sections). (D, E) Dendritic complexity of the hippocampus (D) and prefrontal cortex (E) were significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group (** p < 0.01, n = 9–13 image sections), and this decrease was blocked by treadmill exercise pretreatment both in the hippocampus (D) and prefrontal cortex (E) (** p < 0.01, n = 9–13). 

Treadmill exercise increased the dendritic complexity in the hippocampus (D) and prefrontal cortex (E) in non-Tg mice (* p < 0.05, n = 9–13 image sections). Each data set was obtained from 3 mice.

3.5. Treadmill Exercise Improved the Numbers of Dendritic Spines of the Hippocampus and Prefrontal Cortex in 3×Tg-AD Mice

Dendritic spines are the original sites of neuronal excitatory synaptic transmission, and their morphology and structure are dynamic both under normal conditions in vivo and under conditions of synaptic plasticity [55]. 

Previous studies have shown that the dynamics of dendritic spines are associated with learning and memory, while thin, mushroom, and stubby spines have different roles in learning and memory [56]. 

We further analyzed the dendritic spine numbers of the secondary dendrites and categorized them into three types (thin, mushroom, and stubby) based on the morphological characteristics of the spine head, neck, and length (see Section 2, Figure 6A). 

Two-way ANOVA revealed that genotype and treadmill exercise had significant effects on the numbers of spines both in the hippocampus (genotype: F1,31 = 140.0, p < 0.001; treadmill exercise: F1,31 = 36.8, p < 0.001; genotype × treadmill exercise interaction: F1,31 = 9.4, p = 0.005; Figure 6B) and prefrontal cortex (genotype: F1,31 = 89.4, p < 0.001; treadmill exercise: F1,31 = 31.5, p < 0.001; genotype × treadmill exercise interaction: F1,31 = 6.5, p = 0.017; Figure 6C). 

Tukey's post hoc tests indicated that the numbers of spines of the hippocampus and prefrontal cortex were significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group (p < 0.001; Figure 6B, C). 

Treadmill exercise pretreatment prevented the decrease in the number of spines both in the hippocampus and prefrontal cortex in 3×Tg-AD mice (p < 0.001; Figure 6B, C). Meanwhile, treadmill exercise increased the number of spines of the hippocampus (p = 0.043; Figure 6B) and prefrontal cortex (p = 0.039; Figure 6C) in non-Tg mice. 

Furthermore, the number of spines of the hippocampus and prefrontal cortex were significantly decreased in the 3×Tg-AD exercise group compared to the non-Tg control group (p < 0.001; Figure 6B, C). 

Spines are considered thin if the length is greater than the neck diameter and the diameters of the head and neck are similar [47]. Thin spines emerge and disappear over a few days and concentrate biochemical signals such as Ca2+, providing the synaptic specificity required for learning [56,57]. 

Two-way ANOVA revealed that genotype and treadmill exercise had significant effects on the thin spines both in the hippocampus (genotype: F1,31 = 74.3, p < 0.001; treadmill exercise: F1,31 = 39.4, p < 0.001; genotype × treadmill exercise interaction: F1,31 = 3.5, p = 0.073; Figure 6D) and prefrontal cortex (genotype: F1,31 = 39.3, p < 0.001; treadmill exercise: F1,31 = 27.4, p < 0.001; genotype × treadmill exercise interaction: F1,31 = 4.4, p = 0.044; Figure 6E). Tukey's post hoc tests indicated that the thin spines of the hippocampus and prefrontal cortex were significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group (p < 0.001; Figure 6D, E). 

Treadmill exercise pretreatment blocked the decrease in the number of spines both in the hippocampus and prefrontal cortex in 3×Tg-AD mice (p < 0.001; Figure 6D, E). 

Meanwhile, treadmill exercise increased the number of spines of the hippocampus (p = 0.004; Figure 6D) and prefrontal cortex (p = 0.036; Figure 6E) in non-Tg mice.

Figure 6. Treadmill exercise improves the dendritic spine numbers of the hippocampus and prefrontal cortex in 3×Tg-AD mice. (A) Representative Golgi staining images of the secondary dendrites of the CA1 pyramidal neurons in the hippocampus and layer V pyramidal neurons prefrontal cortex in non-Tg control, non-Tg exercise, 3×Tg-AD control, and 3×Tg-AD exercise mice. 

The dendritic spines in rectangles, triangles, and circles are thin, mushroom, and stubby dendritic spines, respectively. (B, C) The spines numbers of the hippocampus (B) and prefrontal cortex (C) were significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group (*** p < 0.001, n = 8), and this decrease was blocked by treadmill exercise pretreatment both in the hippocampus (B) and prefrontal cortex (C) (*** p < 0.001, n = 8 dendrites, respectively). 

Treadmill exercise pretreatment increased the spine numbers of the hippocampus (B) and prefrontal cortex (C) in non-Tg mice (** p < 0.01, n = 8 dendrites, respectively). (D, E) 

The thin spines of the hippocampus (D) and prefrontal cortex (E) were significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group (*** p < 0.001, n = 8), and this decrease was blocked by treadmill exercise pretreatment both in the hippocampus (D) and prefrontal cortex (E) (*** p < 0.001, n = 8). 

Treadmill exercise pretreatment increased the spine numbers of the hippocampus (D; ** p < 0.01, n = 8) and prefrontal cortex (E; * p < 0.05, n = 8) in non-Tg mice. (F–I) The mushrooms (** p < 0.01, n = 8), and stubby spines (*** p < 0.001, n = 8) of the prefrontal cortex (G, I) were significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group, and this decrease was blocked by treadmill exercise pretreatment both in the prefrontal cortex (*** p < 0.001, n = 8). 

Treadmill exercise pretreatment increased the mushrooms (** p < 0.01, n = 8) and stubby spines (* p < 0.05, n = 8) of the prefrontal cortex (G, I) in non-Tg mice. 

There were no significant effects on the mushrooms and stubby spines of the hippocampus between non-Tg control, non-Tg exercise, 3×Tg-AD control, and 3×Tg-AD exercise mice (F, H; p > 0.05, n = 8). Each data set was obtained from 3 mice.

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