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 4

Spines are classified as mushrooms if the diameter of the head is greater than the diameter of the neck [47]. Mushroom spines are the more stable and persist for months. 

In recent years, repeated environmental changes and increasing pressure in life have made more and more people feel lost, tired, and even exhausted. In this situation, many people can't help but sigh: How long can we survive in such a living state?

However, after many studies, it has been shown that there is no direct correlation between surviving for a few months and memory. In other words, the hardships we have experienced will not cause our memory to decline. On the contrary, the right way of thinking and a positive attitude can not only strengthen our memory but also enable us to better face future challenges.

First of all, a positive attitude is the cornerstone of keeping a healthy memory. When we have a positive mindset, our brains will also be full of energy. We will be more focused and more creative. Several studies have shown that positive emotions and mindsets can even improve vision problems under computer screens. If we can devote ourselves to life and move forward positively, we will naturally not be far from success.

Next, moderate exercise can also help us maintain a good memory. Physical activity can improve the health of the body and brain. Physical exercise can promote metabolism and consume excess negative energy. During physical activity, our brain will receive more oxygen, which is helpful for our brain and can also enhance our memory.

In addition, learning new things and maintaining curiosity can also help us improve memory health. Learning and exploration keep our brain cells active and active. When we explore new areas, our brains will be challenged and the vitality of memory will be enhanced.

In short, the environment and difficulties we are in will not hurt our memory. On the contrary, maintaining a positive attitude, moderate exercise, and learning new things can help us make our memory stronger. If we have a positive attitude and maintain curiosity, I believe we can survive longer in this challenging world. It can be seen that we need to improve our memory. Cistanche can significantly improve memory because Cistanche can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche can also improve blood flow and promote oxygen delivery, which can ensure that the brain gets sufficient nutrition and energy, thereby improving brain vitality and endurance.

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They can be shifted from thin spines by long-term potentiation (LTP) [56,58]. Two-way ANOVA indicated that genotype and treadmill exercise had no significant effects on the mushroom spines in the hippocampus (genotype: F1,31 = 6.2, p = 0.019; treadmill exercise: F1,31 = 1.4, p = 0.245; genotype × treadmill exercise interaction: F1,23 = 0.06, p = 0.803; Figure 6F). 

For the prefrontal cortex, two-way ANOVA indicated that genotype and treadmill exercise had significant main effects on the number of mushroom spines (genotype: F1,31 = 23.3, p < 0.001; treadmill exercise: F1,31 = 24.4, p < 0.001 Figure 6G), but there was not a significant interaction between genotype and treadmill exercise on the number of mushroom spines (genotype × treadmill exercise interaction: F1,31 = 0.1, p = 0.723; Figure 6G). 

Tukey's post hoc tests indicated that the number of mushroom spines was significantly decreased in the prefrontal cortex in the 3×Tg-AD control group compared to the non-Tg control group (p = 0.001; Figure 6G), and treadmill exercise increased the number of mushroom spines in the prefrontal cortex (p < 0.001; Figure 6G) in 3×Tg-AD mice. 

Meanwhile, treadmill exercise increased the mushroom spines (p = 0.003; Figure 6G) of the prefrontal cortex in non-Tg mice. Spines are considered stubby if the length and width are equal [47]. 

Stubby spines are viewed as an immature type that is prevalent during early postnatal development and shows relative scarcity in the mature brain [59]. Stubby spines may participate in the homeostatic regulation of calcium and the control of neuronal excitability [60]. 

Two-way ANOVA indicated that genotype and treadmill exercise had no significant effects on the stubby spines in the hippocampus (genotype: F1,31 = 5.8, p = 0.023; treadmill exercise: F1,31 = 0.6, p = 0.430; genotype × treadmill exercise interaction: F1,31 = 0.01, p = 0.810; Figure 6H). 

However, two-way ANOVA indicated that genotype and treadmill exercise had significant main effects on the number of stubby spines (genotype: F1,31 = 31.1, p < 0.001; treadmill exercise: F1,31 = 13.4, p = 0.001; Figure 6I), and there was not a significant interaction between genotype and treadmill exercise on the number of stubby spines (genotype × treadmill exercise interaction: F1,31 = 0.4, p = 0.522; Figure 6I) in the prefrontal cortex. 

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

Treadmill exercise increased the number of stubby spines in the prefrontal cortex (p = 0.005; Figure 6I) in 3×Tg-AD mice. Meanwhile, treadmill exercise increased the number of stubby spines (p = 0.042; Figure 6I) of the prefrontal cortex in non-Tg mice.

4. Discussion

Here, we have demonstrated that six-month-old 3×Tg-AD mice exhibited impaired spatial working memory and treadmill exercise pretreatment prevented the decline in spatial working memory in 3×Tg-AD mice. 

Treadmill exercise pretreatment led to increases in synapse numbers, synaptic structural parameters, the expression of Syn, the axon length, dendritic complexity, number of the dendritic spines, and restoration of structural synaptic plasticity of the hippocampus and prefrontal cortex in 3×Tg-AD mice. Meanwhile, treadmill exercise pretreatment enhanced synaptic plasticity by increasing synapse numbers, the axon length, dendritic complexity, and number of dendritic spines in the hippocampus and/or prefrontal cortex of non-Tg mice. 

Our findings suggest that exercise may serve as an effective intervention in the early stage to delay the progression of AD. We found that the percentage of working memory errors was significantly increased in the 3×Tg-AD control group compared to the non-Tg control group, but no significant effects on the percentage of reference memory errors. 

Short-term memory and synaptic function loss are the initial and most common presenting signs of memory impairment and cognitive decline [35]. As the disease progresses, people gradually experience long-term memory loss leading to problems with multitasking and abstract thinking. Thus, this result suggests that six-month-old 3×Tg-AD mice are still in the early stages of AD progression. 

Moreover, 12 weeks of treadmill exercise pretreatment led to a significant decrease in the percentage of working memory errors on day 5 and day 6 of the acquisition session under the eight-arm radial maze test. 

The eight-arm radial maze is one of the most common paradigms to assess spatial working memory and spatial reference memory [61]. Spatial working memory is often used synonymously with short-term memory, but working memory allows for the manipulation of stored information, whereas short-term memory only refers to the short-term storage of information [62]. 

Spatial reference memory refers to long-term memories that are required for remembering information [63]. Furthermore, previous studies demonstrated that treadmill running reverses cognitive declines in 3×TgAD mice in the Morris water maze task, which tests hippocampal-dependent learning, including the acquisition of spatial memory and long-term spatial memory [64–66]. 

Taken together, six-month-old 3×Tg-AD mice exhibited diminished spatial working memory, and the robust decrease of working memory errors under the eight-arm radial maze test indicates that treadmill exercise pretreatments prevent a decline in spatial learning and memory in the early stages of 3×Tg-AD mice. 

We examined the potential mechanisms that might underline the treadmill exercise-induced decrease of working memory errors in six-month-old 3×Tg-AD mice. Structural synaptic plasticity is manifested as changes in the number and size of synapses, the length of the synaptic active zone, the width of the synaptic cleft, synaptic curvature, and the thickness of the postsynaptic density [67]. 

It is believed that structural synaptic plasticity within the hippocampus and prefrontal cortex forms the cellular basis of learning and memory, which depends on different pre- and post-synaptic neuronal mechanisms [5,6]. We found that treadmill exercise pretreatments led to an increase in the synapse numbers both in the hippocampus and prefrontal cortex of six-month-old 3×Tg-AD mice. 

Meanwhile, treadmill exercise pretreatments increased the synapse numbers both in the hippocampus and prefrontal cortex in non-Tg mice. The length of the synaptic active zone reflects the presynaptic neuronal mechanisms of structural synaptic plasticity, while the synaptic curvature and the thickness of the postsynaptic density reflect the postsynaptic neuronal mechanisms of structural synaptic plasticity [68,69]. 

The synaptic cleft is the structure responsible for the neurotransmitter transmission between presynaptic and postsynaptic neurons, and optimal shortening of the synaptic cleft may have an adaptive function of optimizing synaptic strength [37,38]. 

Indeed, we found that treadmill exercise pretreatments remarkably increased the length of the synaptic active zone, the synaptic curvature, and the thickness of the postsynaptic density, shortening the width of the synaptic cleft in the hippocampus and prefrontal cortex of six-month-old 3×Tg-AD mice. Syn is a marker protein of presynaptic vesicles of the nerve cells [70], while the PSD95 is a pivotal postsynaptic scaffolding protein that modulates the postsynaptic response to the presynaptic release of glutamate by regulating the anchoring of glutamate receptors to the PSD [71]. 

Previous studies have shown that Syn and PSD95 were downregulated in the cerebral cortex of seven-month-old 3×Tg-AD mice and were recovered by six months of voluntary exercise treatment [72]. 

Consistent with this study [72], we found that treadmill exercise facilitated the expression of Syn in the hippocampus and prefrontal cortex of six-month-old 3×Tg-AD mice. 

The Aβ and tau-induced disruption of synaptic function is also manifest as impaired LTP/LTD induction and network oscillations [37,73,74]. Meanwhile, Treadmill exercise decreases the APP, BACE-1, and Aβ burden in both the hippocampus and cortex in AD model mice [24,27]. 

It is thus likely that exercise-induced increases in the synapse numbers, synaptic structure, and the level of Syn lead to the enhanced efficacy of neurotransmitter release and prevention of the decline in spatial working memory of three×Tg-AD mice. 

Axons, dendrites, and dendritic spines constitute the structural basis of synaptic plasticity. The axon is functionally specialized to transmit signals, whereas the dendrites are specialized to receive signals [75,76]. In vivo, imaging indicated axonal abnormalities and dendritic breakage around amyloid plaques in a 4–12-month-old double transgenic APP/PS1 mouse model of AD and 3×Tg-AD mice [73,74]. 

In vitro studies using Aβ1–42 and oligomeric Aβ have revealed that 60 h Aβ treatment resulted in the degeneration of both the axons, neuronal somata, and neuronal network dynamics [77,78]. Previous studies have shown that treadmill running alleviated Aβ deposition and the level of tau in the hippocampus and cerebral cortex in 3×Tg-AD mice and high-fat diet-fed rats [66,67,79]. 

We found 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. 

Treadmill exercise pretreatment increased the axon length and dendritic complexity in the hippocampus and prefrontal cortex in 3×Tg-AD mice. Treadmill exercise pretreatment likely maintains axon length and dendritic complexity in the hippocampus and prefrontal cortex in 3×Tg-AD mice. Meanwhile, treadmill exercise pretreatment increased the axon length and dendritic complexity in the hippocampus and prefrontal cortex in non-Tg mice, indicating that exercise induces an increased surface area to facilitate interactions with other neurons and leads to enhanced structural synaptic plasticity in non-Tg mice. Dendritic spines increase the surface area for possible synaptic connections. 

Changes in the shape, size, and number of synaptic spines are thought to underlie memory formation and are observed in a variety of neurodegenerative diseases, including AD and Parkinson's disease [80–82]. In vitro studies found that 48-h treatment with 0.5–1.0 µM Aβ1–42 reduced the dendritic spine/synapse density in hippocampal cultures up to a maximum of ~40% [83]. 

Here, we have shown that the number of total dendritic spines of the hippocampus and prefrontal cortex was significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group. Treadmill exercise pretreatment blocked the decrease in the number of spines in the hippocampus and prefrontal cortex in 3×Tg-AD mice. 

However, the number of spines of the hippocampus and prefrontal cortex was significantly decreased in the 3×Tg-AD exercise group compared to the non-Tg control group, suggesting that 12 weeks of treadmill exercise pretreatment partially restored the loss of dendritic spines in six-month-old 3×Tg-AD mice. On the other hand, treadmill exercise increased the number of spines of the hippocampus and prefrontal cortex in non-Tg mice. Previous studies have shown that the dynamics of dendritic spines are associated with learning and memory, and thin, mushroom, and stubby spines have different roles in learning and memory [56]. 

Thin spines are more dynamic than mushroom spines, respond to synaptic activity, and are believed to be "learning spines", responsible for forming new memories during the synaptic plasticity process, accompanied by head enlargement [56,79]. Mushroom spines form strong synaptic connections, have the longest lifetime, and are therefore thought to be sites of long-term memory storage [56,79]. 

Stubby spines are viewed as an immature type that is prevalent during early postnatal development and show relative scarcity in the mature brain [59]. Indeed, we found that the thin spines (hippocampus and prefrontal cortex), mushroom spines (prefrontal cortex), and stubby spines (prefrontal cortex) were significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group. 

This regional difference may be due to the time course of Aβ deposition, which initiates in the neocortex and progresses to the hippocampus in 3×Tg-AD mice [17]. 

Therefore, the dendritic spines of the neocortex are more severely impaired. Treadmill exercise pretreatment blocked the decrease in the number of thin spines, mushroom spines, and stubby spines both in the hippocampus and prefrontal cortex in 3×Tg-AD mice, while treadmill exercise increased the thin spines of the hippocampus and prefrontal cortex, mushroom spines of the prefrontal cortex, and stubby spines of the prefrontal cortex in nonTg mice. 

A strong positive correlation between dendritic spine density in the hippocampus and memory has been demonstrated using the fear conditioning paradigm, Morris water maze, and object placement behavioral assessments [84]. 

Treadmill exercise pretreatment likely potentiates synaptic connections via an increase in dendritic spines. Such mechanisms might explain why treadmill exercise pretreatment prevents a decline in spatial working memory in 3×Tg-AD mice. Exercise intervention is thought to be a safe and economical choice as a therapeutic or preventative strategy against several diseases. As such, exercise may serve as a promising preventive intervention to alter the progression of AD.

Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/cells11020244/s1, Figure S1: Full-length Western blots of the Syn and PSD95 expression data shown in Figure 4.
Author Contributions: Conceptualization, L.M., J.C., Q.-S.L., and L.Z.; methodology, L.M., J.C., B.G., L.Y., and C.L.; software, L.M., J.C., B.G., L.Y., and C.L.; validation, L.M., J.C., B.G., L.Y., and C.L.; formal analysis, L.M., J.C., B.G., L.Y., and C.L.; investigation, L.M., J.C., B.G., L.Y., and C.L.; resources, L.M., J.C., and B.G.; data curation, L.M., J.C., and B.G.; writing-original draft preparation, L.M., J.C., Q.-S.L., and L.Z.; writing-review and editing, L.M., J.C., B.G., L.Y., C.L., Q.-S.L., and L.Z.; visualization, Q.-S.L. and L.Z.; supervision, Q.-S.L. and L.Z.; project administration, Q.-S.L. and L.Z.; funding acquisition, L.Z. All authors have read and agreed to the published version of the manuscript.

Funding: This work was supported by the National Natural Science Foundation of China (31571229) to L.Z.

Institutional Review Board Statement: Animal maintenance and use were by protocols approved by the Institutional Animal Care and Use Committee of the Beijing Sport University.

Informed Consent Statement: Not applicable.

Data Availability Statement: The data presented in this study are available on request from the corresponding author.

Conflicts of Interest: The authors declare no conflict of interest.

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