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

Abstract

Alzheimer's disease (AD) is characterized by deficits in learning and memory. A pathological feature of AD is the alterations in the number and size of synapses, axon length, dendritic complexity, and dendritic spine numbers in the hippocampus and prefrontal cortex. Treadmill exercise can enhance synaptic plasticity in mouse or rat models of stroke, ischemia, and dementia. The aim of this study was to examine the effects of treadmill exercise on learning and memory, and structural synaptic plasticity in 3×Tg-AD mice, a mouse model of AD. Here, we show that 12 weeks treadmill exercise beginning in three-month-old mice improves spatial working memory in six-month-old 3×Tg-AD mice, while non-exercise six-month-old 3×Tg-AD mice exhibited impaired spatial working memory. To investigate potential mechanisms for the treadmill exercise-induced improvement of spatial learning and memory, we examined structural synaptic plasticity in the hippocampus and prefrontal cortex of six-month-old 3×Tg-AD mice that had undergone 12 weeks of treadmill exercise. We found that treadmill exercise led to increases in synapse numbers, synaptic structural parameters, the expression of synaptophysin (Syn, a presynaptic marker), the axon length, dendritic complexity, and the number of dendritic spines in 3×Tg-AD mice and restored these parameters to similar levels of non-Tg control mice without treadmill exercise. In addition, treadmill exercise also improved these parameters in non-Tg control mice. Strengthening structural synaptic plasticity may represent a potential mechanism by which treadmill exercise prevents decline in spatial learning and memory and synapse loss in 3×Tg-AD mice.

Keywords: 3×Tg-AD mice; dendritic spines; exercise; memory; structural synaptic plasticity; synaptic.

Figure 1

Treadmill exercise prevents decline in spatial learning and memory in 3×Tg-AD mice. (A) Timeline of treadmill exercise or non-exercise control, behavioral test and histological test. (B) The percentage of working memory errors on day 5 and day 6 was significantly increased in the 3×Tg-AD mice compared to the Non-Tg control group (B; ** p < 0.01, n = 10 mice). The increase in the percentage of working memory errors was prevented by treadmill exercise pretreatments (B; ## p < 0.01, n = 10 mice). (C) There were no significant effects on the percentage of reference memory errors between Non-Tg control, Non-Tg exercise, 3×Tg-AD control, and 3×Tg-AD exercise mice (C; p > 0.05, n = 10 mice).

Figure 2

Treadmill exercise increases synapse numbers of hippocampus and prefrontal cortex in 3×Tg-AD mice. (A) Representative electron microscope imaging of hippocampus and prefrontal cortex in non-Tg control, non-Tg exercise, 3×Tg-AD control, and 3×Tg-AD exercise mice. The synapses are marked by the blue arrowheads. Red box represents an enlarged synapse. An expanded, high magnification view of synapses in the prefrontal cortex is shown in the red square box in the bottom. (B,C) The synapse numbers of 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 = 6 image sections), and this decrease was blocked by treadmill exercise pretreatment both in the hippocampus (B) and prefrontal cortex (C) (*** p < 0.001, n = 6 image sections). Treadmill exercise pretreatment increased the synapse numbers of the hippocampus (B) and prefrontal cortex (C) in non-Tg mice (** p < 0.01, n = 6 image sections). Each data set was obtained from 4 mice.

Figure 3

Treadmill exercise improves synaptic structural parameters of the hippocampus and prefrontal cortex in 3×Tg-AD mice. (A) A representative measurement of synaptic structural parameters. The formula R = a/2 + b²/8a, where b is the line joining the two ends of the postsynaptic thickening and a is the perpendicular distance from the postsynaptic membrane to b, was used to determine synaptic curvature. (B,C) The length of the synaptic active zone of hippocampus (B) and prefrontal cortex (C) was significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group (*** p < 0.001, n = 12–15 synapses), and this decrease was blocked by treadmill exercise pretreatment both in the hippocampus and prefrontal cortex (*** p < 0.001, n = 12–15). (D,E) The width of the synaptic cleft of the hippocampus (D) and prefrontal cortex (E) was significantly increased in the 3×Tg-AD control group compared to the non-Tg control group (*** p < 0.001, n = 12–15 synapses), and this increase was blocked by treadmill exercise pretreatment both in the hippocampus (D; *** p < 0.001, n = 12–15 synapses) and prefrontal cortex (E; ** p < 0.01, n = 12–15). (F,G) The synaptic curvature of the hippocampus (F; *** p < 0.001, n = 12–15), and prefrontal cortex (G; ** p < 0.01, n = 12–15) was 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 (F; ** p < 0.01, n = 12–15) and prefrontal cortex (G; *** p < 0.001, n = 12–15). (H,I) The thickness of postsynaptic density of hippocampus (H) and prefrontal cortex (I) was significantly decreased in the 3×Tg-AD control group compared to the non-Tg control group (*** p < 0.001, n = 12–15), and this decrease was blocked by treadmill exercise pretreatment both in the hippocampus (H; ** p < 0.01, n = 12–15) and prefrontal cortex (I; *** p < 0.001, n = 12–15). Each data set consisted of 12 and 15 synapses from 4 mice each group.

Figure 4

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

Figure 5

Treadmill exercise enhance the axon length and dendritic complexity of the Hippocampus and prefrontal cortex in 3×Tg-AD mice. (A) Representative Golgi staining images of 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 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 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.

Figure 6

Treadmill exercise improves the dendritic spines numbers of 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 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 spines 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 spines 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 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.