Effects of exercise intensity on the reactive astrocyte polarization in the medial prefrontal cortex
Article information
Abstract
[Purpose]
Physical exercise contributes to neuroplasticity by promoting cognitive functions, such as learning and memory. The astrocytic phenotype is closely associated with synaptic plasticity. This study aimed to determine whether astrocyte polarization and synaptic alterations in the medial prefrontal cortex (mPFC) are affected differently by high- and moderate-intensity exercise.
[Methods]
Mice were subjected to moderate-(MIE) and high-intensity treadmill running (HIE). Memory capacity was assessed using the novel object recognition and modified Y-maze tests. For immunohistochemistry, c-Fos-positive cells were counted in the mPFC. Using western blot analysis, astrocyte phenotype markers were quantified in whole-cell lysates, and synaptic molecules were determined in the synaptosomal fraction.
[Results]
Exercise lengthened the approach time to novel objects regardless of intensity in the NOR test, whereas MIE only improved spatial memory. Exercise induced c-Fos expression in the anterior cingulate cortex (ACC) and c-Fos-positive cells were higher in MIE than in HIE in the ACC area. In the prelimbic/infralimbic cortex region, the number of c-Fos-positive cells were enhanced in MIE and decreased in HIE mice. The A1 astrocyte marker (C3) was increased in HIE mice, while the A2 astrocyte markers were enhanced in exercised mice, regardless of the intensity. In the synaptosomal fraction, synaptic proteins were elevated by exercise regardless of intensity.
[Conclusion]
These results suggest that exercise intensity affects neuronal plasticity by modulating the reactive state of astrocytes in the mPFC.
INTRODUCTION
Memory depends on the encoding, storage, and subsequent retrieval of information in the brain. This process is governed by multiple interconnected structures in the brain, including the medial prefrontal cortex (mPFC) and hippocampus (HP) [1,2]. In general, the HP participates in the storage and recall of information during the early stage of the memory process, whereas the mPFC, including the anterior cingulate cortex (ACC), prelimbic cortex (PrL), and infralimbic cortex (IL), contributes to the consolidation and retrieval of remote memories [3,4]. Studies using immediate-early gene mapping and whole-brain region inhibition have indicated a predominant contribution of the mPFC to later memory consolidation [5-8].
Astrocytes, the most abundant non-neuronal cell type, play an indispensable role in controlling homeostasis and providing trophic support to the central nervous system through neurotransmission, neurotrophic support, neuronal synaptogenesis, and immune function [9-11]. A single astrocyte interconnects 100,000–2,000,000 neuronal synapses in mice and humans [12,13]. Moreover, astrocytes strongly contribute to neuroplasticity, a process characterized by neurogenesis, synaptogenesis, angiogenesis, and release of neurotrophic factors [11,14,15]. Functional and structural changes in neuroplasticity occur during learning and memory development. Based on these findings, astrocytes can alter the morphological and functional characteristics of neurons in response to stimuli through synaptic activity.
Physical exercise is well-known to contribute to neuroplasticity by promoting cognitive function [16]. Mounting evidence suggests that physical exercise facilitates neurogenesis, growth factor production, neuronal excitability, and axonal outgrowth in the brain [16-18]. Numerous studies have shown that astrocytes play important roles in brain circuits that establish long-term adaptations to various physiological and pathological conditions, including exercise [19-21]. Physical exercise can induce astrocyte activation, as indicated by increased astrocyte proliferation and the release of various trophic factors and gliotransmitters [22-24]. These changes are implicated in neuroplasticity and related behavioral outcomes, such as learning and memory. In moderate-intensity exercise, the most investigated exercise regimen, the extended prefrontal and medial frontal regions of the brain are more active in the aerobic group than in the anaerobic group, including adult neurogenesis, long-term potentiation, and memory function [25-27]. This implies that exercise intensity has different effects on neuronal plasticity in the temporal and frontal lobes. However, most studies have focused on neuronal characteristics, and the relationship between exercise intensity and astrocytic characteristics remains poorly understood. In this study, we explored the reactive A1/A2 astrocyte phenotype using high- (HIE) and moderate-intensity (MIE) treadmill running.
METHODS
Experimental mice and experimental procedure
Seven-week-old male C57BL/6 mice were obtained from Daehan Biolink Co., Ltd. (Eumsung, Chungbuk, Korea) and housed in clear plastic cages under specific pathogen-free conditions with a 12:12-h light-dark cycle (lights on at 08:00 and off at 20:00). Mice had free access to standard irradiated chow (Purina Mills, Seoul, Korea). All experimental procedures involving animals were approved by the Animal Care and Use Committee of Ewha Women’s University.
In this study, the exercise intensity protocol (high- and moderate-intensity) was modified according to a previous study [28], in which the intensity was set according to the VO-2max degree (moderate, > 50–60%; high, 75–85%). All mice were acclimated to treadmill running (Myung Jin Instruments Co., Seoul, Korea) by pre-exercise (Pre-Ex) at 5–8 m/min for 20 min/day for 5 days (each group N = 12). For the exercise regimen, the treadmill running speed was initially 10 m/min for 5 min and then increased as follows: moderate intensity 8–10 m/min for 20 min, high intensity 19–25 m/min for 20 min, and 5–8 m/min for 5 min. Finally, the speed was increased to 1 m/min weekly (moderate) and 2 m/min weekly (high).
Behavioral test
For the novel object recognition test, the mice were handled and habituated to an empty open-field arena (35 × 35 × 19; the arena was black inside) on days 1 and 2 (twice/day, 15 min/trial). On day 3 (acquisition session), mice were placed in an arena with two identical objects (Falcon tissue culture flasks filled with sand) and allowed to explore the objects for 10 min (5 min/trial, twice/day, 15-min inter-trial interval). Following a 1-day delay, one of the familiar objects used previously was replaced with a novel object (an interlocking tower of Lego pieces with different shapes and colors) for 5 min. The exploration ratio (%) was expressed as the percentage of time spent exploring each object divided by the total exploration time. A sample phase trial and a test phase trial separated by an inter-trial interval comprised two trials of the modified Y-maze test. In the sample phase trial, each mouse was placed individually in the maze with one of the three arms closed. For 5 min, the mice were allowed to explore the other two arms. Thirty minutes after the sample phase trial, the animal was placed in the maze with all three arms open and allowed to explore the arms freely. The closed arm that was opened during the test phase trial was defined as the new arm. Animal behavior was video recorded for subsequent analysis. The percentage of time spent in the new arm and the total number of arm entries were analyzed using the EthoVision 17 program (Noldus, Wageningen, Netherlands).
Synaptosome extraction and western blot analyses
Tissues dissected from mice were homogenized. The procedures for synaptosome fractionation and western blot analysis have been described previously by our group [29]. Western blotting was performed using the standard protocol and the optical density of each band was quantified using ImageJ software. The primary antibodies used were as follows: anti-GFAP antibody was obtained from Novus Biologicals (Centennial, CO 80112, USA); anti-pentraxin 3 (PTX3), anti-GDNF, anti-BDNF, and anti-β-actin antibodies were obtained from Abcam (Cambridge, MA); anti-complement C3, anti-S100A10, anti-synapsin-1, and PSD-95 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).
Immunohistochemistry
Brain sections were subjected to endogenous peroxidation inactivation with 3% hydrogen peroxide and non-specific binding was blocked with 4% bovine serum albumin. The sections were incubated overnight with primary antibodies and then with biotinylated secondary antibodies for 1 h at 25°C the following day. The sections were subsequently incubated with an avidin-biotin-horseradish peroxidase complex reagent solution for 1.5 h, and a peroxidase reaction was performed using diaminobenzidine tetrahydrochloride. Digital images of the immunohistochemistry were captured using a Leica DM750 microscope (Leica Microsystems). Quantification was performed using ImageJ (NIH, Bethesda, MD, USA).
Statistical analysis
Significant differences between groups were determined using independent t-tests and one-way analysis of variance (SPSS for Windows, version 18.0; Chicago, IL, USA). Post hoc comparisons were performed using Tukey’s test. All values were reported as the mean ± standard error. Statistical significance was set at p < 0.05.
RESULTS
Moderate-intensity, but not high-intensity, exercise enhanced spatial memory
Using the protocol shown in Fig. 1A, we first assessed memory consolidation and spatial memory after exercise. In the NOR test, in the 24-h retention stage, the time spent exploring the novel object was greater than the time spent exploring the familiar object in the CON, MIE, and HIE groups (Fig. 1B; CON t14 = -2.48, p < 0.05; MIE t14 = -6.00, p < 0.01; HIE t14 = -4.42, p < 0.01; CON familiar, 43.39 ± 3.19, novel, 55.61 ± 3.19; MIE familiar, 38.02 ± 2.82, novel, 61.98 ± 2.82; HIE familiar, 42.08 ± 2.53, novel, 57.92 ± 2.53). In modified Y-maze test, the novel arm entry of MIE mice was much than that of CON and HIE mice, and no significant difference between group was observed (Fig. 1C; entry F2, 21 = 3.52, p < 0.05; time spent F2, 21 = 1.57, p > 0.05; CON entry, 34.57 ± 2.43, time, 35.96 ± 3.60; MIE, entry, 41.57 ± 1.70, time, 37,54 ± 2.93; HIE, entry, 35.82 ± 1.74, time, 36.04 ± 2.38).
The effects of exercise intensity on memory and neuronal activity in the mPFC.
A. The experimental procedure. B–C. The quantitative analysis of exploration ratio in NOR test (B), and novel object entry and time (C). D. The photogram showing the area measured. E–F. Photomicrograph showing c-Fos-positive cells (E) and quantitative analysis (F). Data are presented as mean ± standard error. * and ** denote vs. CON p < 0.05 and p < 0.01, respectively. ## vs. MIE p < 0.05 and p < 0.01, respectively.
c-Fos expression was enhanced by MIE, and not HIE in ACC and PrL/IL region
Exercise enhanced c-Fos expression relative to the control group, in which the expression in the MIE group was higher than that in the ACC (Fig. 1E, F; F2, 15 = 21.96, p < 0.01; CON 297.84 ± 10.77; MIE, 428.09 ± 17.77; HIE, 340.74 ± 13.04). Moreover, c-Fos expression in MIE was higher than that in the control and HIE groups, and its expression in HIE was lower than that in the control group (Fig 1E, F; F2, 15 = 12.15, p < 0.01; CON 137.65 ± 6.87; MIE, 164.81 ± 8.17; HIE, 107.41 ± 9.47).
Exercise intensity differently regulated A1 and A2 astrocytic marker protein
Exercise induced GFAP expression regardless of intensity, and HIE enhanced the expression of GFAP more than MIE (Fig. 2A, B; F2, 12 = 15.79, p < 0.01; CON 1.00 ± 0.06; MIE, 1.39 ± 0.07; HIE, 1.74 ± 0.13). C3 expression was enhanced by HIE compared to that by CON and MIE (Fig. 2A, B; F2, 12 = 17.47, p < 0.01; CON 1.00 ± 0.05; MIE, 1.00 ± 0.05; HIE, 1.66 ± 0.14). Exercise induced S100A10 expression, and MIE enhanced protein expression more than HIE (Fig. 2A, B; F2, 12 = 12.42, p < 0.01; CON 1.00 ± 0.06; MIE, 1.82 ± 0.19; HIE, 1.32 ± 0.06). PTX3 expression pattern was similar to that of S100A10 (Fig. 2A, B; F2, 12 = 16.04, p < 0.01; CON 1.00 ± 0.07; MIE, 1.84 ± 0.17; HIE, 1.20 ± 0.06). The expression pattern of GDNF was similar to that of PTX3 (Fig. 2A, B; F2, 12 = 16.86, p < 0.01; CON, 1.00 ± 0.03; MIE, 1.84 ± 0.17; HIE, 1.20 ± 0.06).
The effects of exercise intensity on astrocyte phenotype markers and synaptic proteins in the mPFC.
A. Representative images of western blot for GFAP, C3, S100A10, PTX3, and GDNF. B. The quantitative analysis of GFAP, C3, S100A10, PTX3, and GDNF. C. Representative images of western blot for BDNF, synapsin-1, and PSD-95. D. The quantitative analysis of BDNF, synapsin-1, and PSD-95. Data are presented as mean ± standard error. * and ** denote vs. CON p < 0.05 and p < 0.01, respectively. ## vs. MIE p < 0.05 and p < 0.01, respectively.
Exercise intensity differently induced synaptic proteins in synaptosomal fraction
Exercise induced BDNF (CON 1.00 ± 0.04; MIE, 1.70 ± 0.13; HIE, 1.33 ± 0.03), synapsin-1 (CON 1.00 ± 0.06; MIE, 2.02 ± 0.24; HIE, 1.24 ± 0.06), and PSD-95 expression (CON 1.00 ± 0.04; MIE, 1.83 ± 0.15; HIE, 1.43 ± 0.04) regardless of intensity, in which MIE enhanced more those proteins than that of HIE (Fig. 2C, D; BDNF F2, 12 = 13.38, p < 0.01; synapsin-1 F2, 12 = 14.30, p < 0.01; PSD-95 F2, 12 = 20.19, p < 0.01).
DISCUSSION
This study showed that moderate- and high-intensity exercise induced different neuronal activities and astrocytic phenotypes in the mPFC, along with different spatial memory capacities.
Physical exercise facilitates synaptic plasticity for activity-dependent synaptic transmission by upregulating synaptic molecules, including PSD-95, synapsin-1, and vascular endothelial cell growth factor [30-32]. This change in the mPFC and HP is responsible for cognitive processes, such as learning and memory. Exercise intensity is a key component in determining exercise-induced physiological efficacy and may be implicated in discrete neural circuitry control. A study reported that low- and high-intensity exercises differently stimulate functional connectivity in networks associated with cognitive and emotional processes, respectively33. Moreover, prefrontal cortex (PFC) activity is significantly higher under moderate- and high-intensity conditions, and not under control or low-intensity conditions [34,35]. The findings from these studies supported our interpretation that the expression pattern of c-Fos protein, which is expressed specifically during neuronal activation, emerged differently according to exercise intensity.
Given that astrocytes participate in synaptic activity related to memory processes, exercise-induced synaptic changes are attributed to the astrocyte-neuron interactions. Physical exercise facilitates the astrocyte population and activity in the HP, PFC, striatum, and entorhinal cortex, which are responsible for improving neuronal activity and plasticity [36,37]. This supports our finding that exercise increases GFAP levels in the mPFC and improves memory capacity, including memory consolidation and spatial memory. Reactive astrocytes exert both beneficial and deleterious effects on the brain. A1 astrocytes produce and secrete neurotoxins and pro-inflammatory factors that cause neuronal damage and eliminate synapses [38]. The A2 astrocyte phenotype induces neurotrophic and anti-inflammatory genes that promote neuronal survival and growth of neurons [39]. Moreover, exercise enhances astrocyte-neuron crosstalk by regulating trophic factors such as BDNF and GDNF [40]. In this study, HIE-induced higher C3 levels (an A1 astrocyte marker) and lower S100A10, PTX3, and GDNF levels (A2 astrocyte markers) compared to MIE corresponded well with synaptic markers (synapsin-1 and PSD-95) and BDNF expression in the synaptosome fraction from the mPFC tissue. These results provide evidence that exercise intensity differentially regulates the astrocyte phenotype, contributing to synaptic formation, in which MIE is more effective in enhancing synapse formation in the mPFC and subsequently improving memory function. In this study, the differential behavioral consequences and synaptic protein expression according to exercise intensity may be attributed to synaptic plasticity induced by astrocytic metabolic reprogramming. Some studies have proposed that the energy metabolic reprogramming of inflammatory/immune cells participates in the transfer of M1 (highly glycolytic and pro-inflammatory) toward the M2 phenotype (highly oxidative and anti-inflammatory) [41,42]. Therefore, exercise intensity is likely to affect astrocytic metabolic reprogramming, and subsequently induce changes in synaptic function and behavioral outcomes. However, further studies are required to address this issue.
In conclusion, exercise intensity can affect astrocyte phenotype, thereby modulating neuronal activity, synapse formation, and subsequent behavioral outcomes.