Attention deficit/hyperactivity disorder (ADHD) is a neurobehavioral disorder of cognition. We investigated the effects of treadmill exercise on Purkinje cell and astrocytic reaction in the cerebellum of the ADHD rat. Adult male spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKYR) weighing 210± 10 g were used. The animals were randomly divided into four groups (n= 15): control group, ADHD group, ADHD and methylphenidate (MPH)-treated group, ADHD and treadmill exercise group. The rats in the MPH-treated group as a positive control received 1 mg/kg MPH orally once a day for 28 consecutive days. The rats in the treadmill exercise group were made to run on a treadmill for 30 min once a day for 28 days. Motor coordination and balance were determined by vertical pole test. Immunohistochemistry for the expression of calbindinD-28 and glial fibrillary acidic protein (GFAP) in the cerebellar vermis and Western blot for GFAP, Bax, and Bcl-2 were conducted. In the present results, ADHD significantly decreased balance and the number of calbindin-positive cells, while GFAP expression and Bax/Bcl-2 ratio in the cerebellum were significantly increased in the ADHD group compared to the control group (
Attention deficit/hyperactivity disorder (ADHD) is one of the most common childhood neurobehavioral disorders characterized by hyperactivity, impulsivity, and attention deficit (
The cerebellum is as a key anatomical structure underlying normal attentional and cognitive control mechanisms. Previous studies have shown that the cerebellum is thought to play a crucial role in cognitive function, particularly in learning, attention, and emotional regulation (
Purkinje neurons in the cerebellum play crucial role in movement, coordination, and cognitive function including motor learning (
In clinic, pharmacological stimulants such as norepinephrine agonists (
Many studies have reported that exercise improves motor performance, learning, memory, and cognitive function in animals (
Although the beneficial effects of physical exercise on abnormal brain function are well documented, there is no available study for the effect of exercise on ADHD in relation with Purkinje cells and astrocytic reaction in the cerebellum. Therefore, we investigated the effects of treadmill exercise on Purkinje cell loss and astrocyte activation in the cerebellum of the ADHD rat.
Adult male spontaneously hypertensive rats (SHR) weighing 210±10 g were used as the ADHD animal model, because the SHR displays the major symptoms of ADHD such as hyperactivity. SHR was selected only when showed hyperactivity in open field test. Meanwhile, Wistar-Kyoto rats (WKYR) weighing 210±10 g were used as the control in this study, according to the previous study (
For the vertical pole test, the animal was placed face up on a cloth-tape-covered pole (3.0 cm diameter, 150 cm length), which was held in a horizontal position, then the pole was gradually lifted to a vertical position and the time a rat stayed on the pole was recorded for a maximum of 180 sec. In this test, the animal with deficits in motor coordination and balance will fall off the pole (
The rats in the treadmill exercise group were subjected to run on a treadmill for 30 min once a day, five times a week, for 28 days. Exercise load for the running group consisted of running at a speed of 2 m/min for the first 5 min, at a speed of 5 m/min for the next 5 min, and then at a speed of 8 m/min for the last 20 min, with the 0° inclination. This intensity corresponded to the low-intensity treadmill exercise (% maximal oxygen consumption) of rats in this age (
The rats were sacrificed 29 days after the starting of experiment, immediately after vertical pole test. To begin the sacrifice process, animals were fully anesthetized using Zoletil 50® (10 mg/kg, i.p.; Vibac Laboratories, Carros, France). The anesthetized rats were transcardially perfused with 50 mM phosphate-buffered saline (PBS), and fixed with a freshly prepared solution consisting of 4% paraformaldehyde (PFA) in 100 mM phosphate buffer (PB) at pH 7.4. Cerebellums were dissected, post fixed in the same fixative overnight, and transferred to 30% sucrose solution for cryoprotection. Sagittal sections of 40 μm thickness in each section of cerebellum were made with a freezing microtome (Leica, Nussloch, Germany).
For the detection of calbindin-positive and glial fibrillary acidic protein (GFAP)-positive cells in the cerebellar vermis, immunohistochemistry was performed. Briefly, the sections were incubated in PBS for 10 min and washed 3 times with PBS, and then incubated in 1% hydrogen peroxide (H2O2) for 30 min. Next, the sections were incubated overnight with mouse anti calbindin D-28k antibody (Sigma Chemical Co., St. Louis, MO, USA) at a dilution of 1:1,000 for visualization of Purkinje cells or with mouse anti-GFAP antibody (Chemicon, Temecula, CA, USA) at a dilution of 1:2,000 for visualization of astrocytes. The sections were then incubated for 1 h with anti-mouse secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA). Bound secondary antibody was then amplified with a Vector Elite ABC kit® (Vector Laboratories). The sections were subsequently incubated with avidin-biotin-peroxidase complex (1:100; Vector Laboratories) for 1 h at room temperature. Immunoreactivity was visualized by incubating the sections in a solution consisting of 0.05% 3,3-diaminobenzidine (DAB) and 0.01% H2O2 in 50 mM Tris buffer (pH 7.6) for approximately 3 min. The sections were then mounted on gelatin-coated glass slides. The slides were air-dried overnight at room temperature, and the coverslips were mounted using Permount®.
Brains were collected at lobules VIII–X areas of the cerebellum, and then were immediately frozen at −70°C. Cerebellums were homogenized on ice, and lysed in a lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 mM EGTA, 1.5 mM MgCl2 ·6H2O, 1 mM sodium orthovanadate, and 100 mM sodium flouride. Protein content was measured using a Bio-Rad colorimetric protein assay kit (Hercules, CA, USA). Protein (30 μg) was separated on SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane. Mouse-actin antibody (1:3,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse GFAP antibody (1:1,000; Santa Cruz Biotechnology), mouse Bax antibody (1:1,000; Santa Cruz Biotechnology), and mouse Bcl-2 antibody (1:1,000; Santa Cruz Biotechnology) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-mouse antibody for actin, AP, Bax and Bcl-2 (1: 3,000; Vector Laboratories) were used as the secondary antibodies. Experiments were performed in normal laboratory conditions and at room temperature, except for the transferred membranes. Transferred membranes were performed at 4°C with the cold pack and pre-chilled buffer. Band detection was performed using the enhanced chemiluminescence (ECL) detection kit (Santa Cruz Biotechnology). To compare the relative expression of proteins, the detected bands were calculated densitometrically using Image-Pro® Plus software (Media Cybernetics, Bethesda, MD, USA).
To quantify the number of Purkinje neurons three and four non-adjacent vermis sections per animal were chosen. Using Image-Pro® Plus software (Media Cybernetics, Bethesda, MD), a 1,000 μm contour line was drawn along the Purkinje neuron layer on both sides of the fissure or gyrus. The number of Purkinje neurons for each sample was calculated as the average number of Purkinje neurons. Before beginning the image analysis, the light source was adjusted to the brightness generating the best possible contrast between positive and negative staining astrocytes. Quantification for Purkinje neurons was conducted in a blinded manner. All data are presented as the mean standard error of the mean (SEM). Statistical analysis for Purkinje cell quantification was done by ANOVA following Duncan’s post hoc test using SPSS computer software (Version 18.0). The differences were considered significant at
The vertical pole test was performed for 4 weeks after starting of experiment. The time score of the vertical pole test are presented in
The photomicrographs of the Purkinje neuronal cells in the gyrus of lobules VIII of the cerebellar vermis (dotted rectangle) are presented in
The photomicrographs of GFAP-positive cells in both the granule layers of cerebellar vermis are presented in
The level of Bax and Bcl-2 protein in the cerebellum were analyzed by Western blotting (
The cerebellum is a major structures involved in motor learning, motor coordination and control by maintaining a balance and muscle tension (
Although the precise mechanism on occurrence of cerebellar dysfunction by ADHD was not fully understood to date, previous studies reported the relevance that the Purkinje cells of cerebellar cortex is closely related to the motor function (
It has been widely accepted that regular exercise plays a crucial role to enhance brain structure and cognitive function. Regular exercise increased neuronal survival, learning, and memory (
Although the interesting results of treadmill exercise on ADHD, there was limitation of the current study. Primary limitation is that there is no criteria on the level of balance ability for ADHD animal model. For this reason, the present behavioral results did not explain directly whether the decreased level of balance ability in ADHD group is associated with symptoms of ADHD or not. It is required to examine various types of behaviors for searching adequate behavior test. In sum, treadmill exercise might exert ameliorating effect on ADHD through reduction of Purkinje cell loss and astrocytic reaction in the cerebellum.
This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2011-327-G00121).
No potential conflict of interest relevant to this article was reported.
Effects of treadmill exercise on vertical pole test. (CON) control group, (ADHD) attention deficit/hyperactivity disorder group, (ADHD+MPH), attention deficit/hyperactivity disorder and methylphenidate-treated group, (ADHD+EXE), attention deficit/hyperactivity disorder and treadmill exercise group. *
Effects of treadmill exercise on surviving Purkinje cells in the cerebellular vermis. (A) Photomicrographs of a sagittal section Purkinje cells in the cerebellum. The sections were stained for Purkinje cells immunoreactivity with calbindin D-28k (brown). The scale bar represents 200 μm. (B) The mean number of calbindin-positive Purkinje cells in each group. (CON) control group, (ADHD) attention deficit/hyperactivity disorder group, (ADHD+MPH), attention deficit/hyperactivity disorder and MPH-treated group, (ADHD+EXE), attention deficit/hyperactivity disorder and treadmill exercise group. *
Effects of treadmill exercise on activation of the reactive astrocytes in the cerebellular vermis. (A) Photomicrographs of GFAP-positive reactive astrocytes in molecular layer of the cerebellum. The sections were stained for GFAP immunoreactivity (brown). The scale bar represents 200 μm. (B) Western blot analysis of GFAP protein in the cerebellar vermis. (C) The mean optical density of reactive astrocytes in each group. (CON) control group, (ADHD) attention deficit/hyperactivity disorder group, (ADHD+MPH), attention deficit/hyperactivity disorder and MPH-treated group, (ADHD+EXE), attention deficit/hyperactivity disorder and treadmill exercise group. *
Effects of treadmill exercise on the expression of Bcl-2 and Bax protein. (A) Western blot analysis of Bcl-2 and Bax protein in the cerebellum. (B) The mean optical density of Bax expression in each group. (C) The mean optical density of Bcl-2 expression in each group. (D) The Bax/Bcl-2 ratio in each group. (CON) control group, (ADHD) attention deficit/ hyperactivity disorder group, (ADHD+MPH), attention deficit/hyperactivity disorder and MPH- treated group, (ADHD+EXE), attention deficit/hyperactivity disorder and treadmill exercise group. *