Neuroscience Letters 504 (2011) 3539

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Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet

Treadmill exercise and methylphenidate ameliorate symptoms of attention decit/hyperactivity disorder through enhancing dopamine synthesis and brain-derived neurotrophic factor expression in spontaneous hypertensive rats
Hong Kim a , Hong-Im Heo a , Dong-Hyun Kim b , Il-Gyu Ko b , Su-Shin Lee b , Sung-Eun Kim b , Bo-Kyun Kim b , Tae-Woon Kim b , Eun-Sang Ji b , Jae-Deung Kim b , Mal-Soon Shin b , Young-Woong Choi c , Chang-Ju Kim b,
a

Department of Oriental Sports Medicine, Daegu Haany University, Gyeongsan 712-715, Republic of Korea Department of Physiology, College of Medicine, Kyung Hee University, #1 Hoigi-dong, Dongdaemoon-gu, Seoul 130-701, Republic of Korea c Department of Biochemistry and Molecular Biology, College of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea
b

a r t i c l e

i n f o

a b s t r a c t
Attention decit/hyperactivity disorder (ADHD) is a developmental disorder of cognition. Behavioral symptoms of ADHD are inattention, hyperactivity, and impulsivity. We investigated the effects of treadmill exercise and methylphenidate (MPH) on activity and spatial learning memory in relation to dopamine synthesis and brain-derived neurotrophic factor (BDNF) expression using spontaneously hypertensive adult male rats. The rats in the MPH-treated group received 1 mg/kg MPH orally once a day for 28 days. The rats in the treadmill exercise group were made to run on a treadmill for 30 min once a day, ve times a week, for 28 days. Activity was determined by an open-eld test and spatial learning memory was evaluated by an 8-arm maze test. Immunohistochemistry and Western blotting were conducted to examine the levels of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of dopamine, and BDNF. The rats in the ADHD group showed hyperactivity and spatial learning memory decit. Reduction of TH in the striatum and substantia nigra and BDNF in the hippocampus was observed of the rats in the ADHD group. Treadmill exercise and MPH alleviated the ADHD-induced hyperactivity and spatial learning memory impairment. Expressions of TH and BDNF in the ADHD rats were also increased by both treadmill exercise and MPH. These ndings provide a possibility that exercise may be used as an effective therapeutic intervention for ADHD patients as MPH treatment. 2011 Elsevier Ireland Ltd. All rights reserved.

Article history: Received 26 April 2011 Received in revised form 24 August 2011 Accepted 24 August 2011 Keywords: Attention-decit hyperactivity disorder Treadmill exercise Methylphenidate Spatial memory Hyperactivity

Attention decit/hyperactivity disorder (ADHD) is a developmental disorder of cognition, and its primary behavioral symptoms are inattention, hyperactivity, and impulsivity. It affects approximately 35% of children, and 6080% of affected people persist into adolescence and adulthood. The symptoms of ADHD are suggested to be closely associated with hypo-function of dopaminergic system in some brain regions [5,26,27]. It has been widely accepted that dopamine plays a crucial role for the modulation of neuroendocrine functions, cognition, attention, reward, and behaviors including motor activity. Dysfunction of dopamine signaling is implicated in the brain disorders such as Parkinsons disease and ADHD [31,32]. Several studies showed that dysfunction of dopamine signaling in the midbrain substantia nigra (SN) is one of the most possible mechanisms of the behavioral symptoms of ADHD [5,31]. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamine

Corresponding author. Tel.: +82 2 961 0407; fax: +82 2 964 2195. E-mail address: changju@khu.ac.kr (C.-J. Kim). 0304-3940/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.08.052

neurotransmitters such as dopamine, epinephrine, and norepinephrine. More specically, it converts l-tyrosine to ldihydroxyphenylalanine (l-DOPA), which is rate-limiting in dopamine synthesis. TH activity is progressively decreased according to the loss of dopamine neurons in the SN [11]. TH immunohistochemistry is widely used to detect the injury or death of dopaminergic bers and cell bodies [15]. SN is the crucial part of dopamine production and signaling, and neurons originating in the SN project abundantly to the striatum. ADHD is treated primarily with pharmacological stimulants such as guanfacine, similar norepinephrine agonists [1], and methylphenidate (MPH), indirect dopaminergic agonist. MPH enhances dopamine function through inhibiting re-uptake and facilitating dopamine release [7,19]. Repeated MPH treatment during early development can modify a number of cognitive, behavioral and brain processes within sufcient therapeutic doses in ADHD [6]. MPH treatment transiently modied hippocampalsensitive learning without cognitive impairment in rats [4]. MPH affects both dopaminergic and noradrenergic systems, and chronic administration of MPH provoked the decit of spatial memory [28].

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MPH did not improve interference control allowing the successful maintenance of information during working memory task in young patients with ADHD [25]. MPH is the main drug for the ADHD patients, however, MPH sometimes exerts no effectiveness or induces side effect, often restrain their continued use in children [1]. Many studies have reported that exercise improves motor performance, learning and memory, and cognitive function [16,18]. Animal studies showed that exercise-induced increase in dopamine level might improve some symptoms caused by neurodegenerative disorders, such as Parkinsons disease [32]. Exercise also enhances recovery from nigrostriatal dopamine injury and changes the dopaminergic neurotransmission in nigrostriatal system [22]. In ADHD children, physical movement improved working speed and social behavioral problems, and diminished hyperactivity [20]. Several factors such as genes, neurotransmitters, and neurotrophins have been suggested to be involved in the benecial effects of exercise for brain functions. It is widely known that neurotrophins including nerve growth factor (NGF), basic broblast growth factor (bFGF), insulin-like growth factor I (IGF-I), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5, NT-6, and NT-7 play key roles in neuronal survival, differentiation, connectivity, and plasticity [14,29]. Of them, BDNF is considered to be implicated in the pathophysiology of several neuropsychiatric disorders [12]. Reduction of BDNF level in the hippocampus impairs learning and memory performance in animals [21]. Regular treadmill exercise increased BDNF level in the hippocampus with or without brain diseases [10,30]. Despite a lot of studies on the effect of physical exercise on abnormal brain function, there is no available study for the effect of exercise on the symptoms of ADHD in relation with dopamine and BDNF expressions in the brain. Therefore, we investigated the effects of treadmill exercise on hyperactivity, spatial learning memory, dopamine synthesis, and BDNF expression in the ADHD rats. 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 inattention, hyperactivity, and impulsiveness [26]. SHR was selected only when showed hyperactivity in open eld test. Meanwhile, Wistar-Kyoto rats (WKYR) weighing 210 10 g were used as the control in this study, according to the previous study [26]. The rats were housed under controlled temperature (20 2 C) and lighting conditions (07:0019:00), with food and water made available ad libitum throughout the experiments. Prior to the exercise and drug treatment, we tested the effects of different amount of treadmill exercise on the hyperactivity, as pre-test. Treadmill exercise was conducted as 10 min per day, 30 min per day, and 60 min per day for 10 days. Animals were randomly divided into four groups (n = 15): control group, ADHD group, ADHD and MPH-treated group, ADHD and treadmill exercise group, and ADHD and MPH-treated with treadmill exercise group. All animal procedures were performed in accordance with the animal care guidelines of the National Institutes of Health (NIH) and the Korean Academy of Medical Sciences. The rats in the MPH-treated group received 1 mg/kg MPH (Ritalin , Novartis Co., Bassel, Switzerland) orally once a day for 28 consecutive days, as previous study [19]. The rats in the treadmill exercise group were subjected to run on a treadmill for 30 min once a day, ve times a week, for 28 days. Exercise load for the running group consisted of running at a speed of 2 m/min for the rst 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 [2]. To evaluate activity, open eld test was performed as previously described [8]. Activity score in the open eld test was determined three times: one day before, 14 days after, and 28 days after the

starting of experiment. For open eld tests, the animals were randomly assigned to an order of testing and placed in a white square open eld arena (100 cm 100 cm) made of wood. As previous method [8], it was enclosed with 40 cm high walls and placed under strong illumination (200 lux). The arena was divided into 25 squares (each square is 20 cm 20 cm), dened as 9 central and 16 peripheral squares. The animal was placed in the center of the arena and left free to explore the environment for 1 min. After that time, the number of squares that the rat crossed was recorded for 5 min. Spatial learning memory was tested using a radial 8-arm maze apparatus as previously described [18]. Radial 8-arm maze apparatus consisted of a central octagonal plate (30 cm in diameter) and 8 radiating arms (50 cm in length and 10 cm in width). The apparatus was placed 1 m above the oor. A small receptacle lled with water (3 cm in diameter and 1 cm in depth) was located at the end of the arms. The rats were trained three times before the spatial learning test. The rats were deprived of water for 24 h and were allowed to explore the water for 5 min. Test was conducted 29 days after the starting of experiment. The time spent in seeking water at the end of the arms was counted. The test was terminated when a rat found water in all 8 arms or when >8 min elapsed. Re-entry into the previously visited arms was counted as an error. In addition, the number of correct choice before the rst error was counted. The rats were sacriced 29 days after the starting of experiment, immediately after radial 8-arm maze test. To begin the sacrice 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 xed with a freshly prepared solution consisting of 4% paraformaldehyde (PFA) in 100 mM phosphate buffer (PB) at pH 7.4. Brains were dissected, post-xed in the same xative overnight, and transferred to 30% sucrose solution for cryoprotection. Coronal sections of 40 m thickness were made with a freezing microtome (Leica, Nussloch, Germany). TH immunohistochemistry was performed as previously described [17]. Free-oating tissue sections were incubated overnight with mouse anti-TH antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) and the sections were then incubated for 1 h with biotinylated anti-mouse secondary antibody (1:2000, Vector Laboratories, Burlingame, CA). The sections were subsequently incubated with avidinbiotin-peroxidase complex (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% H2 O2 in 50 mM Trisbuffer (pH 7.6) for 3 min. The sections were then washed three times with PBS and mounted onto gelatin-coated slides. Western blot for BDNF was conducted as previously described [18]. The tissues were homogenized with lysis buffer, then ultracentrifuged at 50,000 rpm for 1 h. Protein (40 g) was separated on SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane (Schleicher & Schuell GmbH, Dassel, Germany). Mouse actin antibody (1:2000; Santa Cruz Biotech) and rabbit BDNF antibody (1:1000; Santa Cruz Biotech) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-mouse antibody for actin and anti-rabbit antibody for BDNF (1:2000; Vector Laboratories) were used as secondary antibodies. The area of striatum and SN from each slice was measured using Image-Pro Plus computer-assisted image analysis system (Media Cyberbetics Inc., Silver Spring, MD) attached to a light microscope (Olympus, Tokyo, Japan). The number of TH-positive cells in the SN was counted hemilaterally through a light microscope (Olympus). TH-immunoreactive ber density in the striatum was measured in 100 m 100 m square images of the striatum using an image analyzer (Multiscan, Fullerton, CA). To estimate TH-staining density, optical densities were corrected for the nonspecic background density, which was measured in the completely denervated

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parts of the striatum. TH-positive ber density ratios in the striatum were calculated as follows: optical density in the lesion side/optical density in the intact side. BDNF bands were calculated densitometrically using Image-Pro Plus computer-assisted image analysis system (Media Cyberbetics Inc.). Statistical analysis was performed using SPSS 12.0 statistical software (SPSS Inc., Chicago, IL). The difference among the groups was determined by one-way ANOVA followed by Duncans post hoc test, and the results are expressed as the mean standard error of the mean (S.E.M.). Signicance was set as P < 0.05. In the pre-test, treadmill exercise for 30 min per day showed most potent alleviating effect on hyperactivity in the ADHD rats (Fig. 1A). The activity score in the open eld test are presented in Fig. 1(B). The results showed that activity in the open eld test was signicantly higher in the rats of the ADHD group than that of the control group (P < 0.05). In contrast, activity was decreased by treadmill exercise and MPH treatment, as duration-dependently (P < 0.05). The numbers of correct and error in the radial 8-maze test are presented in Fig. 1(C and D). The rats in the ADHD group showed signicantly lower number of correct and higher number of error as compared to the rats in the control group (P < 0.05). However, treadmill exercise and MPH treatment increased the correct number and reduced error number in the rats of the ADHD group (P < 0.05). Photomicrographs of TH-immunoreactive bers in the striatum are presented in Fig. 2(A) . The results showed that TH ber expression in the striatum was signicantly decreased in the rats of the ADHD group than that of the control group (P < 0.05). In contrast, treadmill exercise and MPH treatment signicantly increased TH ber expression in the rats of the ADHD group (P < 0.05). Photomicrographs of TH-positive cells in the SN are presented in Fig. 2(B). The results showed that TH expression in the SN was signicantly decreased in the rats of the ADHD group than that of the control group (P < 0.05). In contrast, treadmill exercise and MPH treatment signicantly increased TH expression in the rats of the ADHD group (P < 0.05). The level of BDNF protein in the hippocampus was analyzed by Western blotting (Fig. 2C). The results showed that BDNF expression in the hippocampus was signicantly decreased in the rats of ADHD group than that of the control group (P < 0.05). In contrast, treadmill exercise and MPH treatment signicantly enhanced BDNF expression in the rats of the ADHD group (P < 0.05). SHR is the most commonly accepted animal model for ADHD, because SHR strain shows hyperactivity and spatial working memory decit [26]. We used SHR for ADHD, and this SHR displayed hyperactivity in the open eld test and spatial learning memory decit in the radial 8-arm maze task, as we expected. WKYR, used as the control group, did not show any behavior symptoms of ADHD. Dopamine is an important neurotransmitter in the regulation of attention and cognitive function [27,31]. Sagvolden et al. [27]

Fig. 1. Effects of treadmill exercise on activity and spatial learning memory. (A) Effects of different amount of treadmill exercise on the hyperactivity. (CON) control group, (ADHD) attention decit/hyperactivity disorder group, (ADHD-10 min-EX) attention decit/hyperactivity disorder and treadmill exercise for 10 min

# P < 0.05 compared to the 1st determination in each group. P < 0.05 compared to the 2nd determination in each group. (C) Number of correct in the radial 8arm maze test. (D) Number of error in the radial 8-arm maze test. (CON) control group, (ADHD) attention decit/hyperactivity disorder group, (ADHD-MPH) attention decit/hyperactivity disorder and methylphenidate-treated group, (ADHD-EX) attention decit/hyperactivity disorder and treadmill exercise group. *P < 0.05 compared to the control group. # P < 0.05 compared to the ADHD group.

group. # P < 0.05 compared to the ADHD group. P < 0.05 compared to the ADHD10 min-EX group. (B) Activity in the open eld test. ( ) Control group, ( ) attention decit/hyperactivity disorder group, ( ) attention decit/hyperactivity disorder and methylphenidate-treated group, ( ) attention decit/hyperactivity disorder and treadmill exercise group. *P < 0.05 compared to the control group.

group, (ADHD-30 min-EX) attention decit/hyperactivity disorder and treadmill exercise for 30 min group, (ADHD-60 min-EX) attention decit/hyperactivity disorder and treadmill exercise for 60 min group. *P < 0.05 compared to the control

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reported that hypofunction of dopamine system induces altering of reinforcement of behavior and deciency in extinction of previously reinforced behavior. These conditions give rise to delay aversion, development of hyperactivity in novel situations, impulsiveness, decient sustained attention, increased behavioral variability [27]. Volkow et al. [31] suggested that suppressed dopamine activity in caudate is associated with inattention in the adult with ADHD. May studies have suggested the possibility that dysfunction of dopamine signaling in the midbrain is one of the main mechanisms of hyperactivity and memory decit [5]. The present results showed that expressions of TH in the striatum and SN were significantly decreased in the rats of the ADHD group, suggesting that hyperactivity and memory decit in the ADHD rats are associated with down-regulation of dopamine in the striatum and SN. BDNF is a member of the neurotrophic family involved in neuronal survival and differentiation, and it is implicated in learning process due to its involvement in long-term potentiation in the hippocampus [23]. BDNF regulates synaptic plasticity, and decit in BDNF induced impairment of hippocampal long-term potentiation. This decit in synaptic function was amended by the exogenous application or overexpression of BDNF [24]. BDNF expression in the hippocampus was also suppressed in the old-aged rats with disturbance of short-term and spatial learning memories [18]. The present results showed that the expression of BDNF in the hippocampus was signicantly decreased in the rats of the ADHD group, suggesting that spatial learning memory decit in the ADHD rats is partly due to the decreased BDNF expression in the hippocampus. Physical movement improved working speed and social behavior problems and diminished hyperactivity in ADHD children [20]. In the present results, treadmill exercise showed ameliorating effect on ADHD-induced hyperactivity and spatial learning memory decit. Hattori et al. [13] reported that exercise activates the dopaminergic system and increase the availability of dopamine in the brain. Increased dopamine synthesis by exercise enhanced the survival of dopaminergic neurons in SN and treadmill running also alleviated some symptoms of Parkinsons disease [32]. Recovery of injured nigrostriatal dopamine neurons in 6-hydroxydopamine (6OHDA)-induced Parkinsons rats was facilitated by wheel running [22]. The present results showed that treadmill exercise increased TH expression in the striatum and SN of the ADHD rats. Ferris et al. [9] suggested that exercise directly enhances brain health and functions through a BDNF-mediated mechanism. There are many reports that treadmill exercise increases BDNF expression in the hippocampus [10,30]. Treadmill exercise also alleviated ageinduced decrease of BDNF expression in the hippocampus [18]. The present results showed that treadmill exercise increased BDNF in the hippocampus of the ADHD rats. MPH treatment enhanced attention and working memory, and increased dopamine release in prefrontal cortex and nucleus accumbens of rats [3]. On the other hand, chronic exposure of MPH in juvenile rats impaired spatial memory in the Morris water maze by decreasing BDNF expression and increasing acetylcholinesterase in prefrontal cortex [28]. Another study also showed that MPH could not correct working memory decit in ADHD [25]. The effects of MPH treatment for ADHD have still controversy, however, MPH is the most commonly prescribed medication for the ADHD patients.

Fig. 2. Effects of treadmill exercise on tyrosine hydroxylase (TH) and brain-derived neurotrophic factor (BDNF) expressions. (A) TH-immunoreactive bers in the striatum. The scale bar represents 40 m. (B) TH-positive cells in the substantia nigra. The scale bar represents 100 m. (C) Expression of BDNF in the hippocampus.

(CON) Control group, (ADHD) attention decit/hyperactivity disorder group, (ADHD-MPH) attention decit/hyperactivity disorder and methylphenidatetreated group, (ADHD-EX) attention decit/hyperactivity disorder and treadmill exercise group, (ADHD-MPH-EX) attention decit/hyperactivity disorder and methylphenidate-treated with treadmill exercise group. *P < 0.05 compared to the control group. # P < 0.05 compared to the ADHD group.

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In this study, treadmill exercise and MPH alleviated the ADHDinduced hyperactivity and spatial learning memory impairment. Expressions of TH and BDNF in the ADHD rats were also increased by treadmill exercise and MPH. Combination of MPH and treadmill exercise did not show potentiating effect on the expressions of TH and BDNF in the ADHD rats. These ndings provide a possibility that exercise may be used as an effective therapeutic intervention for ADHD patients as MPH treatment. Acknowledgement This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (313-20082-G00020). References
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