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Manipulation of dopamine metabolism contributes to attenuating innate high locomotor activity in ICR mice
Accepted Manuscript Title: Manipulation of dopamine metabolism contributes to attenuating innate high locomotor activity in ICR mice Author: Takeshi Yamaguchi Mao Nagasawa Hiromi Ikeda Momoko Kodaira Kimie Minaminaka Vishwajit S. Chowdhury Shinobu Yasuo Mitsuhiro Furuse PII: DOI: Reference:
S0166-4328(16)31153-6 http://dx.doi.org/doi:10.1016/j.bbr.2017.04.001 BBR 10791
To appear in:
Behavioural Brain Research
Received date: Revised date: Accepted date:
28-11-2016 27-2-2017 1-4-2017
Please cite this article as: Yamaguchi T, Nagasawa M, Ikeda H, Kodaira M, Minaminaka K, Chowdhury VS, Yasuo S, Furuse M, Manipulation of dopamine metabolism contributes to attenuating innate high locomotor activity in ICR mice, Behavioural Brain Research (2017), http://dx.doi.org/10.1016/j.bbr.2017.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Manipulation of dopamine metabolism contributes to attenuating innate high
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locomotor activity in ICR mice
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Takeshi Yamaguchi1, Mao Nagasawa1, Hiromi Ikeda1, Momoko Kodaira1, Kimie
Laboratory of Regulation in Metabolism and Behavior, Graduate School of
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Minaminaka1, Vishwajit S. Chowdhury2, Shinobu Yasuo1, Mitsuhiro Furuse1*
Bioresource and Bioenvironmental Science, Kyushu University, Fukuoka 812-8581,
Division for Experimental Natural Science, Faculty of Arts and Science, Kyushu
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Japan
Correspondence should be addressed to:
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University, Fukuoka 819-0395, Japan
Mitsuhiro Furuse, Ph.D.
Laboratory of Regulation in Metabolism and Behavior, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan TEL: (+81)-92-642-2953 FAX: (+81)-92-642-2954 E-mail: [email protected]
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Abstract
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Attention-deficit hyperactivity disorder (ADHD) is defined as attention deficiency, restlessness and distraction. The main characteristics of ADHD are hyperactivity, impulsiveness and carelessness. There is a possibility that these abnormal behaviors, in
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particular hyperactivity, are derived from abnormal dopamine (DA) neurotransmission.
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To elucidate the mechanism of high locomotor activity, the relationship between innate activity levels and brain monoamines and amino acids was investigated in this study.
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Differences in locomotor activity between ICR, C57BL/6J and CBA/N mice were determined using the open field test. Among the three strains, ICR mice showed the greatest amount of locomotor activity. The level of striatal and cerebellar DA was lower
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in ICR mice than in C57BL/6J mice, while the level of L-tyrosine (L-Tyr), a DA precursor, was higher in ICR mice. These results suggest that the metabolic conversion
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of L-Tyr to DA is lower in ICR mice than it is in C57BL/6J mice. Next, the effects of
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intraperitoneal injection of (6R)-5, 6, 7, 8-tetrahydro-L-biopterin dihydrochloride (BH4) (a co-enzyme for tyrosine hydroxylase) and L-3,4-dihydroxyphenylalanine (L-DOPA)
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on DA metabolism and behavior in ICR mice were investigated. The DA level in the brain was increased by BH4 administration, but the increased DA did not influence behavior. However, L-DOPA administration drastically lowered locomotor activity and increased DA concentration in several parts of the brain. The reduced locomotor activity may have been a consequence of the overproduction of DA. In conclusion, the high level of locomotor activity in ICR mice may be explained by a strain-specific DA metabolism.
Keywords: Locomotor activity, dopamine metabolism, mice, L-DOPA, BH4
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1. Introduction
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Abnormal locomotor activity is considered a sign of a disease syndrome. For
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instance, attention-deficit hyperactivity disorder (ADHD), characterized by impulsivity,
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inattentiveness and hyperactivity, is a behavioral disorder causing lack of attention,
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distraction and lack of composure [1,2]. The symptoms of ADHD generally occur
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during infancy, and are found in approximately 8-12 % of children [1]. ADHD patients
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have a high risk of other impairments of brain functions supervening, such as learning
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disorder and anxiety disorders [1,2]. Methylphenidate (MPH), a dopamine (DA)
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reuptake inhibitor, has been used to cure ADHD patients by suppressing the
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hyperactivity, though the detailed mechanism of ADHD symptoms is still unknown.
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MPH is a psychostimulant drug which has side effects on appetite and sleeping [3].
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Thus, it is necessary to develop other strategies with fewer or no side effects, using
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mild agents such as nutrients in the form of amino acids, and targeting the factors
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regulating the ADHD symptoms, including innate locomotor activity. In fact,
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supplementation of L-tyrosine (L-Tyr) has been found to reduce locomotor activity in
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Roborovskii hamsters [4].
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Various animal models exhibiting hyperactivity were considered suitable models
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of an ADHD. One of the models is a spontaneously hypertensive rat (SHR) that has the
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characteristic of developing high blood pressure naturally and that exhibits ADHD-like
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symptoms, including hyperactivity, in a novel environment [5]. In the SHR, it is
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suggested that the disrupted DA neurotransmission is related to the abnormal behaviors
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[5]. In fact, DA in the brain controls various behaviors (e.g. activity, attention and
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working memory) [5,6], and dysfunctions in DA systems are believed to be one of the
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causes of hyperactivity in ADHD patients [7,8]. Relationships between activity levels
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and brain DA levels have also been examined using hamsters. Roborovskii hamsters
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exhibit higher locomotor activity and lower dopamine (DA) levels in the whole brain 3
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compared with Djungarian hamsters [9]. Chronic supplementation of L-Tyr, a
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precursor of DA, lowered the locomotor activity in Roborovskii hamsters in their home
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cages [4]. These results suggest that nutritional factors that can modify the brain DA
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metabolism are useful for the regulation of locomotor activity in mice.
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Hyperactivity is a problematic behavior not only in humans but also in domestic
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and companion animals [10,11]. Considering the mouse to be a mammalian animal
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model, we chose mice to study locomotor activity and the brain DA metabolism. The
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first aim of the present study was to clarify the relationship between locomotor activity
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and the brain DA metabolism in three mouse strains, CBA/N, ICR, and C57BL/6J mice.
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Previous reports have shown the differences between the three strains [12,13]. Notably,
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ICR mice have a higher litter size and growth rate, with vigorous activity [14]. The
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second aim was to control locomotor activity by supplementation with a coenzyme or
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with a drug that modifies the DA metabolism. The initial step of DA synthesis is the
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metabolism from L-Tyr to L-dihydroxyphenylalanine (L-DOPA), catalyzed by the
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enzyme tyrosine hydroxylase (TH), a rate-limiting enzyme for DA synthesis [15].
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Thereafter, L-DOPA is rapidly decarboxylated to DA by aromatic L-amino acid
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decarboxylase (AADC). This study used the coenzyme of TH, (6R)-5, 6, 7,
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8-tetrahydro-L-biopterin dihydrochloride (BH4), and L-DOPA as enhancers of the DA
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contents.
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2. Materials and methods
2.1.
Animals
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Male 3-week-old CBA/N mice (n = 10), ICR mice (n = 10) and C57BL6/J mice (n
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= 10) were purchased from Japan SLC, Hamamatsu, Japan. For the experiments
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involving either BH4 or L-DOPA administration, male 3-week-old ICR mice were 4
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purchased from Japan SLC. In each experiment, mice were subjected to behavioral test
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and administration of drugs after 1 week of acclimation starting at 4 weeks old. Mice
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were housed in a plastic cage (5 animals per cage) under a light/dark cycle (lights on at
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08:00, lights off at 20:00) at a room temperature of 23ºC, and had ad libitum access to
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food (MF; Oriental Yeast, Tokyo, Japan) and water. The experimental procedures
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followed the Guidelines for Animal Experiments of the Faculty of Agriculture and the
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Graduate School of Kyushu University, as well as Japanese Law (No. 105) and a
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Notification (No. 6) by the Japanese Government.
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2.2. Procedures
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2.2.1. Experiment 1: Strain-dependent differences in locomotor activity and DA
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metabolism
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After the mice had been acclimated for 1 week, the locomotor activity of three
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strains of mice in a novel environment (n = 10) was recorded by employing the open
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field test (OFT) as described below. After 24 h, the locomotor activity of the mice was
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again observed in a second OFT. Five days after the second test, the animals were
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euthanized by cervical dislocation under anesthesia with isoflurane (Escain®, Mylan,
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Osaka, Japan) and decapitated to collect samples of brain parts (from the cerebellum,
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hippocampus, cerebral cortex, striatum, thalamus, hypothalamus and brain stem). The
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samples were frozen in liquid nitrogen and stored at -80ºC until analysis took place.
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The monoamine and amino acid concentrations in the brain regions were analyzed.
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2.2.2. Experiment 2: The effects on behavior and DA metabolism of a DA
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metabolism-related coenzyme or of a drug
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In the experiment involving BH4 administration, after 1 week of acclimation, the
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mice were divided into three groups (n = 10) — one control group and two BH4 groups.
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The control group and the BH4 groups were intraperitoneally administered saline or
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BH4 (25 mg or 50 mg/10 ml/kg, Wako Pure Chemical Industries, Ltd., Osaka, Japan),
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respectively. The dose of BH4 was decided according to the report by Homma et al.
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[16]. The behavior of the mice was then observed using the OFT at 1 h post
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administration as described below. Brain samples were obtained immediately after the
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OFT and analyzed as described in Experiment 1.
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In the experiment involving L-DOPA administration, male 3-week-old ICR mice
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were acclimated for 1 week, and then their behavior was observed using the OFT.
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Because there was a possibility of the presence of mice in each group with uneven
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level of locomotor activity, we first analyzed the locomotor activity in L-DOPA
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experiment before the acclimation, and subsequently mice were divided into three
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groups with similar level of activity. Then they were divided into three groups (n = 10)
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based on the total distance detected in the OFT — one control group and two L-DOPA
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groups. The L-DOPA groups were intraperitoneally administered L-DOPA (50 or 250
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mg/10 ml/kg, Wako Pure Chemical Industries) with carbidopa (50 mg/10 ml/kg, Wako
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Pure Chemical Industries) [17]. The carbidopa, an inhibiter of amino acid
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decarboxylase, does not enter the brain through the blood-brain barrier. Therefore, the
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carbidopa restrains the peripheral metabolism of L-DOPA, and L-DOPA can enter into
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the brain easily without modification. The behavior of the mice was then observed
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using the OFT at 30 min post administration as described below. Then brain samples
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were obtained and stored as described above.
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2.3. Open field test (OFT)
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This test was performed as a means of evaluating locomotor activity, exploratory 6
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activity and anxiety-like behavior in a novel environment. The mice were individually
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transferred to the open field arena from the home cages. The arena was a square box
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(Experiment 1, 40 cm × 40 cm, and height 40 cm) or a circular arena (Experiment 2,
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diameter 60 cm and height 35 cm), both made of wood and covered with a black
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plastic sheet to show up the white color of the mice. The test started when the animal
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was placed at the center of the box or arena under light (100 lux). Its behavior in the
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open field was digitally recorded for 5 min. After each test, the field was cleaned with
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an ethanol-water solution. The computer-based video tracking system that we used to
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analyze the activity in the OFT is only able to recognize mice with light-colored fur.
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C57BL/6J and CBA/N mice had dark fur, so we were not able to use the automatic
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video tracking system on these mice. Instead, we had to analyze the OFT manually for
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the C57BL/6J and CBA/N mice, as well as for the ICR mice in Experiment 1, using a
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square field to count the number of line crossings. In Experiment 2, we analyzed the
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ICR mice with light-colored fur in the circular arena using the video tracking system.
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In Experiment 1, the behavior in the open field was analyzed manually. The arena
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was divided equally into 25 sections (5 x 5 squares); the inside 9 squares were defined
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as the inner area and the outside 16 were defined as the outer area. The number of line
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crossings was evaluated as a parameter of motor activity, and the number of entries
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into the inner area was evaluated as a parameter of anxiety-like behavior [18,19]. The
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number of times rearing occurred was counted as a parameter of exploratory behavior
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[18], and latency prior to the first entry into the outer area was evaluated as an
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anxiety-like behavior. Latency prior to the first entry into the outer area includes
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freezing behaviors related to anxiety-like behavior caused by the novelty of the field
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[20]. In Experiment 2, the inside diameter of 40 cm was defined as the inner area. The
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following behavioral categories were analyzed automatically with a computer-based
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video tracking system (ANY-maze; Stoelting, Illinois, United States): total distance
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moved (motor activity); number of entries into the central area; time spent in the
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central area; and latency prior to the first entry into the outer area (anxiety-like
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behavior). A diagram of the study design, including an outline of the objective of each
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experiment, has been shown elsewhere (Fig. S2).
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2.4. Analysis of monoamines in the brain
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Levels of monoamines and their metabolites were determined by electrochemical
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detection using high performance liquid chromatography (HPLC). DA, serotonin
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(5-HT),
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dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-HT metabolite
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5-hydroxyindoleacetic
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3-methoxy-4-hydroxyphenylglycol (MHPG) were measured. In brief, the brain tissues
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(the cerebellum, hippocampus, cerebral cortex, striatum, thalamus, hypothalamus and
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brain stem) were homogenized and deproteinized in 0.2 M perchloric acid containing
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100 µM EDTA 2Na. The homogenates were left on ice for 30 min to deproteinize.
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Then, the homogenates were centrifuged at 20,000 × g for 15 min at 4ºC. After
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centrifugation, the supernatants were filtered through a 0.20 µm filter. These solutions
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were stocked. The stocked solutions were adjusted to approximately pH 3.0 by adding
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6 µl of 1 M sodium acetate per 50 µl of solution. 30 µl was injected into the HPLC
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system (Eicom, Kyoto, Japan) with a 150 mm x 3.0 mm ODS column (SC-5ODS,
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Eicom, Kyoto, Japan) at an applied potential of + 0.75 V. The mobile phase (pH 3.5)
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consisted of 0.1 M aceto-citric acid buffer, 17% methanol, 190 mg/l sodium 1-octane
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sulfonate and 5 mg/l EDTA 2Na at a flow rate of 0.5 ml/min. The values were
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expressed as pg/mg wet tissue.
(NE),
(5-HIAA)
(E),
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metabolite
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2.5. Analysis of free amino acids in the brain
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The concentration of free amino acids was analyzed by ultra performance liquid
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chromatography (UPLC) (using the AcquityTM UPLC system, comprised of Waters
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Binary Solvent Manager, Waters Sample Manager and Waters FLR Detector) with an
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ACCQTAGTM ULTRA C18 1.7 µm 2.1 x 100 mm column (Waters Corporation, USA).
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The same samples from the cerebellum, hippocampus and striatum that were used in
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the HPLC analysis were used in the UPLC analysis. Each 20 µl sample from the brain
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parts was dissolved with 2 µl of 1 M NaOH and vortexed. The excitation and emission
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wavelengths required for fluorescent detection of amino acids were 350 nm and 450
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nm, respectively. The system was operated with a flow rate of 0.25 ml/min at 30ºC.
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The UPLC gradient system (A = 50 mM sodium acetate (pH 5.9), B = methanol) was
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10-20% B over 3.2 min, 20% B for 1 min, 20-40% B over 3.6 min, 40% B for 1.2 min,
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40-60% B over 3.8 min, 60% B for 1 min, and 60-10% B over 0.01 min. Just before
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the analysis took place in the UPLC, each sample (10 µl) was transferred to a UPLC
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tube, and NAC/OPA (20 µl) and a borate buffer (70 µl) were added; then it was left for
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2 min in a dark room. The same method was used for the standard solutions containing
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16 L-amino acids, 16 D-amino acids, glycine, taurine and so on. The amino acid
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concentrations in the brains were expressed as pmol/mg wet tissue.
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2.6. Statistical analysis
In Experiment 1, the data from the OFT were analyzed using a repeated-measures
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two-way ANOVA and Tukey-Kramer as a post hoc test. The amino acid and
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monoamine data were analyzed using a one-way ANOVA and Tukey-Kramer as a post
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hoc test. In Experiment 2, regression analysis was applied to the data. In the
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experiment involving L-DOPA administration, the data were analyzed using a one-way
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ANOVA, as in Experiment 1. Differences with P<0.05 were considered significant.
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Data were expressed as means ± SEM. 9
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3. Results
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3.1. Experiment 1: Strain-dependent differences in locomotor activity and DA
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metabolism
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Fig. 1 shows the results from the first and second OFT. Line crossing in ICR mice
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was significantly (P < 0.05) higher than it was in the other two strains, and CBA/N
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mice exhibited the lowest values (Fig. 1 A). The number of entries into the inner area
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and the number of times rearing occurred were also significantly (P < 0.05) higher in
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ICR mice than in CBA/N and C57BL/6J mice (Figs. 1 B and C). CBA/N mice reared
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the lowest number of times. In ICR mice, the number of entries into the inner area was
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significantly lower in the second OFT than in the first OFT. Fig. 1 D shows latency
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prior to the first entry into the outer area. The value was significantly (P < 0.05) higher
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in CBA/N mice than in the other two strains in the first OFT. The latency prior to the
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first entry into the outer area was significantly (P < 0.05) lower in the second OFT than
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in the first OFT in CBA/N mice. As a result, it was confirmed that ICR mice had the
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greatest amount of locomotor activity.
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Fig. 2A shows differences between the three strains in DA levels in the cerebellum,
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striatum, brain stem and thalamus. DA levels in the cerebellum were significantly (P <
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0.05) lower in ICR mice than in the other two strains. Of the three strains, C57BL/6J
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mice showed the highest DA levels in the striatum and the lowest DA levels in the
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brain stem. The changes in DOPAC and HVA, the DA metabolites, in the three strains
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are shown in Figs. 2B and C, respectively. In the cerebellum, the DOPAC level was
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significantly (P < 0.0001) lower in ICR and C57BL/6J mice than in CBA/N mice. In
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the brain stem, the DOPAC level was highest in CBA/N mice and lowest in C57BL/6J
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mice. In the thalamus and striatum, the levels of HVA were significantly (P < 0.01) 10
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higher in C57BL/6J mice than in the other two strains. In the cerebellum, the levels of
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HVA were highest in C57BL/6J mice and lowest in ICR mice. In the brain stem, the
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level of NE in the CBA/N mice was significantly (P < 0.05) higher than in the ICR and
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C57BL/6J mice (Table S1). The serotonergic metabolites were not significantly
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different in any of the brain parts (Table S1). The findings indicated that the DA
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metabolism of ICR mice tends to be low.
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The L-Tyr level in the cerebellum was significantly (P < 0.005) higher in ICR
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mice than in the other strains (Fig. 3). In the striatum, the L-Tyr levels in ICR mice
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tended to be higher than in the other strains (P = 0.0881). In the cerebellum, levels of
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L-Arginine (L-Arg), L-Serine (L-Ser), L-Aspartate (L-Asp), and L-Alanine (L-Ala)
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were significantly (P < 0.05) higher in ICR mice than in the other strains (Table S2). In
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the striatum and hippocampus, the L-Ala level was higher in ICR mice than in either of
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the other two strains (Table S2). In sum, it was confirmed that the concentration of
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L-Tyr, a substrate of DA, tended to be high in ICR mice.
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3.2. Experiment 2: The effects on the behavior and DA metabolism of ICR mice of a
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DA metabolism-related coenzyme or of a drug
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The injection of BH4 did not affect total distance moved, entry into the inner area,
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or time spent in the inner area (data not shown). Fig. 4 shows the relationship between
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administered BH4 concentrations and levels of DA, HVA, and MHPG. The levels of
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DA in the cerebellum and hippocampus were significantly (P < 0.05) increased with
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the increase of BH4 (Fig 4 A). The levels of HVA, a DA metabolite, in the cerebellum,
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and of MHPG, an NE metabolite, in four regions of the brain, were also significantly (P
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< 0.05) increased with the increase in BH4 (Fig. 4 B and C). The contents of the 5-HT
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in those four brain regions did not have any correlation with the BH4 concentrations
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(data not shown). Amino acid levels in the cerebellum and hippocampus did not have 11
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any significant correlation with the BH4 concentrations (data not shown). In sum, the
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administration of BH4 did not affect locomotor activity, but it did enhance the DA
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metabolism.
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Fig. 5 shows the results of the OFT that were due to the influence of L-DOPA
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administration. L-DOPA administration (50 and 250 mg/kg) significantly (P < 0.05)
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decreased the total distance moved. The number of entries into the inner area was also
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significantly (P < 0.05) decreased by L-DOPA administration (50 and 250 mg/kg).
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Amount of time spent in the inner area was significantly (P < 0.05) decreased and
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latency prior to the first entry into the outer area was significantly (P < 0.05) increased
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in the 250 mg/kg L-DOPA group, but not in the 50 mg/kg L-DOPA group. It was
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mainly found that the administration of L-DOPA decreased locomotor activity in ICR
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mice.
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Fig. 6 shows the results relating to the DA level in four regions of the brain. In the
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L-DOPA administration groups (50 and 250 mg/kg), the levels of DA were
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significantly (P < 0.05) increased in the cerebellum (Fig. 6 A), the hippocampus (Fig. 6
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B), the striatum (Fig. 6 C), and the prefrontal cortex (Fig. 6 D). The DA metabolites
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and L-DOPA levels in several regions of the brain were also significantly (P < 0.05)
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increased by L-DOPA administration (Table S3). In the prefrontal cortex, the 5-HT
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level was significantly (P < 0.05) decreased by the administration of L-DOPA (Table
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S4). The 5-HT metabolite, 5-HIAA, was also significantly (P < 0.05) decreased in the
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hippocampus, the striatum, and the prefrontal cortex by the administration of L-DOPA
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(Table S4). Taken together, these results confirmed that the administration of L-DOPA
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increased the DA concentration in some regions of the brain.
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4. Discussion
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The aims of the present study were to investigate the differences in locomotor 12
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activity in three different mouse strains and find out the underlying mechanism of high
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locomotor activity in mice. High locomotor activity was found in ICR mice, and
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differences in DA metabolism were confirmed in the three strains studied. The effects
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of enhanced DA metabolism on locomotor activity were investigated concurrently.
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In Experiment 1, we examined the differences between three mouse strains in
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terms of behavior in the OFT. Of the three strains studied, ICR mice showed the
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greatest amount of locomotor activity, the least anxiety-like behavior (judged by the
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high number of entries they made into the inner area), and the greatest amount of
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exploratory behavior (judged by high amounts of rearing). The high values for
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inner-area entry and rearing may be considered incidental, a result of the greater
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amount of behavioral activity in ICR mice compared with the other two strains.
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However, when C57BL/6J and CBA/N mice were compared, there was a difference in
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number of line crossings in the first OFT, but there was no difference between these
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two strains in terms of inner-area entries. Therefore, we consider that difference in
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number of inner-area entries (an anxiety-like behavior) did not depend on difference in
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number of line crossings. Moreover, inner-area entry and line crossing were
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significantly higher for ICR mice than they were for C57BL/6J mice (Fig. S1). These
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results indicate that the low incidence of anxiety-like behavior and the high amount of
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innate locomotor activity in a novel environment that are found in ICR mice are
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exhibited independently. Based on the information of mice producers (Harlan Sprague
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Dawley, Inc. and Japan SLC, Inc.) and our observation, ICR mice are tame in nature,
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which may be one of the characteristics linked with their lower amount of anxiety.
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However, these behavioral differences may depend on the novelty of the open field. It
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has been reported that CBA/N mice exhibited a high amount of anxiety-like behavior
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compared with C57BL/6J mice in the OFT when a 10-min test period was used [21].
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Hence, if the OFT had been carried out for more than 5 min in the present study, the
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locomotor activity of the ICR mice may have decreased gradually, and that of the
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CBA/N mice may have increased gradually, as reported by the previous study [22].
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Therefore, the results of the present study suggest that the high amount of activity in
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the ICR mice and the low amount of activity in the CBA/N mice were acute behavioral
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responses to a novel environment. In contrast, the change in locomotor activity in the
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longer OFT was found to be small in C57BL/6J mice and the fecal pellet output less
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than occurred with other strains [22]. These findings suggest that the emotional
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behavior exhibited in the novel field depends greatly on the strains and the
305
experimental conditions used. In the present study, we also investigated learning ability
306
(whether mice recognized the field when it had already been seen once) by performing
307
the OFT twice. As shown in the Morris water maze test [23], spatial or circumstantial
308
learning can be examined by repeated observation in mice. Thus, we thought that if
309
mice could memorize the field in the first OFT, they may show learning-dependent
310
behavior in the second OFT. The latency prior to the first entry into the outer area in
311
CBA/N mice was greatly decreased in the second OFT. It is suggested that the CBA/N
312
mice recognized and memorized well, judging by the latency results.
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In the present study, 4-week-old juvenile mice were used to investigate locomotor
314
activity since ADHD is often observed in early childhood [2]. It has also been reported
315
that at 4 weeks old, the development of the DA receptors in mice is at a similar stage to
316
that of rats in the pubertal period [24]. This was the reason for choosing to use
317
4-week-old mice in the present study.
318
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When the contents of amino acids and monoamines in the brains of ICR mice and
319
C57BL/6J or CBA/N mice were compared in Experiment 1, the DA contents were
320
found to be lower in ICR mice than in C57BL/6J mice in the cerebellum and the
321
striatum. However, L-Tyr content in the cerebellum was higher in ICR mice than in
322
C57BL/6J mice. These data may suggest that the metabolism from L-Tyr to DA is
323
lower in ICR mice than in C57BL/6J mice. Furthermore, it was also found that the
324
ratio of DA and L-Tyr was lower in ICR mice (Fig. S3). The cerebellum is important 14
Page 14 of 29
for various behaviors, including cognitive functions, fear, and motor activity [25]. The
326
striatum is identified as a major brain region associated with behavioral alteration in
327
ADHD patients [1]. Therefore, we speculate that a low DA level in these brain regions
328
may be related to the difference between strains in terms of locomotor activity. That is,
329
the high locomotor activity found in ICR mice is related to lower DA contents in the
330
cerebellum and the striatum compared with C57BL/6J mice. Thus, in Experiment 2, we
331
investigated whether an enhancement of DA contents could decrease the high
332
locomotor activity of ICR mice. BH4 dose-dependently increased DA levels in the
333
cerebellum and the hippocampus, indicating that the metabolism of DA was enhanced
334
by BH4 administration. This finding is also supported by the increased levels of HVA
335
and MHPG found in several parts of the brain with increased levels of BH4, as
336
observed in previous studies [26]. Changes in the contents of DA and DA metabolites
337
differed among brain regions after BH4 administration, suggesting that the effect of
338
BH4 on the DA metabolic rate is region-specific. However, locomotor activity and
339
anxiety-like behavior were not modified by BH4 administration. This may be because
340
there was only a slight increase in DA levels after BH4 administration, and the increase
341
in DA levels brought about by BH4 injection may not have been sufficient to alter
342
locomotor activity and anxiety-like behavior.
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To enhance the brain DA metabolism strongly, instead of BH4 administration,
344
L-DOPA was administered to mice as a DA enhancer. Previous studies have confirmed
345
that administration of L-DOPA can clearly increase DA content and its release in the
346
brain [27,28]. In the present study, L-DOPA administration increased the DA level in
347
the brain in all regions examined, and locomotor activity in the OFT was decreased.
348
DA stimulants decreased locomotor activity in ADHD model mice [29], suggesting
349
that lower DA neurotransmission causes hyperactivity. The results obtained here were
350
consistent with the findings of the previous report. However, we only observed the DA
351
contents in the brain, and did not study the DA transmission system, so we did not look 15
Page 15 of 29
at DA release in the synaptic cleft, DA receptor activation, or DA transporter activity.
353
We need to examine these pathways in future to address how DA metabolism is related
354
to high locomotor activity in ICR mice. In particular, receptor and transporter activity
355
are worth addressing, because it has been reported that ADHD patients have an
356
abnormality in the genetic transfiguration of the dopamine D4 receptor [30] and in the
357
density of dopamine transporter 1 (DAT1) [31]. There has also been a report that the
358
DA projection path in different parts of the brain becomes atrophied in ADHD patients
359
[32,33]. Thus, anatomical analysis of the brain DA systems in ICR mice would also be
360
an interesting topic for a future study.
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The interpretation of our data depends on the notion that the increase in DA as a
362
result of L-DOPA administration was too substantial, and the effect appears to be in the
363
pharmacological range, but not in the physiological range. We could speculate that the
364
substantial increase in DA disrupted the balance of the neural system in the brain.
365
Therefore, there was a possibility that in the present study, the excessive upsurge of
366
DA may have caused a substantial decrease in locomotor activity in the mice. It has
367
been reported that an increase in DA has an adverse effect on synaptic functions in
368
mice [34]. It has also been suggested that an appropriate balance between the
369
dopaminergic and serotonergic systems is important for the normal expression of
370
emotional behavior [35]. The multiple neurotransmission systems are related to each
371
other in a complex way [29]. Thus, it is necessary to verify whether the increased level
372
of DA as a result of L-DOPA administration has any detrimental effect on mice. It has
373
been reported that DA and 5-HT levels in the brain could be regulated by various
374
methods, such as use of stimulants or exercise [36]. In the present study, L-DOPA
375
tended to decrease the 5-HT (or 5-HIAA, a 5-HT metabolite) content in some parts of
376
brain, while the DA level increased considerably. Therefore, L-DOPA acted as a strong
377
precursor to the production of abundant levels of DA without having a great effect on
378
the 5-HT metabolism. In a future study, we should elucidate more clearly the
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mechanism involved, or the relationship between behavior and the brain monoamine
380
neurotransmission systems, by observing the monoamine balance in the brain at the
381
same time as we observe emotional behavior.
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In conclusion, locomotor activity in a novel environment was higher in ICR mice
383
than in CBA/N and C57BL/6J mice, and the cause for the high activity may be
384
explained by a strain-specific DA metabolism.
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Acknowledgments
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Part of this project has been supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 26560058 to MF).
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508 509
Figure legends
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Fig. 1. The differences in behavior between the mouse strains in the first and second
512
open field test over 5 min. The number of mice used in each group was 10. Results are
513
expressed as means ± S.E.M. Groups with different letters are significantly different (P
514
< 0.05).
us
cr
511
515
Fig. 2. The differences in monoamine levels in each part of the brain. (A) DA, (B)
517
DOPAC, and (C) HVA levels are shown. The number of mice used in each group was
518
10. Results are expressed as means ± S.E.M. Groups with different letters are
519
significantly different (P < 0.05).
520
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516
Fig. 3. The results concerning L-Tyr levels in the cerebellum and striatum. The number
522
of mice used in each group was 10. Results are expressed as means ± S.E.M. Groups
523
with different letters are significantly different (P < 0.05).
te
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524
d
521
525
Fig. 4. The effect of BH4 injection on monoamine levels in each part of the brain in
526
ICR mice. (A) DA, (B) HVA, and (C) MHPG levels are shown. The number of mice
527
used in each group was 10. Results are expressed as means ± S.E.M.
528 529
Fig. 5. The effect of L-DOPA on behavior of mice in the open field test over 5 min.
530
The number of mice used in each group was 10. There were three groups—con: control
531
group; low: low concentration L-DOPA administration group; high: high concentration
532
L-DOPA administration group. Results are expressed as means ± S.E.M. Groups with
533
different letters are significantly different (P < 0.05).
534 22
Page 22 of 29
Fig. 6. The effect of L-DOPA on levels of DA in some parts of the brain (cerebellum,
536
hippocampus, striatum, prefrontal cortex) as a result of the administration of L-DOPA.
537
The number of mice used in each group was 10. Results are expressed as means ±
538
S.E.M. Groups with different letters are significantly different (P < 0.05).
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an
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23
Page 23 of 29
300 200
b bc
c
100
bc
0
Second
cr a
b bc c
c
First
Second
te
d
First
CBA/N C57BL/6J ICR
a
an
ab
70 60 50 40 30 20 10 0
The result of ANOVA Strain P < 0.05 Day P < 0.05 Interaction P < 0.05
us
CBA/N C57BL/6J ICR
a
Rearing (counts)
400
B
The result of ANOVA Strain P < 0.05 Day P < 0.05 Interaction P < 0.05
M
Line crossing (counts)
A
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Fig. 1
C
D The result of ANOVA Strain P < 0.05 Day P < 0.05 Interaction P < 0.05
40 30
a
20 10
CBA/N C57BL/6J ICR b
c
c
0
First
c c
Second
60 Latency time (s)
Ac ce p
Inner area entry (counts)
The result of ANOVA Strain P < 0.05 Day P < 0.05 Interaction P > 0.05
CBA/N C57BL/6J ICR
50 40
a
30 20 10 0
b b
b
First
b
b
Second
Page 24 of 29
Fig. 2
Striatum 50
2000 a
DA (pg/mg)
8
4
0
0
0
2000
a b
1500
20
1000 10
500
0
Ac ce p
0
Cerebellum
C 40
1500
b
140 120 100 80 60 40 20 0
Striatum
a
30
a
d
b
a
a
c
1000
b
0
0
Thalamus 250
a b
200
120 b
a a
100 50 0
150
a
a
150
c
Brain stem
Thalamus 400
a ab
a b
300
b
b
90 200
20 10
5
Brain stem
te
DOPAC (pg/mg)
Striatum
M
Cerebellum
10
an
10
20
a
15
20
500
30
HVA (pg/mg)
b
b
2
B
ab
30 1000
a
a
b
c
6
25 a
40
a
1500
b
Thalamus
cr
10
Brain stem
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Cerebellum
us
A
500
60 30
0
0
100 0
Page 25 of 29
Striatum
200
200 b
a
b
150 100
an
100
a
us
150
a
50
M
50
0
te
d
0
Ac ce p
L-Tyr (pmol/mg)
a
cr
Cerebellum
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Fig. 3
Page 26 of 29
60
4
40 20
P < 0.05
0
P < 0.05
0 0
25
50
0
25
M
600 500 400 300 200 100 0
y = 1.074x + 200.54 R² = 0.3336
d
300
P < 0.005
100 0
0
25
50
te
200
20 10
50
Striatum
y = 0.9053x + 474.66 R² = 0.3144 400
y = 0.0667x + 17.324 R² = 0.1615
P < 0.05
0
300
25
50
BH4 concentration (mg/kg)
Brain stem
Hippocampus
y = 0.446x + 170.43 R² = 0.328
y = 0.8515x + 186.42 R² = 0.2243
300 200
200
100
100 P < 0.001
P < 0.005
0
25
P < 0.05
0
0 50
0
25
50
0
25
50
Ac ce p
MHPG (pg/mg)
Cerebellum
30
0
BH4 concentration (mg/kg)
C
HVA (pg/mg)
6 2
y = 0.2185x + 26.396 R² = 0.1956
80
Cerebellum
us
y = 0.0221x + 3.051 R² = 0.138
8
B
Hippocampus
cr
Cerebellum
an
DA (pg/mg)
A
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Fig. 4
BH4 concentration (mg/kg)
Page 27 of 29
B
20 15 10
b
5
b
0 50 mg/kg 250 mg/kg
saline
b b
50 mg/kg 250 mg/kg
a ab
50 40
Ac ce p
Inner area times (s)
60
30 20 10
b
0
saline
50 mg/kg 250 mg/kg
D
6
a
5 Latency time (s)
70
te
C
d
M
saline
a
an
Total distance (m)
25
40 35 30 25 20 15 10 5 0
cr
a
Inner area entries (counts)
30
us
A
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Fig. 5
4
ab
3 2 1
b
0 saline
50 mg/kg 250 mg/kg
Page 28 of 29
1400
600 a
DA (pg/mg)
b
100 c
0
3000 2000 1000
d
D
a
b
0
600
b
400 200
c
0 saline
50 mg/kg 250 mg/kg
Prefrontal cortex
4000
a
Ac ce p
4000
te
Striatum
5000 DA (pg/mg)
50 mg/kg 250 mg/kg
M
200
saline
800
an
300
a
3500 DA (pg/mg)
DA (pg/mg)
1000
400
6000
a
us
1200
500
C
Hippocampus
B
cr
Cerebellum
A
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Fig. 6
3000 a
2500 2000 1500
b
1000 500 0
saline
50 mg/kg 250 mg/kg
saline
50 mg/kg 250 mg/kg
Page 29 of 29
S0166-4328(16)31153-6 http://dx.doi.org/doi:10.1016/j.bbr.2017.04.001 BBR 10791
To appear in:
Behavioural Brain Research
Received date: Revised date: Accepted date:
28-11-2016 27-2-2017 1-4-2017
Please cite this article as: Yamaguchi T, Nagasawa M, Ikeda H, Kodaira M, Minaminaka K, Chowdhury VS, Yasuo S, Furuse M, Manipulation of dopamine metabolism contributes to attenuating innate high locomotor activity in ICR mice, Behavioural Brain Research (2017), http://dx.doi.org/10.1016/j.bbr.2017.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Manipulation of dopamine metabolism contributes to attenuating innate high
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locomotor activity in ICR mice
cr
Takeshi Yamaguchi1, Mao Nagasawa1, Hiromi Ikeda1, Momoko Kodaira1, Kimie
Laboratory of Regulation in Metabolism and Behavior, Graduate School of
an
1
us
Minaminaka1, Vishwajit S. Chowdhury2, Shinobu Yasuo1, Mitsuhiro Furuse1*
Bioresource and Bioenvironmental Science, Kyushu University, Fukuoka 812-8581,
Division for Experimental Natural Science, Faculty of Arts and Science, Kyushu
d
2
M
Japan
Correspondence should be addressed to:
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*
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University, Fukuoka 819-0395, Japan
Mitsuhiro Furuse, Ph.D.
Laboratory of Regulation in Metabolism and Behavior, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan TEL: (+81)-92-642-2953 FAX: (+81)-92-642-2954 E-mail: [email protected]
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Abstract
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Attention-deficit hyperactivity disorder (ADHD) is defined as attention deficiency, restlessness and distraction. The main characteristics of ADHD are hyperactivity, impulsiveness and carelessness. There is a possibility that these abnormal behaviors, in
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particular hyperactivity, are derived from abnormal dopamine (DA) neurotransmission.
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To elucidate the mechanism of high locomotor activity, the relationship between innate activity levels and brain monoamines and amino acids was investigated in this study.
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Differences in locomotor activity between ICR, C57BL/6J and CBA/N mice were determined using the open field test. Among the three strains, ICR mice showed the greatest amount of locomotor activity. The level of striatal and cerebellar DA was lower
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in ICR mice than in C57BL/6J mice, while the level of L-tyrosine (L-Tyr), a DA precursor, was higher in ICR mice. These results suggest that the metabolic conversion
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of L-Tyr to DA is lower in ICR mice than it is in C57BL/6J mice. Next, the effects of
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intraperitoneal injection of (6R)-5, 6, 7, 8-tetrahydro-L-biopterin dihydrochloride (BH4) (a co-enzyme for tyrosine hydroxylase) and L-3,4-dihydroxyphenylalanine (L-DOPA)
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on DA metabolism and behavior in ICR mice were investigated. The DA level in the brain was increased by BH4 administration, but the increased DA did not influence behavior. However, L-DOPA administration drastically lowered locomotor activity and increased DA concentration in several parts of the brain. The reduced locomotor activity may have been a consequence of the overproduction of DA. In conclusion, the high level of locomotor activity in ICR mice may be explained by a strain-specific DA metabolism.
Keywords: Locomotor activity, dopamine metabolism, mice, L-DOPA, BH4
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1. Introduction
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Abnormal locomotor activity is considered a sign of a disease syndrome. For
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instance, attention-deficit hyperactivity disorder (ADHD), characterized by impulsivity,
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inattentiveness and hyperactivity, is a behavioral disorder causing lack of attention,
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distraction and lack of composure [1,2]. The symptoms of ADHD generally occur
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during infancy, and are found in approximately 8-12 % of children [1]. ADHD patients
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have a high risk of other impairments of brain functions supervening, such as learning
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disorder and anxiety disorders [1,2]. Methylphenidate (MPH), a dopamine (DA)
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reuptake inhibitor, has been used to cure ADHD patients by suppressing the
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hyperactivity, though the detailed mechanism of ADHD symptoms is still unknown.
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MPH is a psychostimulant drug which has side effects on appetite and sleeping [3].
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Thus, it is necessary to develop other strategies with fewer or no side effects, using
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mild agents such as nutrients in the form of amino acids, and targeting the factors
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regulating the ADHD symptoms, including innate locomotor activity. In fact,
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supplementation of L-tyrosine (L-Tyr) has been found to reduce locomotor activity in
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Roborovskii hamsters [4].
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Various animal models exhibiting hyperactivity were considered suitable models
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of an ADHD. One of the models is a spontaneously hypertensive rat (SHR) that has the
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characteristic of developing high blood pressure naturally and that exhibits ADHD-like
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symptoms, including hyperactivity, in a novel environment [5]. In the SHR, it is
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suggested that the disrupted DA neurotransmission is related to the abnormal behaviors
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[5]. In fact, DA in the brain controls various behaviors (e.g. activity, attention and
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working memory) [5,6], and dysfunctions in DA systems are believed to be one of the
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causes of hyperactivity in ADHD patients [7,8]. Relationships between activity levels
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and brain DA levels have also been examined using hamsters. Roborovskii hamsters
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exhibit higher locomotor activity and lower dopamine (DA) levels in the whole brain 3
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compared with Djungarian hamsters [9]. Chronic supplementation of L-Tyr, a
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precursor of DA, lowered the locomotor activity in Roborovskii hamsters in their home
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cages [4]. These results suggest that nutritional factors that can modify the brain DA
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metabolism are useful for the regulation of locomotor activity in mice.
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Hyperactivity is a problematic behavior not only in humans but also in domestic
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and companion animals [10,11]. Considering the mouse to be a mammalian animal
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model, we chose mice to study locomotor activity and the brain DA metabolism. The
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first aim of the present study was to clarify the relationship between locomotor activity
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and the brain DA metabolism in three mouse strains, CBA/N, ICR, and C57BL/6J mice.
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Previous reports have shown the differences between the three strains [12,13]. Notably,
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ICR mice have a higher litter size and growth rate, with vigorous activity [14]. The
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second aim was to control locomotor activity by supplementation with a coenzyme or
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with a drug that modifies the DA metabolism. The initial step of DA synthesis is the
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metabolism from L-Tyr to L-dihydroxyphenylalanine (L-DOPA), catalyzed by the
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enzyme tyrosine hydroxylase (TH), a rate-limiting enzyme for DA synthesis [15].
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Thereafter, L-DOPA is rapidly decarboxylated to DA by aromatic L-amino acid
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decarboxylase (AADC). This study used the coenzyme of TH, (6R)-5, 6, 7,
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8-tetrahydro-L-biopterin dihydrochloride (BH4), and L-DOPA as enhancers of the DA
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contents.
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2. Materials and methods
2.1.
Animals
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Male 3-week-old CBA/N mice (n = 10), ICR mice (n = 10) and C57BL6/J mice (n
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= 10) were purchased from Japan SLC, Hamamatsu, Japan. For the experiments
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involving either BH4 or L-DOPA administration, male 3-week-old ICR mice were 4
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purchased from Japan SLC. In each experiment, mice were subjected to behavioral test
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and administration of drugs after 1 week of acclimation starting at 4 weeks old. Mice
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were housed in a plastic cage (5 animals per cage) under a light/dark cycle (lights on at
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08:00, lights off at 20:00) at a room temperature of 23ºC, and had ad libitum access to
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food (MF; Oriental Yeast, Tokyo, Japan) and water. The experimental procedures
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followed the Guidelines for Animal Experiments of the Faculty of Agriculture and the
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Graduate School of Kyushu University, as well as Japanese Law (No. 105) and a
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Notification (No. 6) by the Japanese Government.
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2.2. Procedures
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2.2.1. Experiment 1: Strain-dependent differences in locomotor activity and DA
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metabolism
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After the mice had been acclimated for 1 week, the locomotor activity of three
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strains of mice in a novel environment (n = 10) was recorded by employing the open
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field test (OFT) as described below. After 24 h, the locomotor activity of the mice was
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again observed in a second OFT. Five days after the second test, the animals were
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euthanized by cervical dislocation under anesthesia with isoflurane (Escain®, Mylan,
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Osaka, Japan) and decapitated to collect samples of brain parts (from the cerebellum,
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hippocampus, cerebral cortex, striatum, thalamus, hypothalamus and brain stem). The
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samples were frozen in liquid nitrogen and stored at -80ºC until analysis took place.
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The monoamine and amino acid concentrations in the brain regions were analyzed.
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2.2.2. Experiment 2: The effects on behavior and DA metabolism of a DA
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metabolism-related coenzyme or of a drug
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In the experiment involving BH4 administration, after 1 week of acclimation, the
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mice were divided into three groups (n = 10) — one control group and two BH4 groups.
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The control group and the BH4 groups were intraperitoneally administered saline or
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BH4 (25 mg or 50 mg/10 ml/kg, Wako Pure Chemical Industries, Ltd., Osaka, Japan),
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respectively. The dose of BH4 was decided according to the report by Homma et al.
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[16]. The behavior of the mice was then observed using the OFT at 1 h post
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administration as described below. Brain samples were obtained immediately after the
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OFT and analyzed as described in Experiment 1.
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In the experiment involving L-DOPA administration, male 3-week-old ICR mice
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were acclimated for 1 week, and then their behavior was observed using the OFT.
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Because there was a possibility of the presence of mice in each group with uneven
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level of locomotor activity, we first analyzed the locomotor activity in L-DOPA
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experiment before the acclimation, and subsequently mice were divided into three
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groups with similar level of activity. Then they were divided into three groups (n = 10)
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based on the total distance detected in the OFT — one control group and two L-DOPA
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groups. The L-DOPA groups were intraperitoneally administered L-DOPA (50 or 250
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mg/10 ml/kg, Wako Pure Chemical Industries) with carbidopa (50 mg/10 ml/kg, Wako
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Pure Chemical Industries) [17]. The carbidopa, an inhibiter of amino acid
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decarboxylase, does not enter the brain through the blood-brain barrier. Therefore, the
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carbidopa restrains the peripheral metabolism of L-DOPA, and L-DOPA can enter into
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the brain easily without modification. The behavior of the mice was then observed
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using the OFT at 30 min post administration as described below. Then brain samples
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were obtained and stored as described above.
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2.3. Open field test (OFT)
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This test was performed as a means of evaluating locomotor activity, exploratory 6
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activity and anxiety-like behavior in a novel environment. The mice were individually
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transferred to the open field arena from the home cages. The arena was a square box
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(Experiment 1, 40 cm × 40 cm, and height 40 cm) or a circular arena (Experiment 2,
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diameter 60 cm and height 35 cm), both made of wood and covered with a black
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plastic sheet to show up the white color of the mice. The test started when the animal
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was placed at the center of the box or arena under light (100 lux). Its behavior in the
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open field was digitally recorded for 5 min. After each test, the field was cleaned with
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an ethanol-water solution. The computer-based video tracking system that we used to
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analyze the activity in the OFT is only able to recognize mice with light-colored fur.
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C57BL/6J and CBA/N mice had dark fur, so we were not able to use the automatic
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video tracking system on these mice. Instead, we had to analyze the OFT manually for
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the C57BL/6J and CBA/N mice, as well as for the ICR mice in Experiment 1, using a
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square field to count the number of line crossings. In Experiment 2, we analyzed the
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ICR mice with light-colored fur in the circular arena using the video tracking system.
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In Experiment 1, the behavior in the open field was analyzed manually. The arena
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was divided equally into 25 sections (5 x 5 squares); the inside 9 squares were defined
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as the inner area and the outside 16 were defined as the outer area. The number of line
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crossings was evaluated as a parameter of motor activity, and the number of entries
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into the inner area was evaluated as a parameter of anxiety-like behavior [18,19]. The
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number of times rearing occurred was counted as a parameter of exploratory behavior
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[18], and latency prior to the first entry into the outer area was evaluated as an
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anxiety-like behavior. Latency prior to the first entry into the outer area includes
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freezing behaviors related to anxiety-like behavior caused by the novelty of the field
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[20]. In Experiment 2, the inside diameter of 40 cm was defined as the inner area. The
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following behavioral categories were analyzed automatically with a computer-based
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video tracking system (ANY-maze; Stoelting, Illinois, United States): total distance
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moved (motor activity); number of entries into the central area; time spent in the
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central area; and latency prior to the first entry into the outer area (anxiety-like
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behavior). A diagram of the study design, including an outline of the objective of each
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experiment, has been shown elsewhere (Fig. S2).
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2.4. Analysis of monoamines in the brain
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Levels of monoamines and their metabolites were determined by electrochemical
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detection using high performance liquid chromatography (HPLC). DA, serotonin
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(5-HT),
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dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-HT metabolite
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5-hydroxyindoleacetic
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3-methoxy-4-hydroxyphenylglycol (MHPG) were measured. In brief, the brain tissues
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(the cerebellum, hippocampus, cerebral cortex, striatum, thalamus, hypothalamus and
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brain stem) were homogenized and deproteinized in 0.2 M perchloric acid containing
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100 µM EDTA 2Na. The homogenates were left on ice for 30 min to deproteinize.
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Then, the homogenates were centrifuged at 20,000 × g for 15 min at 4ºC. After
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centrifugation, the supernatants were filtered through a 0.20 µm filter. These solutions
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were stocked. The stocked solutions were adjusted to approximately pH 3.0 by adding
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6 µl of 1 M sodium acetate per 50 µl of solution. 30 µl was injected into the HPLC
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system (Eicom, Kyoto, Japan) with a 150 mm x 3.0 mm ODS column (SC-5ODS,
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Eicom, Kyoto, Japan) at an applied potential of + 0.75 V. The mobile phase (pH 3.5)
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consisted of 0.1 M aceto-citric acid buffer, 17% methanol, 190 mg/l sodium 1-octane
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sulfonate and 5 mg/l EDTA 2Na at a flow rate of 0.5 ml/min. The values were
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expressed as pg/mg wet tissue.
(NE),
(5-HIAA)
(E),
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NE
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metabolite
metabolite
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norepinephrine
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2.5. Analysis of free amino acids in the brain
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The concentration of free amino acids was analyzed by ultra performance liquid
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chromatography (UPLC) (using the AcquityTM UPLC system, comprised of Waters
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Binary Solvent Manager, Waters Sample Manager and Waters FLR Detector) with an
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ACCQTAGTM ULTRA C18 1.7 µm 2.1 x 100 mm column (Waters Corporation, USA).
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The same samples from the cerebellum, hippocampus and striatum that were used in
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the HPLC analysis were used in the UPLC analysis. Each 20 µl sample from the brain
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parts was dissolved with 2 µl of 1 M NaOH and vortexed. The excitation and emission
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wavelengths required for fluorescent detection of amino acids were 350 nm and 450
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nm, respectively. The system was operated with a flow rate of 0.25 ml/min at 30ºC.
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The UPLC gradient system (A = 50 mM sodium acetate (pH 5.9), B = methanol) was
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10-20% B over 3.2 min, 20% B for 1 min, 20-40% B over 3.6 min, 40% B for 1.2 min,
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40-60% B over 3.8 min, 60% B for 1 min, and 60-10% B over 0.01 min. Just before
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the analysis took place in the UPLC, each sample (10 µl) was transferred to a UPLC
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tube, and NAC/OPA (20 µl) and a borate buffer (70 µl) were added; then it was left for
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2 min in a dark room. The same method was used for the standard solutions containing
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16 L-amino acids, 16 D-amino acids, glycine, taurine and so on. The amino acid
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concentrations in the brains were expressed as pmol/mg wet tissue.
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2.6. Statistical analysis
In Experiment 1, the data from the OFT were analyzed using a repeated-measures
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two-way ANOVA and Tukey-Kramer as a post hoc test. The amino acid and
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monoamine data were analyzed using a one-way ANOVA and Tukey-Kramer as a post
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hoc test. In Experiment 2, regression analysis was applied to the data. In the
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experiment involving L-DOPA administration, the data were analyzed using a one-way
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ANOVA, as in Experiment 1. Differences with P<0.05 were considered significant.
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Data were expressed as means ± SEM. 9
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190 191
3. Results
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3.1. Experiment 1: Strain-dependent differences in locomotor activity and DA
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metabolism
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Fig. 1 shows the results from the first and second OFT. Line crossing in ICR mice
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was significantly (P < 0.05) higher than it was in the other two strains, and CBA/N
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mice exhibited the lowest values (Fig. 1 A). The number of entries into the inner area
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and the number of times rearing occurred were also significantly (P < 0.05) higher in
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ICR mice than in CBA/N and C57BL/6J mice (Figs. 1 B and C). CBA/N mice reared
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the lowest number of times. In ICR mice, the number of entries into the inner area was
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significantly lower in the second OFT than in the first OFT. Fig. 1 D shows latency
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prior to the first entry into the outer area. The value was significantly (P < 0.05) higher
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in CBA/N mice than in the other two strains in the first OFT. The latency prior to the
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first entry into the outer area was significantly (P < 0.05) lower in the second OFT than
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in the first OFT in CBA/N mice. As a result, it was confirmed that ICR mice had the
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greatest amount of locomotor activity.
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Fig. 2A shows differences between the three strains in DA levels in the cerebellum,
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striatum, brain stem and thalamus. DA levels in the cerebellum were significantly (P <
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0.05) lower in ICR mice than in the other two strains. Of the three strains, C57BL/6J
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mice showed the highest DA levels in the striatum and the lowest DA levels in the
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brain stem. The changes in DOPAC and HVA, the DA metabolites, in the three strains
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are shown in Figs. 2B and C, respectively. In the cerebellum, the DOPAC level was
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significantly (P < 0.0001) lower in ICR and C57BL/6J mice than in CBA/N mice. In
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the brain stem, the DOPAC level was highest in CBA/N mice and lowest in C57BL/6J
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mice. In the thalamus and striatum, the levels of HVA were significantly (P < 0.01) 10
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higher in C57BL/6J mice than in the other two strains. In the cerebellum, the levels of
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HVA were highest in C57BL/6J mice and lowest in ICR mice. In the brain stem, the
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level of NE in the CBA/N mice was significantly (P < 0.05) higher than in the ICR and
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C57BL/6J mice (Table S1). The serotonergic metabolites were not significantly
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different in any of the brain parts (Table S1). The findings indicated that the DA
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metabolism of ICR mice tends to be low.
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The L-Tyr level in the cerebellum was significantly (P < 0.005) higher in ICR
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mice than in the other strains (Fig. 3). In the striatum, the L-Tyr levels in ICR mice
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tended to be higher than in the other strains (P = 0.0881). In the cerebellum, levels of
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L-Arginine (L-Arg), L-Serine (L-Ser), L-Aspartate (L-Asp), and L-Alanine (L-Ala)
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were significantly (P < 0.05) higher in ICR mice than in the other strains (Table S2). In
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the striatum and hippocampus, the L-Ala level was higher in ICR mice than in either of
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the other two strains (Table S2). In sum, it was confirmed that the concentration of
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L-Tyr, a substrate of DA, tended to be high in ICR mice.
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3.2. Experiment 2: The effects on the behavior and DA metabolism of ICR mice of a
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DA metabolism-related coenzyme or of a drug
234 235
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The injection of BH4 did not affect total distance moved, entry into the inner area,
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or time spent in the inner area (data not shown). Fig. 4 shows the relationship between
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administered BH4 concentrations and levels of DA, HVA, and MHPG. The levels of
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DA in the cerebellum and hippocampus were significantly (P < 0.05) increased with
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the increase of BH4 (Fig 4 A). The levels of HVA, a DA metabolite, in the cerebellum,
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and of MHPG, an NE metabolite, in four regions of the brain, were also significantly (P
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< 0.05) increased with the increase in BH4 (Fig. 4 B and C). The contents of the 5-HT
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in those four brain regions did not have any correlation with the BH4 concentrations
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(data not shown). Amino acid levels in the cerebellum and hippocampus did not have 11
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any significant correlation with the BH4 concentrations (data not shown). In sum, the
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administration of BH4 did not affect locomotor activity, but it did enhance the DA
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metabolism.
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Fig. 5 shows the results of the OFT that were due to the influence of L-DOPA
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administration. L-DOPA administration (50 and 250 mg/kg) significantly (P < 0.05)
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decreased the total distance moved. The number of entries into the inner area was also
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significantly (P < 0.05) decreased by L-DOPA administration (50 and 250 mg/kg).
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Amount of time spent in the inner area was significantly (P < 0.05) decreased and
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latency prior to the first entry into the outer area was significantly (P < 0.05) increased
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in the 250 mg/kg L-DOPA group, but not in the 50 mg/kg L-DOPA group. It was
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mainly found that the administration of L-DOPA decreased locomotor activity in ICR
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mice.
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Fig. 6 shows the results relating to the DA level in four regions of the brain. In the
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L-DOPA administration groups (50 and 250 mg/kg), the levels of DA were
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significantly (P < 0.05) increased in the cerebellum (Fig. 6 A), the hippocampus (Fig. 6
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B), the striatum (Fig. 6 C), and the prefrontal cortex (Fig. 6 D). The DA metabolites
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and L-DOPA levels in several regions of the brain were also significantly (P < 0.05)
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increased by L-DOPA administration (Table S3). In the prefrontal cortex, the 5-HT
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level was significantly (P < 0.05) decreased by the administration of L-DOPA (Table
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S4). The 5-HT metabolite, 5-HIAA, was also significantly (P < 0.05) decreased in the
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hippocampus, the striatum, and the prefrontal cortex by the administration of L-DOPA
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(Table S4). Taken together, these results confirmed that the administration of L-DOPA
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increased the DA concentration in some regions of the brain.
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4. Discussion
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The aims of the present study were to investigate the differences in locomotor 12
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activity in three different mouse strains and find out the underlying mechanism of high
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locomotor activity in mice. High locomotor activity was found in ICR mice, and
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differences in DA metabolism were confirmed in the three strains studied. The effects
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of enhanced DA metabolism on locomotor activity were investigated concurrently.
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In Experiment 1, we examined the differences between three mouse strains in
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terms of behavior in the OFT. Of the three strains studied, ICR mice showed the
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greatest amount of locomotor activity, the least anxiety-like behavior (judged by the
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high number of entries they made into the inner area), and the greatest amount of
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exploratory behavior (judged by high amounts of rearing). The high values for
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inner-area entry and rearing may be considered incidental, a result of the greater
281
amount of behavioral activity in ICR mice compared with the other two strains.
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However, when C57BL/6J and CBA/N mice were compared, there was a difference in
283
number of line crossings in the first OFT, but there was no difference between these
284
two strains in terms of inner-area entries. Therefore, we consider that difference in
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number of inner-area entries (an anxiety-like behavior) did not depend on difference in
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number of line crossings. Moreover, inner-area entry and line crossing were
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significantly higher for ICR mice than they were for C57BL/6J mice (Fig. S1). These
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results indicate that the low incidence of anxiety-like behavior and the high amount of
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innate locomotor activity in a novel environment that are found in ICR mice are
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exhibited independently. Based on the information of mice producers (Harlan Sprague
291
Dawley, Inc. and Japan SLC, Inc.) and our observation, ICR mice are tame in nature,
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which may be one of the characteristics linked with their lower amount of anxiety.
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However, these behavioral differences may depend on the novelty of the open field. It
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has been reported that CBA/N mice exhibited a high amount of anxiety-like behavior
295
compared with C57BL/6J mice in the OFT when a 10-min test period was used [21].
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Hence, if the OFT had been carried out for more than 5 min in the present study, the
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locomotor activity of the ICR mice may have decreased gradually, and that of the
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CBA/N mice may have increased gradually, as reported by the previous study [22].
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Therefore, the results of the present study suggest that the high amount of activity in
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the ICR mice and the low amount of activity in the CBA/N mice were acute behavioral
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responses to a novel environment. In contrast, the change in locomotor activity in the
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longer OFT was found to be small in C57BL/6J mice and the fecal pellet output less
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than occurred with other strains [22]. These findings suggest that the emotional
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behavior exhibited in the novel field depends greatly on the strains and the
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experimental conditions used. In the present study, we also investigated learning ability
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(whether mice recognized the field when it had already been seen once) by performing
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the OFT twice. As shown in the Morris water maze test [23], spatial or circumstantial
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learning can be examined by repeated observation in mice. Thus, we thought that if
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mice could memorize the field in the first OFT, they may show learning-dependent
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behavior in the second OFT. The latency prior to the first entry into the outer area in
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CBA/N mice was greatly decreased in the second OFT. It is suggested that the CBA/N
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mice recognized and memorized well, judging by the latency results.
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In the present study, 4-week-old juvenile mice were used to investigate locomotor
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activity since ADHD is often observed in early childhood [2]. It has also been reported
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that at 4 weeks old, the development of the DA receptors in mice is at a similar stage to
316
that of rats in the pubertal period [24]. This was the reason for choosing to use
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4-week-old mice in the present study.
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When the contents of amino acids and monoamines in the brains of ICR mice and
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C57BL/6J or CBA/N mice were compared in Experiment 1, the DA contents were
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found to be lower in ICR mice than in C57BL/6J mice in the cerebellum and the
321
striatum. However, L-Tyr content in the cerebellum was higher in ICR mice than in
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C57BL/6J mice. These data may suggest that the metabolism from L-Tyr to DA is
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lower in ICR mice than in C57BL/6J mice. Furthermore, it was also found that the
324
ratio of DA and L-Tyr was lower in ICR mice (Fig. S3). The cerebellum is important 14
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for various behaviors, including cognitive functions, fear, and motor activity [25]. The
326
striatum is identified as a major brain region associated with behavioral alteration in
327
ADHD patients [1]. Therefore, we speculate that a low DA level in these brain regions
328
may be related to the difference between strains in terms of locomotor activity. That is,
329
the high locomotor activity found in ICR mice is related to lower DA contents in the
330
cerebellum and the striatum compared with C57BL/6J mice. Thus, in Experiment 2, we
331
investigated whether an enhancement of DA contents could decrease the high
332
locomotor activity of ICR mice. BH4 dose-dependently increased DA levels in the
333
cerebellum and the hippocampus, indicating that the metabolism of DA was enhanced
334
by BH4 administration. This finding is also supported by the increased levels of HVA
335
and MHPG found in several parts of the brain with increased levels of BH4, as
336
observed in previous studies [26]. Changes in the contents of DA and DA metabolites
337
differed among brain regions after BH4 administration, suggesting that the effect of
338
BH4 on the DA metabolic rate is region-specific. However, locomotor activity and
339
anxiety-like behavior were not modified by BH4 administration. This may be because
340
there was only a slight increase in DA levels after BH4 administration, and the increase
341
in DA levels brought about by BH4 injection may not have been sufficient to alter
342
locomotor activity and anxiety-like behavior.
cr
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d
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To enhance the brain DA metabolism strongly, instead of BH4 administration,
344
L-DOPA was administered to mice as a DA enhancer. Previous studies have confirmed
345
that administration of L-DOPA can clearly increase DA content and its release in the
346
brain [27,28]. In the present study, L-DOPA administration increased the DA level in
347
the brain in all regions examined, and locomotor activity in the OFT was decreased.
348
DA stimulants decreased locomotor activity in ADHD model mice [29], suggesting
349
that lower DA neurotransmission causes hyperactivity. The results obtained here were
350
consistent with the findings of the previous report. However, we only observed the DA
351
contents in the brain, and did not study the DA transmission system, so we did not look 15
Page 15 of 29
at DA release in the synaptic cleft, DA receptor activation, or DA transporter activity.
353
We need to examine these pathways in future to address how DA metabolism is related
354
to high locomotor activity in ICR mice. In particular, receptor and transporter activity
355
are worth addressing, because it has been reported that ADHD patients have an
356
abnormality in the genetic transfiguration of the dopamine D4 receptor [30] and in the
357
density of dopamine transporter 1 (DAT1) [31]. There has also been a report that the
358
DA projection path in different parts of the brain becomes atrophied in ADHD patients
359
[32,33]. Thus, anatomical analysis of the brain DA systems in ICR mice would also be
360
an interesting topic for a future study.
an
us
cr
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352
The interpretation of our data depends on the notion that the increase in DA as a
362
result of L-DOPA administration was too substantial, and the effect appears to be in the
363
pharmacological range, but not in the physiological range. We could speculate that the
364
substantial increase in DA disrupted the balance of the neural system in the brain.
365
Therefore, there was a possibility that in the present study, the excessive upsurge of
366
DA may have caused a substantial decrease in locomotor activity in the mice. It has
367
been reported that an increase in DA has an adverse effect on synaptic functions in
368
mice [34]. It has also been suggested that an appropriate balance between the
369
dopaminergic and serotonergic systems is important for the normal expression of
370
emotional behavior [35]. The multiple neurotransmission systems are related to each
371
other in a complex way [29]. Thus, it is necessary to verify whether the increased level
372
of DA as a result of L-DOPA administration has any detrimental effect on mice. It has
373
been reported that DA and 5-HT levels in the brain could be regulated by various
374
methods, such as use of stimulants or exercise [36]. In the present study, L-DOPA
375
tended to decrease the 5-HT (or 5-HIAA, a 5-HT metabolite) content in some parts of
376
brain, while the DA level increased considerably. Therefore, L-DOPA acted as a strong
377
precursor to the production of abundant levels of DA without having a great effect on
378
the 5-HT metabolism. In a future study, we should elucidate more clearly the
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Page 16 of 29
mechanism involved, or the relationship between behavior and the brain monoamine
380
neurotransmission systems, by observing the monoamine balance in the brain at the
381
same time as we observe emotional behavior.
ip t
379
In conclusion, locomotor activity in a novel environment was higher in ICR mice
383
than in CBA/N and C57BL/6J mice, and the cause for the high activity may be
384
explained by a strain-specific DA metabolism.
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Acknowledgments
389
Part of this project has been supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 26560058 to MF).
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388
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390
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508 509
Figure legends
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Fig. 1. The differences in behavior between the mouse strains in the first and second
512
open field test over 5 min. The number of mice used in each group was 10. Results are
513
expressed as means ± S.E.M. Groups with different letters are significantly different (P
514
< 0.05).
us
cr
511
515
Fig. 2. The differences in monoamine levels in each part of the brain. (A) DA, (B)
517
DOPAC, and (C) HVA levels are shown. The number of mice used in each group was
518
10. Results are expressed as means ± S.E.M. Groups with different letters are
519
significantly different (P < 0.05).
520
M
an
516
Fig. 3. The results concerning L-Tyr levels in the cerebellum and striatum. The number
522
of mice used in each group was 10. Results are expressed as means ± S.E.M. Groups
523
with different letters are significantly different (P < 0.05).
te
Ac ce p
524
d
521
525
Fig. 4. The effect of BH4 injection on monoamine levels in each part of the brain in
526
ICR mice. (A) DA, (B) HVA, and (C) MHPG levels are shown. The number of mice
527
used in each group was 10. Results are expressed as means ± S.E.M.
528 529
Fig. 5. The effect of L-DOPA on behavior of mice in the open field test over 5 min.
530
The number of mice used in each group was 10. There were three groups—con: control
531
group; low: low concentration L-DOPA administration group; high: high concentration
532
L-DOPA administration group. Results are expressed as means ± S.E.M. Groups with
533
different letters are significantly different (P < 0.05).
534 22
Page 22 of 29
Fig. 6. The effect of L-DOPA on levels of DA in some parts of the brain (cerebellum,
536
hippocampus, striatum, prefrontal cortex) as a result of the administration of L-DOPA.
537
The number of mice used in each group was 10. Results are expressed as means ±
538
S.E.M. Groups with different letters are significantly different (P < 0.05).
Ac ce p
te
d
M
an
us
cr
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535
23
Page 23 of 29
300 200
b bc
c
100
bc
0
Second
cr a
b bc c
c
First
Second
te
d
First
CBA/N C57BL/6J ICR
a
an
ab
70 60 50 40 30 20 10 0
The result of ANOVA Strain P < 0.05 Day P < 0.05 Interaction P < 0.05
us
CBA/N C57BL/6J ICR
a
Rearing (counts)
400
B
The result of ANOVA Strain P < 0.05 Day P < 0.05 Interaction P < 0.05
M
Line crossing (counts)
A
ip t
Fig. 1
C
D The result of ANOVA Strain P < 0.05 Day P < 0.05 Interaction P < 0.05
40 30
a
20 10
CBA/N C57BL/6J ICR b
c
c
0
First
c c
Second
60 Latency time (s)
Ac ce p
Inner area entry (counts)
The result of ANOVA Strain P < 0.05 Day P < 0.05 Interaction P > 0.05
CBA/N C57BL/6J ICR
50 40
a
30 20 10 0
b b
b
First
b
b
Second
Page 24 of 29
Fig. 2
Striatum 50
2000 a
DA (pg/mg)
8
4
0
0
0
2000
a b
1500
20
1000 10
500
0
Ac ce p
0
Cerebellum
C 40
1500
b
140 120 100 80 60 40 20 0
Striatum
a
30
a
d
b
a
a
c
1000
b
0
0
Thalamus 250
a b
200
120 b
a a
100 50 0
150
a
a
150
c
Brain stem
Thalamus 400
a ab
a b
300
b
b
90 200
20 10
5
Brain stem
te
DOPAC (pg/mg)
Striatum
M
Cerebellum
10
an
10
20
a
15
20
500
30
HVA (pg/mg)
b
b
2
B
ab
30 1000
a
a
b
c
6
25 a
40
a
1500
b
Thalamus
cr
10
Brain stem
ip t
Cerebellum
us
A
500
60 30
0
0
100 0
Page 25 of 29
Striatum
200
200 b
a
b
150 100
an
100
a
us
150
a
50
M
50
0
te
d
0
Ac ce p
L-Tyr (pmol/mg)
a
cr
Cerebellum
ip t
Fig. 3
Page 26 of 29
60
4
40 20
P < 0.05
0
P < 0.05
0 0
25
50
0
25
M
600 500 400 300 200 100 0
y = 1.074x + 200.54 R² = 0.3336
d
300
P < 0.005
100 0
0
25
50
te
200
20 10
50
Striatum
y = 0.9053x + 474.66 R² = 0.3144 400
y = 0.0667x + 17.324 R² = 0.1615
P < 0.05
0
300
25
50
BH4 concentration (mg/kg)
Brain stem
Hippocampus
y = 0.446x + 170.43 R² = 0.328
y = 0.8515x + 186.42 R² = 0.2243
300 200
200
100
100 P < 0.001
P < 0.005
0
25
P < 0.05
0
0 50
0
25
50
0
25
50
Ac ce p
MHPG (pg/mg)
Cerebellum
30
0
BH4 concentration (mg/kg)
C
HVA (pg/mg)
6 2
y = 0.2185x + 26.396 R² = 0.1956
80
Cerebellum
us
y = 0.0221x + 3.051 R² = 0.138
8
B
Hippocampus
cr
Cerebellum
an
DA (pg/mg)
A
ip t
Fig. 4
BH4 concentration (mg/kg)
Page 27 of 29
B
20 15 10
b
5
b
0 50 mg/kg 250 mg/kg
saline
b b
50 mg/kg 250 mg/kg
a ab
50 40
Ac ce p
Inner area times (s)
60
30 20 10
b
0
saline
50 mg/kg 250 mg/kg
D
6
a
5 Latency time (s)
70
te
C
d
M
saline
a
an
Total distance (m)
25
40 35 30 25 20 15 10 5 0
cr
a
Inner area entries (counts)
30
us
A
ip t
Fig. 5
4
ab
3 2 1
b
0 saline
50 mg/kg 250 mg/kg
Page 28 of 29
1400
600 a
DA (pg/mg)
b
100 c
0
3000 2000 1000
d
D
a
b
0
600
b
400 200
c
0 saline
50 mg/kg 250 mg/kg
Prefrontal cortex
4000
a
Ac ce p
4000
te
Striatum
5000 DA (pg/mg)
50 mg/kg 250 mg/kg
M
200
saline
800
an
300
a
3500 DA (pg/mg)
DA (pg/mg)
1000
400
6000
a
us
1200
500
C
Hippocampus
B
cr
Cerebellum
A
ip t
Fig. 6
3000 a
2500 2000 1500
b
1000 500 0
saline
50 mg/kg 250 mg/kg
saline
50 mg/kg 250 mg/kg
Page 29 of 29