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|Year : 2013
: 15 | Issue : 65 | Page
|Behavioral and plasma monoamine responses to high-speed railway noise stress in mice
Guoqing Di, Lingjiao He
Department of Environmental Science and Institute of Environmental Pollution and Control Technology, Zhejiang University, Hangzhou, China
Click here for correspondence address
|Date of Web Publication||15-Jun-2013|
Studies have reported that railway noise causes stress responses. To evaluate the effects of high-speed railway (HSR) noise on behaviors and plasma monoamines. Institute of cancer research mice were exposed to previously recorded HSR noise for 53 days. The noise was arranged according to the HSR's 24-h traffic number and adjusted to a day-night equivalent continuous A-weighted sound pressure level (Ldn ) of 70 dB (A). The open field test (OFT) and the light/dark box test were applied to observe mice behaviors. High performance liquid chromatography-fluorimetric detection was performed to determine the concentrations of plasma norepinephrine (NE), dopamine (DA), serotonin (5-hydroxytryptamine, 5-HT). Data were analyzed by two-way analysis of variance using SPSS 16.0. After 53 days of noise exposure, center time and the frequency of line crossing of the exposed mice decreased significantly in the OFT compared with the control group. Meanwhile, transitions and the time spent in the lit compartment of the exposed group decreased significantly in the light/dark box test. After 40 days of HSR noise exposure, the concentrations of plasma DA of the exposed group were significantly higher than those of the control group, while the plasma NE and 5-HT concentrations showed no significant difference between the two groups. The behavioral tests indicate that 70 dB (A) HSR noise can result in anxiety-like behaviors in mice. The physiological results show that plasma DA is more sensitive to HSR noise compared with NE and 5-HT.
Keywords: Anxiety, behavior, high-speed railway noise, monoamine
|How to cite this article:|
Di G, He L. Behavioral and plasma monoamine responses to high-speed railway noise stress in mice. Noise Health 2013;15:217-23
| Introduction|| |
Field and laboratory studies have reported that railway noise causes stress responses, leading to physiological and psychological effects such as cardiovascular diseases, sleep disturbance, annoyance and so forth. ,, Stress is expressed in a physiological and behavioral response,  including a series of co-ordinated responses being composed of alterations in behavior, autonomic function and the secretion of multiple hormones.  The level of the responses depends on the characteristics of the stressor and the body.  Therefore, behavior, autonomic nervous, and neuroendocrine systems are regarded as indicators of stress. ,
Numbers of animal behavior models are used to assess stress responses. In this study, the open field test (OFT) and the light/dark box test were selected to measure mice behaviors, which are widely used to evaluate stress and anxiety. , Specifically, the OFT, firstly applied to evaluate animals' emotionality in Hall's research,  are mostly used to analyze spontaneous and exploratory behaviors. Center time, grooming, rearing, and defecation are common indexes. The light/dark box test is based on the inborn aversion of rodents to brightly illuminated areas and on the natural tendencies to explore a novel environment.  Transitions between the light and dark chambers and the time spent in the lit compartment are essential features.
Monoamines, taken as important indicators of stress responses, are a collection of biologically active substances, including the catecholamines (norepinephrine [NE], epinephrine, and dopamine [DA]) and indoleamines, (e.g., serotonin). These substances play a critical role in the regulation of physiological processes and the development of psychiatric, endocrine, and cardiovascular diseases. ,,, Studies have shown that many forms of stress such as noise, cold wind, cold, immobilization and foot shock, will activate the autonomic nervous and neuroendocrine systems, and cause alterations in the plasma NE, serotonin (5-HT), and DA levels. ,,,,,
At present, with the development of high-speed rail (HSR) technology, more and more countries are committed to HSR construction. To the end of October 2011, about 15 countries Including China have provided HSR services, and more than 17,000 km of lines were in operation around the world.  The rapid growth of HSR construction makes railway noise a significant problem. As the speed of train increases, the characteristics of railway noise change. HSR (speed ≥ 250 km/h) noise is mainly composed of aerodynamic noise,  which is generally of shorter duration and higher peak sound levels.  Noise with different acoustic properties may cause different actual impacts on the body. ,, Epidemiological investigations and laboratory experiments are two basic methods to study the physiological effects of noise. However, epidemiological investigations are difficult to draw firm conclusions due to the related uncontrollable interference factors and long-term follow-up surveys. On the contrary, the influencing factors in laboratory experiments are relatively controllable. Few studies have investigated the behavioral and physiological effects of HSR noise under controlled laboratory conditions. The objective of this study was to explore stress responses caused by HSR noise. Both behavioral (the OFT and the light/dark box test) and physiological (plasma NE, DA, 5-HT concentrations) criteria were evaluated.
| Methods|| |
Long-term exposure to high-intensity noise may injure the body. Considering the auditory system of mice have some similarities to that of humans, and are commonly used as an experimental model for studying human auditory system,  in this study, 120 male Institute of Cancer Research (ICR) mice (the Experimental Animal Center of Zhejiang University, Hangzhou, China) were chosen as subjects. The mice were 5 weeks of age weighing 25-30 g on arrival in the laboratory. They were randomly divided into two groups, the control group (CG, n = 60) and the experimental group (EG, n = 60). Mice were housed five per cage and maintained under constant temperature (22 ± 2°C) and a 12 h/12 h light/dark cycle (light on at 8:00 a.m.) at 50-60% relative humidity. Food and water were available ad libitum. All procedures were performed in line with the guidelines established by the National Institutes of Health Guide for the Care and use of Laboratory Animals.
Nowadays, a few of countries such as France, Belgium, and Italy and regions like Taiwan have developed emission standards for HSR noise. For instance, in Belgium, the day-time and night-time hour equivalent sound level limits are 60 dB (A) and 50 dB (A), respectively, at the distance of 25 m away from the railroad track. France developed the corresponding emission standards for different functional areas, all of which are lower than those of conventional railway. China has not specifically issued HSR noise emission standards yet and currently adheres to Railway boundary noise limits and measurement methods for conventional railway, which states that the day-time and night-time hour equivalent sound level limits are 70 dB (A) at the boundary (30 m away from the railroad outside tracks' center line) for existing railways, including the renovation and expansion of an existing railway. For newly built railways, the day-time and night-time hour equivalent sound level limits are 70 dB (A) and 60 dB (A), respectively, at the boundary. In order to investigate the impacts of 70 dB (A) HSR noise on the body, we used a four-channel dynamic signal analyzer (Photon II, Royston, England) to record HSR noise and background noise at the boundary and played it in the laboratory through a dodecahedron non-directional sound source (Nor270, Norsonic, Lierskogen, Norway). The day-night equivalent continuous A-weighted sound pressure level (Ldn ) of the EG was adjusted to 70 dB (A) according to the HSR 24-h traffic number. The equivalent continuous A-weighted sound pressure level (LAeq ) of the background noise was no more than 35 dB (A), which was the intensity presented to the CG.
After 3 days of adaptation in the laboratory, the EG was exposed to the HSR noise, while the CG was not exposed. The behavioral tests (the OFT and the light/dark box test) were conducted at 19:00 on days 1, 8, 18, 28, 38, 53 after the initial noise exposure. Blood collection from the same mice examined in the behavioral tests was carried out 2 days later (days 3, 10, 20, 30, 40). Blood drawing was done at the same time of the day. Ten mice from each group were randomly selected for each experiment.
Open field test
The open field is an enclosure with surrounding walls that prevent rodents from escaping. The size of the open field varies from one to another.  In this study, testing was performed in a well illuminated wooden cage (48 cm × 48 cm × 25 cm) with a plane black floor. The floor was divided into 64 grids (6 cm × 6 cm) by white lines. We termed the grids along the walls and the rest grids as "peripheral area" and "center area", respectively. The animal was placed in the center of the open field at the beginning of the test and its behaviors were recorded by a camera. Center time, the frequency of line crossing, grooming, and rearing was analyzed by reviewing the first 5-min behaviors of the mice in the videos.
Light/dark box test
The light/dark box test was firstly described by Crawley and Goodwin.  The box consists of two chambered arena, where two-thirds of the area was illuminated and one-third was darkened. Now, many authors have used it with several structural modifications.  In this study, the size of the box was 50 cm × 25 cm × 30 cm. It was divided into two parts: Two-thirds was illuminated by 20-W red bulb at the height of 30 cm above the floor; one-third was painted black, covered by the lid and separated from the lit compartment with a partition containing an opening at the floor. The animal was placed in the center of the lit compartment facing away from the hole at the start of the test. The time spent in the lit compartment and the number of transitions between the two compartments during the first 5 min was measured.
Determination of plasma monoamine levels
1.0 ml of blood was sampled by eyeball removal and immediately transferred into a 1.5-ml micro-tube. Twenty microliter of 10 g/ml disodium ethylene diamine tetraacetic acid (EDTA) was added to the blood and oscillated to mix for 15 min. Then the mixture was centrifuged at 3000 r/min for 20 min at 4°C. Next 150 μl of supernatants was extracted from the sample, to which 150 μl of 5% perchloric acid was added. The mixture was shaken and left at room temperature for 20 min to fully precipitate the plasma proteins. After centrifuged (10,000 r/min) for 15 min, supernatants were filtered with 0.45 μm membrane filters. The high-performance liquid chromatography-fluorimetric detection was used to determine the plasma catecholamine levels. 
All data are presented as mean ± SEM. The comparison between the two groups at different exposure duration was analyzed by two-way analysis of variance. The results were considered significant when P < 0.05.
| Results|| |
The behavioral tests
Behavioral data were obtained by three individuals working independently, and their mean values were taken as the final results shown in [Table 1].
|Table 1: Results from the behavioral tests in experimental group (n=10) and control group (n=10)|
Click here to view
As shown in [Table 1], center time in the OFT showed no significant difference between the two groups on days 1 and 8; with increasing duration of HSR noise exposure, center time of the EG was significantly less than that of the CG (P < 0.05); on day 53, EG still spent less time in center area compared with the CG (P < 0.01). The line crossing frequency of the EG was lower than that of the CG after 28 days of noise exposure and showed significance after 53 days of exposure (P < 0.01). The results of grooming frequency showed that the grooming frequency of the EG was generally lower over the duration of noise exposure. Meanwhile, the rearing frequency of the EG was lower than that of the CG except the 8 th day and showed significant differences after 28 days of noise exposure.
The results of the light/dark box test showed that the EG spent more time in the lit compartment and the number of transitions between the two compartments increased during the first few days of noise exposure compared with the CG, but the differences were not significant (P > 0.05). With increasing duration of noise exposure, the time spent in the lit compartment and the number of transitions of the EG decreased compared with the CG and showed significant differences on the 53 rd day of noise exposure (P < 0.05).
Levels of plasma monoamine
The mean plasma monoamine (NE, DA, 5-HT) concentrations of the two groups are shown in [Figure 1]. Over the duration of noise exposure, there was no significant difference in the plasma NE [Figure 1]a and 5-HT [Figure 1]c concentrations between the two groups. The mean plasma DA concentrations of the EG were significantly higher compared with the CG after 40 days of noise exposure (P < 0.05), as shown in [Figure 1]b.
|Figure 1: Relationship between plasma monoamine concentrations and noise exposure duration. (a) Norepinephrine, (b) Dopamine, (c) 5 - HT. Data were presented as mean ± SEM. "*" indicates significance compared with CG, P < 0.05, using two - way analysis of variance|
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| Discussion|| |
High-speed railway noise and behavior
Stress responses are considered to play an important role in mental disorders.  As emotion is coupled with somatic and visceral responses, psychologists have developed behavioral models to measure the emotionality of animals.  Evans et al. pointed out that chronic noise exposure was associated with cognitive, affective disorders of psychological stress. According to the definition of chronic stress proposed by Burchfield,  chronic stress can be interpreted as a succession of repeated acute stressors. Two animal behavior models, the OFT and the light/dark box test were selected in this study to investigate the effects of HSR noise on emotionality of animals.
In the OFT, activity and center time in the first 5 min are often used to assess some aspects of emotionality like anxiety and depression.  In Denenberg's study,  emotional animals were defined as those having low activity and high defecation scores, which has been accepted by some other authors. , They also pointed out that emotional animals spent less time in the central part of the field and showed lower levels of grooming and rearing compared with non-emotional animals. Lipkind et al. specified that avoidance of the field center was considered anxiety related. In this test, center time and activity of the EG decreased compared with the CG as noise exposure duration increased, indicating that chronic exposure to 70 dB (A) HSR noise may cause anxiety-like response. In the light/dark box test, time spent in the light compartment was suggested as an indicator of anxiety state. , Under normal circumstances, mice spend approximately 60% of their time in the dark chamber.  The number of transitions between the light and the dark chamber was also measured, which has been reported to be an index of exploratory activity. In general, an increase in anxiety is indicated by an animal's tendency to spend less time in the lit compartment and a decrease in the number of transitions.  In this study, the results from the two behavioral models supported the hypothesis that long-term HSR noise exposure will induce anxiety-like reactions in mice. In addition, mice went through their periadolescent (about postnatal day 35), adolescent (about postnatal day 45) and adult (about postnatal day 60) phases ,, over the duration of noise exposure in the present study. Pronounced changes in behaviors from periadolescence through adulthood in mice have been reported. ,, Ontogenetic phases and noise stress are two important factors influencing on behaviors of mice in this study. [Table 1] showed that there was more variability in the control animals among days than between CG and EG on a given day, which indicated that ontogenetic phases influence behaviors of mice more greatly compared with noise stress in the study.
HSR noise and monoamines
Sympathetic-adrenomedullary system is activated in stress responses, resulting in an increase in the secretion of NE and DA. The levels of these two catecholamines are influenced by age greatly, as age is considered to be the main physiological variable affecting the peripheral sympathetic activity.  In the present study, the results showed that the variation trend of plasma NE levels increased with age in both CG and EG, which are consistent with some relevant studies. , While plasma DA levels showed fluctuation with age. There are indications that DA systems undergo substantial reorganization during adolescence, resulting in the variation of DA levels.  A temporary reduced rate of DA release was observed during mice's periadolescence.  Sagiyama et al. reported that elder mice showed more DA levels compared with younger ones.  However, Lee et al.  observed plasma DA levels decreased in elder rats. The conclusions on the variation of plasma DA levels with age have not reached an agreement at the moment. It implies that the fluctuation of plasma DA levels with age could be great and the great variation of plasma DA levels in control group at different times is reasonable in the present study.
NE, synthesized from DA, is widely used as an indicator of stress reactions caused by numerous stressors including noise. , The results suggested that HSR noise of 70 dB (A) had little influence on plasma NE levels in mice in this study. The results of epidemiological studies reveal that the influence of traffic noise is closely related to the types and intensities.  Previously, most studies on this topic focused on aircraft and road traffic noise, paying little attention to railway noise, probably because it is considered to be less annoying. , However, with train speed increasing, the properties of railway noise have altered. HSR noise is known to be sudden and intermittent, which is very similar to aircraft noise. Di et al. measured the concentrations of plasma NE in rats during aircraft noise exposure. They found that the concentrations of plasma NE of rats exposed to aircraft noise of 80 dB (Ldn = 70.3 dB (A)) were significantly higher than those of the control group after 29 days exposure, while the plasma NE concentrations of rats exposed to aircraft noise of 75 dB (Ldn = 65.3 dB (A)) showed no significant difference. The noise intensity (Ldn ) adopted in the present study is close to 70.3 dB (A) used in the aircraft noise study, but the plasma NE concentrations between the two groups showed no significant difference. This difference between above two experimental results may be related to the different experimental subjects. In the present study, ICR mice were used as subjects, while SD rats were used in the study on aircraft noise effect reported by Di et al. Kight et al.  points out that rats are more sensitive to noise than mice. Furthermore, it is known that biological responses to acoustical stress is frequency-dependent.  As a contrast, the frequency distribution of pass-by noise from HSR used in this study and landing and take-off noises from Airbus aircraft used by Di et al.  was analyzed as shown in [Figure 2]. The ratio of the low-frequency sound energy (22.4-224 Hz) to the audible sound energy (20-20,000 Hz) was calculated.  The results are also shown in [Figure 2]. It can be seen that HSR noise has less low-frequency components compared with aircraft noise. Huang et al. reported that noise containing more low-frequency components was more annoying at same A-weighted sound pressure level. Thus, HSR noise with less low-frequency components maybe has a smaller effect on plasma NE levels compared with aircraft noise, which deserves to be further studied.
|Figure 2: 1/3 - octave - band spectrum of high - speed railway noise and aircraft noise|
Click here to view
Plasma DA levels elevate during stress, most from the sympathetic nerve terminals. , The plasma DA concentrations in the experimental mice increased compared with the CG after noise exposure, most probably due to enhanced sympathetic nerve activity. DA is reported to be associated with many clinical diseases such as anxiety, depression. Dopaminergic over activity was found in patients with panic disorder.  In Hamner's study,  Plasma DA levels demonstrated a significant correlation with the anxiety subscale of the Hamilton rating scale for depression score. Combined with the results of the behavioral tests in the present study, we speculate that anxiety-like reactions in mice may be related to abnormal plasma DA levels.
5-HT is synthesized from tryptophan and metabolized to 5-hydroxyindoleacetic acid (5-HIAA). Stress can alter 5-HT, tryptophan, and 5-HIAA concentrations in the central nervous system and in the periphery.  1-h foot shock induced an increase in both plasma and brain 5-HIAA, plasma 5-HT, and tryptophan levels.  Studies investigating the effects of noise on the 5-HT levels showed that varying noise intensity and exposure duration led to different impacts on 5-HT levels.  The results of the plasma 5-HT concentrations in our study suggested that HSR noise of 70 dB (A) had little influence on the plasma 5-HT concentrations in ICR mice.
In summary, this study indicated that HSR noise of 70 dB (A) caused anxiety-like behavior in ICR mice and an increase in plasma DA concentration, which may be one of the reasons for anxiety reactions. In line with the precautionary principle, it is suggested that the emission limit (Ldn ) for HSR noise should be stricter than that for conventional railway noise, and the limit value should not be greater than 70 dB (A).
| Acknowledgment|| |
This study was supported by the National Nature Science Foundation of China (Grant No. 11174251) and in part by the National Public Benefit Research Foundation (Grant No. 200809142). The authors would like to thank Zhou Bin and Jia Li for their assistance with animal feeding.
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Nongshenghuan Building B388, Zijingang Campus, Zhejiang University, Yuhangtang Road 866, Hangzhou, Zhejiang Province 310058
Source of Support: The National Nature Science Foundation of China (Grant No. 11174251) and in part by the National Public Benefit Research Foundation (Grant No. 200809142),, Conflict of Interest: None
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