Alterations of cognitive function and 5-HT system in rats after long term microwave exposure

Alterations of cognitive function and 5-HT system in rats after long term microwave exposure

PHB-10653; No of Pages 11 Physiology & Behavior xxx (2014) xxx–xxx Contents lists available at ScienceDirect Physiology & Behavior journal homepage:...

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PHB-10653; No of Pages 11 Physiology & Behavior xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Physiology & Behavior journal homepage: www.elsevier.com/locate/phb

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Keywords: Microwave Electromagnetic radiation Neurotransmitters Serotonin 5-HT receptor Hippocampus Learning and memory

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The increased use of microwaves raises concerns about its impact on health including cognitive function in which neurotransmitter system plays an important role. In this study, we focused on the serotonergic system and evaluated the long term effects of chronic microwave radiation on cognition and correlated items. Wistar rats were exposed or sham exposed to 2.856 GHz microwaves with the average power density of 5, 10, 20 or 30 mW/cm2 respectively for 6 min three times a week up to 6 weeks. At different time points after the last exposure, spatial learning and memory function, morphology structure of the hippocampus, electroencephalogram (EEG) and neurotransmitter content (amino acid and monoamine) of rats were tested. Above results raised our interest in serotonin system. Tryptophan hydroxylase 1 (TPH1) and monoamine oxidase (MAO), two important rate-limiting enzymes in serotonin synthesis and metabolic process respectively, were detected. Expressions of serotonin receptors including 5-HT1A, 2A, 2C receptors were measured. We demonstrated that chronic exposure to microwave (2.856 GHz, with the average power density of 5, 10, 20 and 30 mW/cm2) could induce dosedependent deficit of spatial learning and memory in rats accompanied with inhibition of brain electrical activity, the degeneration of hippocampus neurons, and the disturbance of neurotransmitters, among which the increase of 5-HT occurred as the main long-term change that the decrease of its metabolism partly contributed to. Besides, the variations of 5-HT1AR and 5-HT2CR expressions were also indicated. The results suggested that in the longterm way, chronic microwave exposure could induce cognitive deficit and 5-HT system may be involved in it. © 2014 Published by Elsevier Inc.

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exposure, since many kinds of neurotransmitters exist in the nervous system and there is tremendous electrical activity in neural transmission [1]. Yet whether the alterations of neurochemistry and electrophysiology have functional consequences after microwave exposure is still under debate in terms of subject species, frequency, intensity, duration of irradiation and so on. To date, a large number of studies have focused on the short term effect of acute microwave exposure. Evidences showed that microwave exposure (2450 MHz) induced deficits in spatial learning ability [2,3]. Nevertheless, few researches about the long term effects of chronic microwave exposure on cognitive functions have been conducted and the results remained controversial [4–6]. The neurotransmitters play important roles in cognitive function. Studies indicated that most classical neurotransmitter systems are

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The increased use of microwaves in communications, industries and medical treatments raises the concern about its effects on health. Microwave with the frequency of 2.856 GHz and high power is widely used in radar and other communication devices. Long term impacts of high power microwave with the frequency of 2.856 GHz radiation on the population who occupationally exposed to radar and other communication devices should be given adequate attention. Accumulative studies show that the nervous system is a sensitive target of electromagnetic

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1. Introduction

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a b s t r a c t

Article history: Received 13 August 2014 Received in revised form 19 November 2014 Accepted 22 December 2014 Available online xxxx

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• Long term microwave exposure induced dose-dependent cognitive function deficit. • Among neurotransmitters, the increase of 5-HT was the main remote effect. • 5-HT system may be involved in microwave-induced cognitive deficit.

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Department of Experimental Pathology, Beijing Institute of Radiation Medicine, Beijing, China Department of Radiation Protection and Health, Beijing Institute of Radiation Medicine, Beijing, China

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Hai-Juan Li a, Rui-Yun Peng a, Chang-Zhen Wang a, Si-Mo Qiao a, Yong-Zou a, Ya-Bing Gao a, Xin-Ping Xu a, Shao-Xia Wang a, Ji Dong a, Hong-Yan Zuo a, Li-Zhao a, Hong-Mei Zhou b, Li-Feng Wang a,⁎, Xiang-Jun Hu a,⁎

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Alterations of cognitive function and 5-HT system in rats after long term microwave exposure

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⁎ Corresponding authors. E-mail addresses: [email protected] (L.-F. Wang), [email protected] (X.-J. Hu).

http://dx.doi.org/10.1016/j.physbeh.2014.12.039 0031-9384/© 2014 Published by Elsevier Inc.

Please cite this article as: H.-J. Li, et al., Alterations of cognitive function and 5-HT system in rats after long term microwave exposure, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.12.039

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using a computer-assisted tracking system (SLY-MWM system, Beijing Sunny Instrument Co. Ltd., Beijing, China), and the average escape latency (AEL) was analyzed. 5 days after the exposure, the platform was removed and the probe test was performed. The rats were placed in a start position which was farthest from the platform quadrant used in navigation test. The time for probe trials lasted 60 s. Percentage of time in the target quadrant was recorded and analyzed. To test the long-term effect of microwave on spatial memory, navigation tests were performed 14 and 28 days after the exposure. The time schedule of MWM test is shown in Fig. 1A.

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2. Material and methods

2.3.2. EEG recording 14 days after microwave exposure, the rats of exposed and sham exposed groups were evaluated under light anesthesia conditions using a four-electrode configuration. The EEG recorded the collective activity of neurons through electrodes placed on the surface of the scalp. The EEG signals were obtained through a BIOPAC MP-150 system (USA) and power spectral analyses were performed on spontaneous EEG segments.

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2.1. Animals and groups

2.4. Hematoxylin and eosin (H&E) staining of the hippocampus

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2.2. Microwave exposure system and dosimetry

14 days, 28 days and 2 months after exposure, rats' brains were removed and fixed in 10% buffered formalin solution. Coronal brain sections (3.5 μm) including the hippocampal area were prepared for H&E staining. The brain sections were deparaffinized and rehydrated with different concentrations of xylene and alcohol, and then dipped in hematoxylin for 5 min, de-stained in 1% hydrochloric acid ethanol for several seconds, and redyed in eosin for 2 min. Following dehydration in an alcohol gradient, xylene clearance and coverslipping, stained brain sections were examined under a microscope and the hippocampi were photographed at a 200× magnification.

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Male Wistar rats weighting 140–160 g (4 weeks old) were obtained from the Laboratory Animal Center of Beijing Institute of Radiation Medicine (Beijing, China) and maintained at 22 ± 2 °C with a 12 hour light–dark cycle. All experiments were performed with the approval of the Institutional Animal Care and Use Committee. 75 rats were randomly divided into control group and microwave exposure groups which were classified into subgroups of different average power densities (5, 10, 20, 30 mW/cm2) with 15 rats in each group.

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Pulsed microwaves at the frequency of 2.856 GHz were generated by the microwave exposure apparatus, which has been described at length in Wang's report [17]. Microwave energy was transmitted by rectangular waveguide and A16-dB standard-gain horn antenna to an electromagnetic shield chamber. The average power densities were measured with a waveguide antenna, the GX12M1CHP power meter (Guanghua Microelectronics Instruments, Hefei, China) and GX12M30A power heads. The whole bodies of Wistar rats were sham exposed or exposed to microwaves with the average power density of 5, 10, 20 or 30 mW/ cm2 respectively for 6 min three times a week up to 6 weeks.

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2.3. Behavioral test and electroencephalogram (EEG) recording

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2.3.1. Morris water maze (MWM) behavioral test The MWM test [18] was implemented to assess cognitive evaluation of rats (spatial learning and long-term memory) and the MWM apparatus used in this study was described in Qiao's study [19]. The MWM consisted of a black circular pool (150 cm in diameter) filled with clear water (23 ± 0.5 °C). The pool was surrounded by thick curtains to hide extra-maze visual cues from the rats. A movable escape platform (12 cm in diameter) was submerged 1.5 cm below the surface of the water in the center of an arbitrarily defined quadrant of the pool and remained in the same position throughout the testing. After the completion of long-term exposure, the MWM training sessions were initiated. Rats were trained to find the submerged escape platform during four consecutive daily sessions. Four trials were given to the rats per day and began by placing the rats into one of the four starting positions in a fixed order. Rats were positioned to face the wall of the pool and released into the water. Each trial had a maximum duration of 60 s and rats that failed to locate the platform in 60 s were guided to the platform. All rats remained on the platform for 10 s before proceeding to the next trial. Rat behavior in the MWM experiments during the training and memory test procedures was digitally recorded

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involved in learning and memory of rats in some way [7]. Whether neurotransmitters were involved in the effect of microwave radiation on cognitive function still needs to be further explored. Serotonergic system includes multiple receptors, including at least 15 serotonin (5-hydroxytryptamine, 5-HT) receptors which were divided into 7 classes (divided from 5-HT1 to 5-HT7 receptors) [8]. Growing number of studies reported that serotonergic systems regulated learning and memory function of animals [9–13] and humans [14, 15]. A capital essence of the modulation is 5-HT receptors' density during memory formation and amnesic states [16]. In this study, we examined whether microwave exposure produces a long term impairment of cognitive function, morphology structure of the hippocampus, related neurochemistry and electrophysiology. According to the results of above experiments, we then assessed the level of 5-HT synthesis and metabolism and measured the expressions of 5-HT1A, 2A, 2C receptors considering their important roles on cognitive function.

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2.5. Measurements of neurotransmitter contents of the hippocampus and 162 cerebrospinal fluid (CSF) 163 2.5.1. Sample processing and pretreatment of samples Rats from microwave exposure and sham exposure groups were anesthetized 14 days, 28 days and 2 months after exposure. CSF samples were obtained by cisternal puncture using a 1 mL syringe. Then brains of the rats were removed and the hippocampi were dissected immediately on the ice and stored at −80 °C. For pretreatment of samples, the hippocampi and CSF were homogenized in 10% salicylsulfonic acid (for amino acid measurements) or 5% perchloric acid (for monoamine measurements). The homogenate was centrifuged at 15,000 rpm for 20 min at 4 °C and the supernatant was transferred to a clean tube for the next test.

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2.5.2. Amino acid neurotransmitter measurements Aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), taurine (Tau) and γ-aminobutyric acid (GABA) in the hippocampus and CSF were measured by high performance liquid chromatography with fluorescence detector (HPLC–FLD) procedure. The HPLC system consisted of a microbore reverse-phase column (particle size 5 μm, 150 mm × 4.6 mm; Model Venusil AA, Bonna-Agela Technologies, China), an Agilent 1100 pump (Agilent Technologies, USA) and a fluorescence detector (Agilent Technologies, USA). The mobile phase (pH 6.8) consisted of 100 mM disodium hydrogen phosphate and 30% methanol. 1 μL sample was derivated with 5 μL o-phthalaldehyde before being injected to the detection system.

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2.5.3. Monoamine neurotransmitter measurements Noradrenalin (NA), homovanillic acid (HVA), dopamine (DA), dihydroxy-phenyl acetic acid (DOPAC), and 5-HT and 5hydroxyindoleacetic acid (5-HIAA) in the hippocampus and CSF were measured by high performance liquid chromatography with electrochemical detector (HPLC–ECD) procedure. The HPLC system

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Please cite this article as: H.-J. Li, et al., Alterations of cognitive function and 5-HT system in rats after long term microwave exposure, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.12.039

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consisted of a microbore reverse-phase column (particle size 10 μm, 250 mm × 4.6 mm; Model C-18, DIKMA Technologies Ltd., China), an Agilent 1100 pump (Agilent Technologies, USA) and an Intro electrochemical detector (Antec Leyden, Netherlands) with VT03 flow cell glassy carbon working electrode set at 700 mV (with respect to an Ag/ AgCl reference electrode). The mobile phase (pH 3.7) consisted of 85 mM citrate, 100 mM sodium acetate, 0.2 mM EDTA, 0.9 mM octylsodium sulfate, and 8% methanol. The volume of injection was 50 μL. 2.6. Measurement of two rate-limiting enzymes (TPH1 and MAO) in 5-HT system Tryptophan hydroxylase 1 (TPH1) expressions in rats' hippocampi of microwave exposure (30 mW/cm2) and sham exposure groups were measured using western blot. After the completion of sampling, bicinchoninic acid (BCA) protein assay (Thermo Scientific, USA) was used for quantification of protein. The western blot process was performed as previously described [20] and the antibody against TPH1 (Bioworld Technology, USA) was at 1:750 dilution. Quantification of the protein band intensity was performed using Quantity One imaging software (Bio-Rad Laboratories, USA). Results were normalized to GAPDH (antibody at 1:5000 dilution, Kangchen Bio-tech, China) staining on the same blot to account for variations in total protein loading. Monoamine oxidase (MAO) activities in rats' hippocampi of microwave exposure (30 mW/cm2) and sham exposure groups were measured using a detection kit (Jiancheng Bioengineering Institute, China). The hippocampi of rats were acquired as described in Section 2.5.1. Then the detection procedure was performed according to the following protocol. Briefly, sample homogenate was prepared

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Fig. 1. Performances of rats in the Morris water maze test. (A) Time schedule of experiments; (B) swimming speed of rats; (C) average escape latencies during navigation test. Statistical significances (repeated measures ANOVA): effect of 10, 20, 30 mW/cm2 group: ⁎P b 0.05 versus the control group, effect of 20 and 30 mW/cm2 group: ○P b 0.05 versus 5 mW/cm2 group, effect of 30 mW/cm2 group: △P b 0.05 versus 10 mW/cm2 group. ★P b 0.05 versus control at a corresponding time point after exposure; (D) percent time in the target quadrant in probe test. ⁎P b 0.05 versus the control group.

and then incubated in reaction buffer containing aniline hydrochloride. The protein concentration was determined using BCA protein assay. The absorbance of supernatant solution was then measured by a microplate reader at 242 nm. MAO activity was calculated and expressed as U/h/mg protein.

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2.7. Assessment of 5-HT1AR, 5-HT2AR, 5-HT2CR expressions

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2.7.1. Western blot The expressions of 5-HT1A, 2A, 2CR of hippocampi and cerebral cortices of rats in microwave exposure (30 mW/cm2) and sham exposure groups were quantitatively assessed by western blot analysis. The preparations, protein quantifications and western blot procedures were the same as those of TPH1 described above. Antibody against 5-HT1AR (Millipore, USA), 5-HT2AR (Santa Cruz, USA) and 5-HT2CR (Santa Cruz, USA) was at 1:400, 1:200, 1:200 dilutions respectively.

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2.7.2. Immunohistochemistry To further clarify the subregions where the expression alterations of 5-HT receptors mainly occurred, immunohistochemistry reactions were performed to identify 5-HT receptor expressions of rats in exposure (30 mW/cm2) and sham exposure groups. Anti-5-HT1AR (at 1:200 dilution) and anti-5-HT2CR (at 1:100 dilution) which were produced in mouse were the primary antibodies, and polink-2 plus polymer horseradish peroxidase (HRP) detection system (ZSGB-BIO, China) for mouse primary antibody was used in this assay. The sections were reacted with diaminobenzidine kit (BOSTER, China) finally and counterstained with hematoxylin. Bright field images were acquired digitally on a microscope. We obtained three to five images each of the CA1, CA2,

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Please cite this article as: H.-J. Li, et al., Alterations of cognitive function and 5-HT system in rats after long term microwave exposure, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.12.039

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2.8. Data analysis

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Statistical analyses were carried out using SPSS 16.0 software. Repeated-measures ANOVA followed by post-hoc test was used for the analysis of AEL in MWM test. Independent-samples T test and one-way ANOVA (with post hoc multiple comparisons) were used to

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In navigation tests, the AEL of rats in 10, 20 and 30 mW/cm2 groups 259 was significantly longer than that of the control group, the AEL of rats in 260 20 and 30 mW/cm2 groups was longer than that of 5 mW/cm2 group, 261

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3.1. Spatial learning and memory ability of rats declined after microwave 257 exposure 258

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compare the differences among sham exposure and exposure groups 254 for other data. The accepted level of significance for all tests was P b 0.05. 255

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and CA3 and cerebral cortex region for each subject. The protein expressions (5-HT1AR and 5-HT2CR) of neurons in rats' hippocampal regions (CA1, CA2, CA3) and cerebral cortex were quantified by integral optical density (IOD) using Image-Pro Plus 6.0 software.

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Fig. 2. Electroencephalogram (A), the amplitude (B), frequency (C) and delta band relative power (D) of rats at 14th day after microwave exposure (0, 5, 10, 20, 30 mW/cm2). ⁎P b 0.05 versus the control group, ○P b 0.05 versus 5 mW/cm2 group, △P b 0.05 versus 10 mW/cm2 group, □P b 0.05 versus 20 mW/cm2 group.

Fig. 3. H&E staining images of rats' hippocampi (CA3 and dentate gyrus) 28 days after microwave exposure (0, 5, 10, 20, 30 mW/cm2). Scale bar = 50 μm.

Please cite this article as: H.-J. Li, et al., Alterations of cognitive function and 5-HT system in rats after long term microwave exposure, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.12.039

H.-J. Li et al. / Physiology & Behavior xxx (2014) xxx–xxx

Contents (ng/mg)

t1:20

⁎ P b 0.05 versus the control group.

0 5 10 20 30 0 5 10 20 30 0 5 10 20 30

28 days

2 months

Glu

Gly

Tau

GABA

12.57 ± 1.21 8.94 ± 0.35⁎ 11.85 ± 0.59 11.14 ± 0.63 13.81 ± 1.31 13.16 ± 0.83 8.96 ± 0.61⁎

55.35 ± 2.63 44.65 ± 1.75⁎ 50.32 ± 4.34 50.43 ± 3.53 56.27 ± 3.24 69.35 ± 3.48 52.57 ± 2.50⁎

10.05 ± 0.61 14.03 ± 2.11 13.20 ± 0.86 12.18 ± 0.59 13.48 ± 0.56 17.50 ± 1.60⁎ 12.17 ± 0.64 13.53 ± 0.61

58.72 ± 3.40 56.09 ± 3.71 65.16 ± 4.55 61.17 ± 2.06 68.74 ± 2.96 77.46 ± 2.38⁎ 67.73 ± 5.86 65.37 ± 2.19

24.99 ± 1.63 20.79 ± 1.37 20.10 ± 2.42 19.24 ± 1.43 22.50 ± 2.40 15.44 ± 0.50 16.59 ± 1.40 14.64 ± 0.56 13.04 ± 0.75⁎ 12.14 ± 0.62⁎ 13.46 ± 0.44 13.84 ± 0.42 15.29 ± 0.68 13.89 ± 0.75 13.95 ± 0.57

58.29 ± 1.91 52.40 ± 3.47 48.85 ± 3.10 54.42 ± 4.40 59.39 ± 4.33 70.89 ± 3.27 54.03 ± 4.63⁎ 56.34 ± 6.78⁎ 49.53 ± 3.62⁎ 53.37 ± 3.53⁎

22.85 ± 2.54 19.55 ± 1.16 27.12 ± 3.02 18.37 ± 1.18 28.64 ± 2.14⁎ 23.46 ± 1.19 21.86 ± 1.82 18.18 ± 1.80 19.78 ± 3.04 20.69 ± 1.44 24.02 ± 1.33 19.43 ± 1.25 28.38 ± 4.25 19.72 ± 2.11 22.96 ± 1.60

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3.2. Alterations of EEG after microwave exposure

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EEG was recorded in rats (Fig. 2A), and the amplitude, frequency and delta band relative power were estimated (Fig. 2B, C, D). 14 days after exposure, the amplitude was increased and the mean frequency was decreased with the delta band relative power being significantly increased (P b 0.05 compared to the control group). In advance, the amplitude and the delta band relative power were higher in 10 mW/ cm2 and 20 mW/cm2 groups than that of the 5 mW/cm2 group (P b 0.05), and they were higher in 30 mW/cm2 than those of other exposure groups (5, 10 and 20 mW/cm2 groups) (P b 0.05) as well.

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3.3. Neuronal degeneration in the hippocampus

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As shown in Fig. 3, in the hippocampus region (especially in CA3 and dentate gyrus subregions), karyopyknosis occurred and the nuclei shrank into blue pieces in exposure groups 14 days, 28 days and 2 months after exposure, among which the most serious injury occurred in 30 mW/cm2 group 28 days after microwave exposure as we presented in Fig. 3. In addition, neuronal degeneration in the cerebral cortex after exposure was also observed in H&E staining slices.

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and the AEL of rats in 30 mW/cm2 groups was longer than that of 10 mW/cm2 group according to repeated-measures ANOVA analysis (P b 0.05) (Fig. 1C). Specifically, at the end of the training sessions (4 days after exposure), the AEL of each group (including the control and exposure groups) was gradually decreased to a stable level, which meant rats had acquired the ability to find the platform, though rats in 10, 20 and 30 mW/cm2 exposure groups developed significant prolongations in AEL compared to the control group (P b 0.05). Rats of all exposure groups behaved longer AEL (P b 0.05) 14 days after exposure. 28 days after exposure, rats in 10, 20 and 30 mW/cm2 exposure groups still developed longer AEL compared to the control group (P b 0.05). In probe test, the percentage of time in the target quadrant of rats in all exposure groups was less than sham exposed rats (P b 0.05) (Fig. 1D). A low percentage of time spent in the platform quadrant is interpreted as a lower level of memory retention. In contrast, the swimming speed did not differ among the groups (P b 0.05) (Fig. 1B).

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cm2 group; Gly (IAA) was lower in 20 mW/cm2 and 30 mW/cm2 groups; and Tau (IAA) was lower in all exposure groups (5, 10, 20 and 30 mW/cm2 groups) than that of the control group as well (P b 0.05). 2 months after exposure, Asp and Glu were higher in 10 mW/cm2 group (P b 0.05). Amino acid contents in rats' CSF were shown in Table 2. Compared to the control group, 14 days after exposure, Asp was lower in 5 mW/cm2 group and higher in 30 mW/cm2 group (P b 0.05). 28 days after exposure, Glu was lower in all exposure groups (P b 0.05). Monoamine contents in rats' hippocampus were shown in Table 3. Compared to the control group, 14 days after exposure, NA was increased in 20 and 30 mW/cm2 groups (P b 0.05). 28 days after exposure, NA was increased in all exposure groups; DA content was increased in 5 and 10 mW/cm2 groups, decreased in 30 mW/cm2 group; DOPAC was increased in 30 mW/cm2 group; and 5-HT was increased in 10, 20 and 30 mW/cm2 groups (P b 0.05). 2 months after exposure, DA was increased in 5, 10 and 20 mW/cm2 groups; DOPAC was decreased in 30 mW/cm2 group; and 5-HT was increased while 5-HIAA was decreased in all exposure groups (P b 0.05). Monoamine contents in rats' CSF were shown in Table 4. Compared to the control group, 14 days after exposure, NA and HVA were increased in 30 mW/cm2 group (P b 0.05) and 5-HIAA was increased in 10 mW/cm2 group (P b 0.05). 28 days after exposure, NA and 5-HIAA were increased in 30 mW/cm2 group (P b 0.05). 2 months after exposure, DOPAC and 5-HIAA were decreased in 30 mW/cm2 group (P b 0.05).

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Table 2 Amino acid contents in rat's CSF after microwave exposure (mean ± SEM).

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3.4. Derangement of neurotransmitter contents in the hippocampus and CSF Amino acid contents in rats' hippocampus were shown in Table 1. Compared to the control group, 14 days after exposure, Asp and Glu (excitatory amino acids, EAA) were lower in 5 mW/cm2 group while GABA (inhibitory amino acid, IAA) was higher in 30 mW/cm2 group (P b 0.05). 28 days after exposure, Asp and Glu were lower in 5 mW/

65.34 ± 2.73 58.50 ± 4.93 62.11 ± 2.85 65.52 ± 7.96 65.27 ± 3.34

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Table 1 Amino acid contents in rat's hippocampus after microwave exposure (mean ± SEM).

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Asp

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Gly

Tau

t2:3 t2:4 t2:5

0.23 ± 0.04 0.13 ± 0.01⁎ 0.15 ± 0.01 0.20 ± 0.01 0.33 ± 0.04⁎ 0.19 ± 0.04 0.25 ± 0.06 0.13 ± 0.03 0.13 ± 0.03 0.21 ± 0.03 0.17 ± 0.02 0.13 ± 0.04 0.16 ± 0.02 0.14 ± 0.02 0.20 ± 0.03

5.03 ± 0.16 5.05 ± 0.12 5.73 ± 0.45 5.22 ± 0.32 4.70 ± 0.33 4.52 ± 0.19 3.37 ± 0.09⁎ 2.77 ± 0.26⁎ 3.52 ± 0.12⁎ 4.02 ± 0.18⁎

6.99 ± 0.40 5.49 ± 0.35 6.32 ± 0.69 6.50 ± 0.58 6.79 ± 0.57 5.51 ± 0.30 4.89 ± 0.38 4.63 ± 0.20 5.18 ± 0.49 5.40 ± 0.36 5.95 ± 0.44 5.47 ± 0.36 6.00 ± 0.22 5.60 ± 0.81 5.95 ± 0.27

3.77 ± 0.44 3.16 ± 0.57 2.59 ± 0.10 2.79 ± 0.29 3.05 ± 0.43 2.40 ± 0.34 2.80 ± 0.45 2.31 ± 0.55 1.77 ± 0.14 2.47 ± 0.14 2.44 ± 0.24 1.84 ± 0.18 2.92 ± 0.37 1.88 ± 0.11 2.31 ± 0.21

t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20

Contents (μg/mL)

5.59 ± 0.26 5.11 ± 0.50 5.43 ± 0.39 4.47 ± 0.66 6.02 ± 0.37

⁎ P b 0.05 versus the control group.

Please cite this article as: H.-J. Li, et al., Alterations of cognitive function and 5-HT system in rats after long term microwave exposure, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.12.039

t2:21

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⁎ P b 0.05 versus the control group.

0 5 10 20 30 0 5 10 20 30 0 5 10 20 30

28 days

2 months

DOPAC

DA

5-HIAA

HVA

5-HT

121.77 ± 8.66 122.28 ± 2.99 178.77 ± 19.33 241.50 ± 12.55⁎ 238.49 ± 31.54⁎

17.94 ± 4.70 15.75 ± 2.83 18.69 ± 4.98 12.52 ± 0.60 11.26 ± 2.69 11.38 ± 1.22 9.99 ± 1.80 13.94 ± 4.08 16.84 ± 4.28 17.05 ± 2.03⁎ 17.38 ± 1.16 14.58 ± 1.48 14.90 ± 2.45 14.94 ± 2.54 14.10 ± 0.83⁎

15.76 ± 3.06 17.35 ± 1.80 15.37 ± 1.00 15.19 ± 1.19 16.42 ± 0.85 14.85 ± 0.74 22.32 ± 2.42⁎ 18.61 ± 1.28⁎

191.21 ± 15.42 143.55 ± 3.66 134.40 ± 23.36 147.04 ± 26.96 142.13 ± 19.95 263.24 ± 15.04 269.49 ± 15.33 305.83 ± 24.87 260.70 ± 34.94 309.78 ± 25.72 268.10 ± 15.12 167.05 ± 13.43⁎ 172.82 ± 18.76⁎ 170.04 ± 16.24⁎ 206.36 ± 15.22⁎

9.47 ± 0.83 10.87 ± 3.18 12.54 ± 5.02 5.64 ± 0.45 5.58 ± 1.63 6.28 ± 1.06 6.08 ± 2.13 8.58 ± 3.52 7.84 ± 2.00 6.14 ± 1.13 5.63 ± 0.94 3.42 ± 1.20 4.73 ± 1.56 4.75 ± 1.76 4.47 ± 0.74

57.35 ± 9.33 47.44 ± 6.26 41.89 ± 1.84 51.89 ± 8.51 51.48 ± 3.68 34.39 ± 5.43 69.71 ± 11.58 108.10 ± 14.68⁎ 76.20 ± 11.84⁎ 68.21 ± 11.33⁎ 36.20 ± 6.53 98.49 ± 8.34⁎ 87.42 ± 7.82⁎ 138.41 ± 11.87⁎ 68.08 ± 11.94⁎

151.20 ± 15.37 281.98 ± 22.72⁎ 268.21 ± 28.99⁎ 255.41 ± 31.01⁎ 242.58 ± 28.75⁎ 230.07 ± 28.69 257.07 ± 53.69 310.96 ± 32.75 307.08 ± 41.13 257.73 ± 21.56

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The results of TPH1 expression and MAO activity were displayed in Fig. 4. TPH1 expressions in the hippocampus did not vary after 30 mW/ cm2 microwave exposure (P N 0.05) as shown in Fig. 4A. While MAO activity was decreased 28 days and 2 months after 30 mW/cm2 microwave exposure (P b 0.05) as shown in Fig. 4B.

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3.6. Alterations of 5-HT1AR, 5-HT2AR and 5-HT2CR expressions

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3.6.1. Results of western blot Western blot results of rats' hippocampi and cerebral cortices were respectively shown in Figs. 5 and 6. There were no alterations of 5-HT2AR expressions either in the hippocampus or cerebral cortex at different time points after exposure (30 mW/cm2) (P N 0.05) (Figs. 5B and 6B). Results presented in Fig. 5A and C indicated the increased expressions of 5-HT1AR and 5-HT2CR 14 days and 2 months after exposure but decreased expressions of 5-HT1AR and 5-HT2CR 28 days after exposure (30 mW/cm2) in the hippocampus (P b 0.05). Fig. 6A and C indicated the increased expressions of 5-HT1AR, and 5-HT2CR in the cerebral cortex 28 days after 30 mW/cm2 microwave exposure (P b 0.05).

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regions) and cerebral cortex which were then quantitatively analyzed by IOD as shown in Figs. 7B and 8B. 5-HT1AR and 5-HT2CR positive immunohistochemistry staining brown particles were observed to exist in the cytoplasm of the cells, especially distributed densely in pyramidal cell layer of CA1, CA2 and CA3 subregions of the hippocampus. Fig. 7 showed that in comparison with the sham exposure rats, the expressions of cytoplasmic 5-HT1AR were significantly increased in CA1, CA2 and CA3 regions of rats' hippocampi 14 days after microwave exposure (30 mW/cm2) (P b 0.05). 28 days after exposure, 5-HT1AR expressions were increased in CA1 and CA2 regions significantly (P b 0.05). 2 months after exposure, 5-HT1AR expression was only increased in the CA3 region (P b 0.05). As for the cerebral cortex region, cytoplasmic expression of 5-HT1AR was increased 28 days after exposure (P b 0.05). The increased expression was represented with the raise of IOD value. Fig. 8 indicated that compared to the control group, the expressions of cytoplasmic 5-HT2CR were significantly increased in the CA3 region 14 days and 2 months after exposure, while 5-HT2CR was decreased in the CA2 region 28 days after exposure (P b 0.05). With the same trend as 5-HT1AR, 5-HT2CR was increased in the cerebral cortex significantly 28 days after exposure (P b 0.05).

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15.12 ± 1.62 10.66 ± 0.56⁎ 11.38 ± 1.54 23.76 ± 1.81⁎ 24.76 ± 3.84⁎ 21.18 ± 0.94⁎

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Table 4 Monoamine contents in rat's CSF after microwave exposure (mean ± SEM).

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23.67 ± 2.17 28.33 ± 8.98 33.08 ± 7.23 23.29 ± 3.17 42.36 ± 5.38⁎ 23.84 ± 4.25 32.61 ± 7.31 35.92 ± 7.75 26.20 ± 3.27 38.57 ± 4.12⁎

6.65 ± 0.52 8.63 ± 0.98 8.69 ± 0.81 6.58 ± 0.55 6.93 ± 0.53 8.26 ± 0.70 8.21 ± 0.48 8.20 ± 0.45 7.12 ± 0.70 9.08 ± 0.81 8.03 ± 0.44 7.71 ± 0.28 7.75 ± 0.76 6.70 ± 0.36 6.89 ± 0.33⁎

73.76 ± 7.75 83.87 ± 7.57 121.99 ± 5.85⁎ 86.20 ± 3.66 74.19 ± 6.63 66.98 ± 2.74 81.91 ± 7.20 79.27 ± 7.03 80.24 ± 7.46 78.57 ± 4.02⁎

10.13 ± 1.49 8.19 ± 0.96 14.51 ± 1.29⁎ 10.45 ± 0.22 9.85 ± 0.22 11.29 ± 0.74 10.69 ± 1.21 10.98 ± 1.85 10.11 ± 0.72 12.38 ± 0.79 13.65 ± 1.01 12.44 ± 0.63 10.99 ± 1.05 11.38 ± 0.91 11.37 ± 0.50

26.36 ± 2.36 20.55 ± 0.39 20.68 ± 0.52 19.26 ± 0.57 30.55 ± 3.76

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The effects of microwave radiation are closely related to many 371 exposure parameters such as frequency, orientation, modulation, 372 power density, duration of exposure, and so on. Researches on chronic 373

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3.6.2. Results of immunohistochemistry Figs. 7A and 8A respectively showed the immunohistochemistry images for 5-HT1AR and 5-HT2CR in the hippocampus (CA1, CA2, CA3

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83.84 ± 3.66 81.21 ± 5.54 73.24 ± 6.38 76.38 ± 5.72 72.33 ± 3.64⁎

Please cite this article as: H.-J. Li, et al., Alterations of cognitive function and 5-HT system in rats after long term microwave exposure, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.12.039

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Fig. 4. A: Western blots of TPH1 expressions in rats' hippocampi of the control group and exposure group (30 mW/cm2). The histogram panels show the semi-quantified data of protein level. Each value is expressed as a ratio versus the level of the control group; B: MAO activity in rats' hippocampi of the control group and exposure group (30 mW/cm2). ⁎P b 0.05 versus the control group.

Fig. 5. Western blots of 5-HT1A, 2A, 2C R expressions in rats' hippocampi of the control group and exposure group (30 mW/cm2). The histogram panels show the semi-quantified data of protein level. Each value is expressed as a ratio versus the level of the control group. ⁎P b 0.05 versus the control group.

Please cite this article as: H.-J. Li, et al., Alterations of cognitive function and 5-HT system in rats after long term microwave exposure, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.12.039

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Electric activity of the brain is a direct reflection of brain functional status and the EEG remains the primary tool for measuring changes in dynamic brain functions [26]. The alterations of EEG can be seen in pathological and cognitive disorders, in which the loss of cognitive (particularly mnemonic) abilities occurred in close relation to the slowing of the EEG [27–30]. Studies found significant effects in the power spectral analysis of EEG frequency after microwave exposure [31,32]. In our study significant effects in the power spectral analysis of EEG were indicated: microwave exposure induced a decrease in frequency, rises in wave amplitude and increases in the relative power of delta band which index the reversal of learning [33]. Our observation is consistent with those in earlier reports. According to Thuroczy's study, whole body exposure of 2.45 GHz continuous wave (30 mW/cm2) for 10 min caused an increase in the total power of the EEG spectrum, local brain exposure of 4 GHz continuous wave induced an increase in the power of EEG delta waves (0.5–4 Hz) [34]. Chizhenkova's data also exhibited an increase of slow waves in rabbits after 2400 MHz CW radiation [35]. The inhibition of the electrical activity of the brain was consistent with the spatial learning and memory retardation in the MWM tests. In addition, delta wave could reflect increased tone of inhibitory monoamine receptor subtypes such as 5-HT1A neuronal assemblies [33], which was consistent with the increase of 5-HT1AR expression after microwave exposure in our study described later. Since the surface EEG predominantly reflected the activity of cortical neurons which close to the electrodes [1], we also measured the alterations of 5-HTR expression in the cerebral cortex besides the hippocampus in this study. Through HPLC measurements, disturbance of amino acid and monoamine in the hippocampus and CSF was conducted. Neurotransmitter systems are involved in learning and memory [7]. Amino acid neurotransmitters include two categories: the excitatory amino acids (mainly comprise Asp and Glu) and the inhibitory amino acids (mainly comprise

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exposure found that deficit in cognitive function could be induced by long-lasting exposure of some dose. For example, 2.45 GHz pulsed MW irradiation exposure (at a power density of 1 mW/cm2 for 3 h/day, for up to 30 days) induced spatial learning and memory impairments in rats [21]. Zhao also revealed that there was a significant decrease in learning and memory activity after 2.856 GHz microwave exposure (6 min daily up to 1 month with the power density of 2.5, 5, and 10 mW/cm2) [22]. Though Cobb's study reported that the exposure of 2450 MHz fields (45 min/day, for 10 days) did not cause decrements in the ability of rats to learn spatial memory task [23]. To meet the needs of occupational exposed populations, studies on long-term effects of high intensity microwave exposure for long durations were necessary. At the early stage of our study, we analyzed cognitive behavioral changes and several related physiological and biochemical indexes in rats exposed to microwaves of four doses 14 days, 28 days and 2 months after exposure. Through MWM results, we found that the microwave exposure caused rats' spatial learning and memory ability retarded and the effect was dose-dependent. Rats in groups of high power density performed lower learning ability and memory retention than those in groups of low power density and sham exposure group. The encoding of information about an animal's spatial word is associated with the hippocampus, which is the main area of the limbic system involved in learning and memory activities [24,25]. Pyramidal cells of the hippocampus are closely related to the formation and maintenance of spatial memory [1]. Variations of the hippocampal structure in the dentate gyrus and CA3 area after exposure was consistent with the MWM test [17]. Correspondingly, we observed that the most serious injury occurred in rats of 30 mW/cm2 group 28 days after exposure and presented recovery trend later. We assumed that the structural injury of the hippocampal CA3 and dentate gyrus might lead to the impairment of spatial learning and memory abilities.

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Fig. 6. Western blots of 5-HT1AR, 5-HT2AR and 5-HT2CR expressions in rats' cerebral cortices of the control group and exposure group (30 mW/cm2). The histogram panels show the semiquantified data of protein level. Each value is expressed as a ratio versus the level of the control group. ⁎P b 0.05 versus the control group.

Please cite this article as: H.-J. Li, et al., Alterations of cognitive function and 5-HT system in rats after long term microwave exposure, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.12.039

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caused by tranylcypromine, resulted in improved cognitive flexibility (ability for reversal learning) [42]. We assumed that the increase of 5-HT was involved in microwave-induced cognitive impairment. However the cognitive impairment which was induced by microwave exposure can hardly be ascribed to a specific transmitter system because of complicated interactions between them. In view of the long-lasting effect of exposure on 5-HT, we chose serotonergic system as our further research target on circumstance of 30 mW/cm2 microwave exposure. The augmentation of 5-HT content could be achieved either by increasing its synthesis or decreasing its metabolism. Our results illustrated that the decrease of MAO activity was consistent with the HPLC results, which showed the increase of 5-HT and decrease of 5-HIAA in the hippocampus. In other words, the metabolic process of 5-HT to 5-HIAA by MAO in the hippocampus was reduced after 30 mW/cm2 microwave exposure. As to 5-HT receptors, we focused on 5-HT1AR, 5-HT2AR and 5-HT2CR subtypes. The increase of 5-HT1AR and 5-HT2CR expressions in the cerebral cortex 28 days after exposure was indicated by both western blot and immunohistochemistry. Otherwise, alterations of 5-HT1AR and 5-HT2CR expression were uneven in different hippocampal subregions. We assumed that serious injury in CA3 and dentate gyrus areas might contribute to the decrease trend of 5-HT1A, 2CR indicated by western blot 28 days after exposure. The general increase and decrease trend of 5-HT2CR were mainly contributed by its increased expression in the CA3 region and decreased expression in the CA2 region respectively. Serotonergic system takes an imperative effect in learning and memory by interacting with other transmitter systems like the cholinergic,

Gly, Tau and GABA). These compounds are highly presented in the central nervous system (CNS) and modify neuronal and behavioral function including cognition [36]. In our study, 14 days after exposure, Asp and Glu (EAA) of the hippocampus declined in groups of low power intensity while GABA (IAA) ascended in groups of high power intensity. Tau in the hippocampus was decreased as well as Glu in the CSF 28 days after exposure. Glu is reported to play pivotal roles in learning and memory process [7]. Thus we assumed that the decline of Glu observed in our study may be involved in cognitive impairment after microwave exposure. Tau induced synaptic potentiation through the activation of a transporting system and its uptake was required for the induction of synaptic plasticity phenomena [37]. A neuroprotective effect of Tau was presented in damaged circumstances. Consequently, we assumed that the decline of Tau observed in our study may also correlate with microwave-induced cognitive impairment. Parallelly, the results of hippocampal monoamine neurotransmitters elucidated that NA disturbance occurred at the earlier stage while 5-HT disturbance occurred as a long-lasting effect after microwave exposure. Aboul's study revealed significant increase of 5-HT content in rats' hippocampi after 1 and 2 months of daily electromagnetic radiation exposure (1800 MHz, power density 0.02 mW/cm2) and the increase recurred after stopping the exposure for 1 month [38]. In agreement with that the increase of 5-HT concentration after microwave exposure was observed in our study. Hippocampal monoamines were related to a variety of behaviors including learning and memory processes [39]. 5-HT had a suppression influence on learning and memory function and negative effects on several pathways which were involved in spatial information processing [40,41]. The drastic reduction in 5-HT concentration, which was

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Fig. 7. A: Immunohistochemistry images for 5-HT1AR of rats' hippocampi (CA1, CA2 and CA3 subregions) and cerebral cortices of the control group and exposure group (30 mW/cm2); B: IOD values analyzed from immunohistochemistry images (A) using image-pro plus software. ⁎P b 0.05 versus the control group.

Please cite this article as: H.-J. Li, et al., Alterations of cognitive function and 5-HT system in rats after long term microwave exposure, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.12.039

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glutamatergic, dopaminergic or GABAergic systems [43]. Serotonin modulates functions of the CNS via binding to specific receptors located in related crucial brain structures like the hippocampus and frontal cortex. 5-HT receptors showed regional distribution in brain areas implicated in learning and memory [44]. 5-HT1AR and 5-HT2A, 2CR, which belong to the G protein-coupled receptor class, have gained much attention concerning to cognitive function. The stimulation or blockade of presynaptic 5-HT1A, 5-HT2A, 2C and other receptors modulated learning and memory processes [44]. 5-HT1AR in the hippocampus has an inhibitory effect on human explicit memory [45]. The stimulation of the receptor caused the impairment of spatial learning capability of rats [46], the agonist for 5-HT1AR impaired the short and long term memory [13] and the antagonist for 5-HT1AR caused memory improvement [47]. Thus we supposed that the increase of 5-HT content and 5-HT1A receptor expression observed in this study may mediate the disruption of spatial learning and memory caused by the microwave exposure. Dissimilarly, 5-HT2CR acted on learning and memory in various ways. Meneses et al. reported that stimulation of 5-HT2CR impaired both short term memory and long term memory and a low dose of 5-HT2CR antagonist impaired both short and long term memory but intermediate or higher doses impaired long term memory [13]. In this study, we found that the expression of 5-HT2CR was increased in the CA3 region 14 days and 2 months after exposure but decreased significantly in the CA2 region 28 days after exposure which may be both the reasons that partly caused microwave exposure-induced cognitive impairment. Further studies are required to confirm our assumptions. In summary, our observations indicated that long-term exposure to microwave (2.856 GHz, with the average power density of 5, 10, 20 and

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Fig. 8. A: Immunohistochemistry images for 5-HT2CR of rats' hippocampi (CA1, CA2 and CA3 subregions) and cerebral cortices of the control group and exposure group (30 mW/cm2); B: IOD values analyzed from immunohistochemistry images (A) using image-pro plus software. ⁎P b 0.05 versus the control group.

30 mW/cm2) could induce dose-dependent deficit of spatial learning and memory accompanied with the inhibition of brain electrical activity, the degeneration of hippocampus neurons, and disturbances of neurotransmitters. Further experiments elucidated that elevated level of metabolism partly contributed to the increase of 5-HT after microwave exposure. Besides aberrant 5-HT1AR and 5-HT2CR expressions were also indications of 5-HT system modulation. We assumed that 5-HT system may be involved in microwave exposure-induced cognitive deficit and further studies are needed to confirm that.

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Conflict of interest statement

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There are none. Acknowledgments

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This study was supported by the National Basic Research Program of 532 China (2011CB503706). 533 References [1] J.A. D'andrea, C.K. Chou, S.A. Johnston, E.R. Adair, Microwave effects on the nervous system, Bioelectromagnetics (Suppl. 6) (2003) S107–S147. [2] H. Lai, Interaction of microwaves and a temporally incoherent magnetic field on spatial learning in the rat, Physiol. Behav. 82 (2004) 785–789. [3] B. Wang, H. Lai, Acute exposure to pulsed 2450-MHz microwaves affects watermaze performance of rats, Bioelectromagnetics 21 (2000) 52–56. [4] H. Nittby, G. Grafstrom, D.P. Tian, L. Malmgren, A. Brun, B.R. Persson, et al., Cognitive impairment in rats after long-term exposure to GSM-900 mobile phone radiation, Bioelectromagnetics 29 (2008) 219–232.

Please cite this article as: H.-J. Li, et al., Alterations of cognitive function and 5-HT system in rats after long term microwave exposure, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.12.039

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Please cite this article as: H.-J. Li, et al., Alterations of cognitive function and 5-HT system in rats after long term microwave exposure, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.12.039

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