Environ Sci Pollut Res DOI 10.

1007/s11356-012-1266-5

ECOTOXICOLOGY AND ENVIRONMENTAL TOXICOLOGY : NEW CONCEPTS, NEW TOOLS

Effects of chronic exposure to radiofrequency electromagnetic fields on energy balance in developing rats
Amandine Pelletier & Stéphane Delanaud & Pauline Décima & Gyorgy Thuroczy & René de Seze & Matteo Cerri & Véronique Bach & Jean-Pierre Libert & Nathalie Loos
Received: 4 July 2012 / Accepted: 16 October 2012 # Springer-Verlag Berlin Heidelberg 2012

Abstract The effects of radiofrequency electromagnetic fields (RF-EMF) on the control of body energy balance in developing organisms have not been studied, despite the involvement of energy status in vital physiological functions. We examined the effects of chronic RF-EMF exposure (900 MHz, 1 Vm−1) on the main functions involved in body energy homeostasis (feeding behaviour, sleep and thermoregulatory processes). Thirteen juvenile male Wistar rats were exposed to continuous RF-EMF for 5 weeks at 24 °C of air temperature (Ta) and compared with 11 non-exposed animals. Hence, at the beginning of the 6th week of exposure, the functions were recorded at Ta of 24 °C and then at 31 °C. We showed that the frequency of rapid eye movement sleep episodes was greater in the RF-EMF-exposed group, independently of Ta (+42.1 % at 24 °C and +31.6 % at 31 °C). The other effects of RF-EMF exposure on several sleep parameters were dependent on Ta. At 31 °C, RF-EMF-exposed animals had a significantly lower subcutaneous tail temperature (−1.21 °C) than controls at all sleep stages; this suggested
Responsible editor: Philippe Garrigues A. Pelletier : S. Delanaud : P. Décima : V. Bach : J.-P. Libert : N. Loos (*) PériTox Laboratory (EA 4285-UMI01), Faculty of Medicine, Jules Verne University of Picardy (UPJV), 3 rue des Louvels, CS 13602, 80036 Amiens cedex 1, France e-mail: nathalie.loos@u-picardie.fr G. Thuroczy : R. de Seze PériTOX Laboratory (EA 4285-UMI01), VIVA/TOXI, National Institute of Industrial Environment and Risks (INERIS), Parc ALATA BP2, 60550 Verneuil-en-Halatte, France M. Cerri Department of Human and General Physiology, Alma Mater Studiorum-University of Bologna, Bologna, Italy

peripheral vasoconstriction, which was confirmed in an experiment with the vasodilatator prazosin. Exposure to RFEMF also increased daytime food intake (+0.22 gh−1). Most of the observed effects of RF-EMF exposure were dependent on Ta. Exposure to RF-EMF appears to modify the functioning of vasomotor tone by acting peripherally through α adrenoceptors. The elicited vasoconstriction may restrict body cooling, whereas energy intake increases. Our results show that RF-EMF exposure can induce energy-saving processes without strongly disturbing the overall sleep pattern. Keywords Radiofrequency electromagnetic field . Sleep . Feeding behaviour . Thermoregulation . Young rat

Introduction Body energy balance is a relevant factor in growing organisms, since energy is required for vital functions, thermoregulation and tissue synthesis (in that order). The regulation of energy balance involves a complicated interaction between energy intake (feeding behaviour), energy saving (sleep), energydissipating mechanisms (vasomotricity) and energy production, all of which are controlled by hypothalamic structures (Blessing 2003; El Hajjaji et al. 2011; Himms-Hagen 1995). Several studies have shown that these functions are in competition; sleep and feeding cannot be performed simultaneously, for example. In the rat, there is a positive correlation between meal size and the durations of both non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS): the larger the energy intake, the longer the rat will sleep (Danguir and Nicolaidis 1985). As far as thermal status and feeding behaviour are concerned, body cooling and body heating stimulate and reduce food intake, respectively (De Vries et al. 1993; El Hajjaji et al. 2011). Thus, an animal in a cold environment consumes extra food

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in order to avoid use of its energy reserves and to have a negative energy balance. It is also well known that there is a strong functional interaction between sleep and thermoregulation in both animals (Parmeggiani 1977) and humans (Libert et al. 1982). On the basis of the scientific literature, it is difficult to draw firm conclusions as to the effects of a mobile phone base station on body energy balance. There are several possible explanations for these contradictory data. Firstly, the frequency and intensity of radiofrequency electromagnetic field (RF-EMF) exposure differ from one study to another and thus preclude direct comparisons. Mostly, experiments have been performed with higher intensities than those commonly found in the environment. Secondly, RF-EMF exposures have always been studied for relatively short periods of time, and the effect of chronic exposure on sleep over several weeks (i.e. reflecting the situation near mobile phone antennae) has never been assessed. Thirdly, sleep analyses have not generally been performed in strictly controlled thermal environments; this represents a potential source of bias because of the strong functional interaction between sleep and thermal stress. Fourthly, energy balance studies have failed to simultaneously take account of the complex functional interaction between sleep, thermoregulation and energy intake. Given that body energy balance is sensitive to a number of influences, it can be hypothesized that RF-EMF exposure can disturb the functional interactions that underlie the management of energy flows. Several thermoregulation studies have found that a RFEMF exposure at 2,450 MHz (specific absorption rate (SAR) between 1.1 and 3.2 Wkg−1) decreases the comfort air temperature (Adair and Adams 1980, 1983). However, the intensity of RF-EMF in these experiments was higher than that of exposure to typical mobile phone antenna and induced hypothalamic heating of 0.2–0.3 °C (Adair et al. 1984). Studies on sleep architecture and electroencephalographic (EEG) activity in adult humans have shown that RFEMF exposure is associated with a decrease in the duration of REMS (Mann and Roschke 1996) or an increase in the spectral power density during NREMS (Borbély et al. 1999). In adult rats exposed to RF-EMF exposure, the total amount of REMS increased (Crouzier et al. 2007b) or the total spectral power density increased (Thuroczy et al. 1994). There is a lack of data on young subjects; sleep, especially, is of particular importance in growing organisms in which the brain maturation process may not have completed. Sleep alterations can disturb the functional organization of cortical neuronal synchronization, body growth and restitution (Horne 1988). With a view to clarifying this matter, we studied the effects of chronic low-level RF-EMF exposure on energy balance in developing rats exposed in strictly controlled ambient temperatures (Ta) of 24 and 31 °C. These two temperatures are in the rat's thermoneutral zone (Kumar et

al. 2009; Romanovsky et al. 2002), within which the animal's cutaneous heat losses can be solely controlled by changes in peripheral vasomotor tone. The RF-EMF parameters were chosen to simulate the chronic exposure commonly encountered for a mobile phone base station. As the mobile phone emission is a clear GSM modulation one eighth of the time, i.e. 576 μs every 4.6 ms, the emission from a base station is continuous, at least for one of the channels of the antenna (control channel or BCCH); other channels have an aleatory emission depending on the traffic and cannot be mimicked by a fixed modulation structure. Then, a continuous emission is satisfactorily representative of that of a base station antenna. This type of exposure is called a continuous wave (CW) exposure. The used intensity around 1 Vm−1 is the maximum emission found at the ground level in front of a base station antenna at a distance between 100 and 200 m from the mast, and this level does not induce body heating. The rats' cortical and subcutaneous tail temperatures were recorded as indexes of the central temperature and peripheral vasomotor tone, respectively. The well-vascularized tail region plays a major role in the maintenance of body homeothermia by modulating peripheral cutaneous blood flow and thus convective and radiative heat losses through the skin (Dawson and Keber 1979). Indeed, the tail can dissipate up to 40 % of the animal's basal metabolic heat production (Young and Dawson 1982). The present study probed the response of this thermoregulatory effector by using the vasodilatator prazosin (a postsynaptic antagonist of the α1 adrenergic receptor) (Langer 1974). The sympathetic adrenergic nervous fibres are primarily involved in the peripheral vasomotor tone, and the α1-adrenoceptor subtype mainly mediates arteriolar vasoconstriction in the rat tail (Roberts et al. 2002). As energy expenditure and energy intake are strongly correlated, feeding behaviour was also recorded as an index of the energy needs required to maintain energy balance and body growth (Strubbe and van Dijk 2002).

Materials and methods Animals and accommodation Young male Wistar rats (aged 3 weeks and weighing 50– 75 g) at the start of the experiment, supplied by Centre d’Elevage René Janvier (Le Genest Saint-Isle, France), were studied in two similar, air-conditioned, sound-proofed climatic chambers (2.4×2.43×1.6 m) with strictly controlled environmental conditions. The animals were divided into two groups: eight rats in one chamber were exposed to RF-EMF (from their arrival in the laboratory onwards), and eight controls in a second chamber were not exposed. Two series of animals were studied: 2×8 exposed rats and

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2×8 control rats. The animals were housed in individual plastic cages (32×35×20.5 cm) within the chamber and maintained under standard environmental conditions in terms of air and wall temperatures (24±1 °C), light (light– dark cycle, 12–12 h, lights on/off at 7 am/7 pm), relative air humidity (40 ± 5 %), air velocity (<0.1 m/s, indicating convection-free conditions) and noise levels (<65 dB). Food (standard AO3 chow—51.7 % carbohydrate, 5.1 % lipid and 21.4 % protein, expressed in percentages of total energy content; SAFE, Augy, France) and tap water were available ad libitum. The rats were handled every day at 6 pm and treated in accordance with French and European guidelines on the care and use of laboratory animals. The rats were acclimatized to the laboratory conditions for 3 weeks prior to surgery. At the end of the study, the animals were euthanized with a 200-mg/kg intraperitoneal injection of pentobarbital sodium (CEVA Santé Animale, Libourne, France). Exposure system The climatic chambers housing the exposed group were equipped with RF-EMF generators (Fig. 1). A radiofrequency power source (type RFS 900–64, RFPA, Artiguesprès-Bordeaux, France) emitting a CW 900-MHz electromagnetic field was connected to a four-output divider which simultaneously supplied four antennae (Kathrein 80010465, Rosenheim, Germany). Each antenna was a broadband, directional, vertically polarized gain antenna designed for indoor radio installations and measured 23×14×5 cm. The antenna's operating frequency bands were 806–960 and 1,710–2,700 MHz. The antennae were located horizontally in the climatic chamber, 80 cm above the exposed rats' boxes. Modelling of the field has been performed without grids and plateaus from a near-field to near-field transformation developed at SUPELEC (Bolomey et al. 2004), to tune a chosen field intensity of 1 Vm−1. The distance between two antennae (48 cm) was chosen to obtain an electromagnetic field that was as homogeneous as possible for each rat. As we did not have access to a numerical computation of SAR by FDTD, the whole-body SAR (defined as
Fig. 1 Radiofrequency exposure system in the climatic chamber. The radiofrequency power source was outside the climatic chamber and connected to a four-output divider, which simultaneously supplied four antennae to expose eight rats

RF power absorbed per unit of tissue mass) was calculated as recommended by Durney et al. (1986). The SAR was estimated at 0.3 mWkg−1 for rats aged 3 weeks and 0.1 mW kg−1 for rats aged 8 weeks (i.e. at the start of the recording period). The RF-EMF exposure level was checked with a frequency-selective radiofrequency dosimeter (EME-SPY 121, Satimo, Plouzané, France) for each box position in both the exposed and sham climatic chambers (Fig. 2a) and a broadband radiation monitor (EMR-200, Narda-STS, Pfullingen, Germany). Frequency-selective fields were measured in each climatic chamber with a log-periodic wideband antenna (HE 200, 20 MHz–3 GHz, Rohde & Schwarz, Meudon-la-Forêt, France) and a spectrum analyzer (FSH3, 9 kHz – 3 GHz, Rohde & Schwarz, Meudon-la-Forêt, France). The system's input and reflected powers were measured with a dual-channel power meter (the NRVD from Rohde & Schwarz, with NRVZ4 and NRVZ5 probes). Measurements have been performed in the actual situation, with the grids at the bottom of the cages and the plateaus hereunder. The dosimeter was placed on the metallic grids at the bottom of the box to record the RF-EMF exposure. As the goal level of 1 Vm−1 has been achieved at ±3 dB, this suggests that the field is only weakly distorted by the metallic elements at the bottom of the cages. In order to assess potential changes in exposure over time, the RF dosimeter recorded a measurement every 4 s for 6 h (Fig. 2b). The RF-EMF was emitted for 23.5 h per day, from the day of installation to the day of euthanasia. Emission was only stopped for 30 min each day (from 6 pm to 6.30 pm) for animal care (Fig. 2b). In the climatic chamber housing the control (non-exposed) group, four white similarly shaped empty boxes were used to mimic the antennae. Surgery After a 3-week exposition period (Fig. 3), the rats were implanted with electrodes and sensors under general anaesthesia which was administered through intraperitoneal injection of 60 mgkg−1 pentobarbital (CEVA Santé Animale,

Environ Sci Pollut Res Fig. 2 a Dosimetry measurements in each rat's box for 6 h (boxes 1 to 8, mean values±SE) for the control group (open circles) and the exposed group (filled circles). b Representative example of the variations in intensity over time in a single box

Fig. 3 Study design

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Libourne, France) so that polysomnographic and thermal parameters could be recorded. Four miniature gold-plated EEG screws (diameter, 1.08 mm; length, 7.8 mm) were inserted in the skull of each animal (above the dura mater) to record the bilateral frontoparietal EEG activity. The electrodes did not penetrate into the brain tissue, in order to avoid any related effect of the RF-EMF on the metal electrodes. Two gold-plated electromyographic (EMG) electrodes were bilaterally inserted into the neck muscles in order to discriminate wakefulness (W) from REMS. To check for possible electrical interference between the RF-EMF and the EEG electrodes, spectral analysis was performed for 10 min in a group of eight exposed rats with and without RF-EMF exposure and during a post-exposure recovery period. There was no specific effect of RF-EMF exposure on the EEG electrodes (F2,126 0 0.31, p 0 0.74) in terms of either the electrode position (right or left, F2,126 0 0.03, p 0 0.97) or the specific frequency bands defined above (F4,126 0 1.41, p 0 0.23) (data not shown). The subcutaneous tail temperature was measured with a thermoprobe (thermistance PB7-43-S1 Shibaura, 10 kΩ at 25 °C; accuracy ±0.1 °C; BFI OPTILAS, Evry, France) inserted into the proximal part of the tail, since this site reflects the overall thermal status of the tail surface (Sapsed-Byrne et al. 1995). An additional thermoprobe was placed under the skull but over the dura mater of the brain. Prior to surgery, the temperature probes were calibrated in a water bath by assessing the linear relationship between probe resistance (in kilohms) and 1 °C temperature increments ranging from 18.0 to 43.0 °C. The absence of cortical temperature variations from the probe placed between two EEG electrodes at the same height over the dura mater showed that the electrical signals were not being amplified. All leads were passed subcutaneously from the dorsal nape and neck to the head and were fixed to a connector held in place on the skull with dental cement (Dentalon, Henri Schein, Alfortville, France). The connector was attached to a commutator suspended over the middle of the cage. Then, the main orientation of the leads coming out from the head of the animal was perpendicular to the polarisation of the electric field coming out from the antennas. All commutators were connected to a monitoring system with an 18-channel rotating connector swivel (SL18C: Plastic One Inc., Phymep, Paris, France). The rat was able to move freely around its cage, and its movements did not interfere with the signals. The temperature probes' positions in situ were visually checked in a postmortem inspection. Measurements of sleep, food and thermal parameters started after 5 weeks of actual or sham RF-EMF exposure (3 weeks of acclimatization and two further weeks of surgery and post-surgery recovery) (Fig. 3).

Experimental protocol and data acquisition At the beginning of the 6th week of exposure to continuous RF-EMF (Fig. 3), the rats' physiological parameters were recorded on two successive days at air temperatures (Ta) of 24±1 and 31±1 °C. The air and wall (Tw) temperatures of the climatic chamber did not differ (i.e. Ta 0 Tw), in order to control the radiative heat exchange. All physiological parameters (polysomnographic parameters, body temperatures and food intake) were continuously recorded from 12 am to 6 pm with three computers located outside the chambers. The computers were radiosynchronized hourly using the 55RS card (BH Technologies, Grenoble, France). The EEG and EMG signals were collected with a 32channel polysomnographic system (model 15, Astromed Grass Polygraph, Astro-med SNC, Trappes, France). Bipolar signals were amplified (×10,000; Neurodata Amplifier System, Astro-med SNC, Trappes, France) and filtered (with pass bands of 0.3–30 and 1–64 Hz for EEG and EMG signals, respectively). The EEG and EMG signals were digitized at a sampling rate of 128 Hz. Body temperatures were recorded every 5 s with an 80channel analogue–digital station (AOIP Instrumentation, Tourcoing, France). Food intake was quantified for each animal using a high-accuracy calibrated electronic scale (Sartorius BL 200; accuracy, ±0.1 g; Labo & Co, Marollesen-Brie, France) connected to a multichannel RS232 recorder (Acksys 8-port RS232, Acksys Communication & Systems, Villepreux, France). Sleep scoring and EEG power spectral analysis Due to signal failures of EEG, polysomnographic data recorded between 12 am and 6 pm were visually scored for 13 of the 16 exposed rats and 11 of the 16 control rats. The data were analysed for W (low-amplitude, highfrequency EEG; high-amplitude EMG), NREMS (high-amplitude, low-frequency EEG; low-amplitude EMG) and REMS (low-amplitude, high-frequency EEG and no EMG muscle tone). We calculated the total amounts of W, NREMS, REMS and total sleep time (TST) (expressed as percentages of the overall analysis time) and the mean durations and frequencies of the W, NREMS and REMS episodes. In our study, active wakefulness corresponds to behaviour activities during which the animals awake, move and/or eat. The bilateral frontoparietal EEG signals were processed with fast Fourier transformation (FFT) by using 8-s periods to obtain a distribution of 1,024 points (PolyVIEW PRO software). The FFT analysis enabled extraction of the power densities in the delta band (δ, 0.75–4.0 Hz), theta band (θ, 5.5–9.0 Hz) and sigma band (σ, 11.0–16.0 Hz). Each

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specific band was analysed for each sleep–wake stage, and the average power density in each frequency band was calculated for W, NREMS and REMS episodes. The spectral power densities were expressed as volts per hertz (square microvolts per hertz). Analysis of body temperatures The subcutaneous tail temperature (Ttail) and the cortical temperature (Tcor) served as indices of the peripheral and central temperatures, respectively. These body temperatures were measured between 12 am and 6 pm and scored for the W, NREMS and REMS stages. Due to probe failures, tail and cortical temperatures were analysed for 10 of the 13 exposed rats and 9 of the 11 control rats. An additional study was performed on five other RFEMF-exposed rats and five other non-exposed rats, in order to analyse the impact of the vasodilatator prazosin (SigmaAldrich, Lyon, France) on Ttail and the peripheral vasomotor tone at 24 and 31 °C. Prazosin was administered by intraperitoneal injection at a dose of 1 mgkg−1, since it is well known that this dose inhibits the peripheral vasoconstrictor tone without inducing major changes in the animals' cortical metabolism and neural glucose levels (Inoue et al. 1991; Larson et al. 1996). The tail temperature was recorded from 12 am to 6 pm. Prazosin was injected at 12 am. For each rat and each thermal condition (24 and 31 °C), the Ttail values were averaged and plotted against time. Feeding behaviour The quantity of food ingested between 12 am and 6 pm was analysed individually in 13 exposed rats and 11 controls using LabView version 7.1 (National Instruments, Nanterre, France). They were the same rats as those for which the EEG signals were analysed. The ingested quantity was corrected for the weight of uneaten food found in the litter and expressed in grams per hour. The energy equivalent was expressed in kilojoules per hour. Statistical analysis All mean values are quoted with their corresponding standard error (±SE). Data were analysed using Statview software (version 5.0, SAS Institute Inc., Cary, NC, USA). The normality of the data distribution was checked with a Kolmogorov–Smirnov test. A one-way analysis of variance with repeated measures was used to test the difference between the two Ta conditions (31 vs. 24 °C), between RF-EMF-exposed animals and controls and between the body temperatures measured for the different vigilance states. Interactions between the RF-EMF exposure and Ta were indicated. When F values were significant, Student's paired and unpaired t tests were calculated.

Student's paired test was used to test the difference between the two ambient temperature conditions and between the body temperatures measured for the different vigilance states. Student's unpaired test was used to test the difference between the exposed group and the control group. The subscripts beneath F and t values correspond to degrees of freedom. To avoid false positives, the threshold for statistical significance was set to p <0.01 after Bonferroni correction. Non-parametric tests were used for the prazosin study, in view of the small number of animals. A Mann–Whitney test was used to probe the difference between exposed and control animals, and a Wilcoxon test was applied to assess the difference between thermal conditions in the presence or absence of prazosin. The threshold for statistical significance was set to p <0.05.

Results Specific effects of ambient air temperature Table 1 shows the parameters related to sleep in the RF-EMFexposed and control groups. The total duration of W was lower at Ta of 31 °C than at 24 °C (−31.2 %, t23 0 7.22, p <0.001), independently of RF-EMF exposure (i.e. there was no significant interaction between RF-EMF and Ta). The lower amount of W at 31 °C was due to greater total amounts of NREMS and REMS (NREMS, +6.3 %, t23 0 5.54; REMS, +20.1 %, t23 0 5.05, p <0.001). Consequently, TST increased (+7.5 %, t23 0 8.22, p <0.001). The mean duration of a REM episode was higher at 31 °C (+10.5 %, t23 0 5.42, p <0.001). The EEG signals' spectral power densities are shown in Table 2. When compared with 24 °C, the spectral power density in σ bands at 31 °C was greater in W (+9.8 %, t23 0 3.24, p <0.01), NREMS (+8.8 %, t23 0 3.96, p <0.001) and REMS (+17.5 %, t23 0 3.46, p <0.01). As expected (Fig. 4), Tcor was greater at 31 °C than at 24 °C (+0.15 °C, F1,17 0 15.57, t56 0 6.07, p <0.001). Table 3 shows that this effect was significant in W and NREMS (W, +0.17 °C, t18 0 4.01; NREMS, +0.18 °C, t18 0 4.05, p <0.001) but not in REMS. Ttail increased with Ta (+1.42 °C, F1,17 0 16.00, t56 0 15.26, p <0.001) in all sleep stages (with no interaction between Ta and sleep stages, F2,34 0 0.16): W (+2.13 °C), NREMS (+1.91 °C) and REMS (+1.94 °C). As shown in Fig. 5, the food intake (i.e. the energy intake) was lower at 31 °C than at 24 °C but did not depend on RF-EMF exposure (−0.28 gh−1, i.e. −4.10 kJh−1, t23 0 8.87, p <0.001). Effects of RF-EMF exposure As shown in Table 1, the only specific effect of RF-EMF (i.e. independently of Ta) was a higher frequency of REMS episodes (with no significant interaction between Ta and RF-

Environ Sci Pollut Res Table 1 Sleep parameters in the control and the RF-EMF-exposed groups at ambient temperatures of 24 and 31 °C Control group 24 °C W Total amount Frequency of episodes Duration of episodes Total amount Frequency of episodes Duration of episodes Total amount Frequency of episodes Duration of episodes Total amount 16.9±0.7 1.2±0.17 9.0±0.4**** 71.4±1.0 12.1±0.5 3.0±0.2 10.5±0.5 3.8±0.1 1.4±0.1 90.2±0.7 31 °C 11.6±0.7 1.8±0.2 5.0±0.6 75.3±0.7 14.2±0.6 2.8±0.1 13.9±0.5 3.8±0.4 2.1±0.1 83.1±0.9 Exposed group 24 °C 18.7±1.1 1.3±0.1*** 6.5±0.3** 68.6±1.0 14.0±0.5**** 2.6±0.1**** 12.9±0.7 5.4±0.2 1.4±0.1 89.2±1.0 31 °C 12.9±1.1 0.9±0.1** 7.0±0.6 73.6±0.9 12.1±0.4* 3.2±0.1 14.2±0.8 5.0±0.3 1.7±0.1 83.7±0.7 F1,22 0 49.38, p <0.001 NS NS F1,22 0 29.12, p <0.001 NS NS F1,22 0 31.66, p <0.001 NS F1,22 0 36.28, p <0.001 F1,22 0 68.76, p <0.001 NS NS NS NS NS NS NS F1,22 0 24.30, p <0.001 NS NS NS F1,22 0 16.44, p <0.001 F1,22 0 22.76, p <0.001 NS F1,22 0 17.68, p <0.001 F1,22 0 15.33, p <0.001 NS NS NS NS Ta effect RF effect RF × Ta interaction

NREMS

REMS

TST

Total amount (expressed as a percentage of recording time), frequency of episodes per hour and the mean duration (in minutes) of W, NREMS and REMS and the TST in the control (n 0 11) and RF-EMF-exposed groups (n 0 13). Values are expressed as mean±SE. The right-hand columns indicate effects due to the ambient temperature (“Ta effect”), effects due to RF-EMF exposure (“RF effect”) and effects of an interaction between RF-EMF exposure and Ta (“RF × Ta interaction”) NS non-significant p <0.01, ** p <0.001—statistical significance of comparisons between control and exposed groups; icance of comparisons between the two ambient temperatures
* ***

p <0.01,

****

p <0.001—statistical signif-

EMF exposure: +42.1 % at 24 °C and +31.6 % at 31 °C, t46 0 5.10, p <0.001). All the other effects of RF-EMF exposure were temperature dependent (i.e. with a significant interaction between Ta and RF-EMF exposure).

At Ta of 31 °C, RF-EMF exposure was associated with a lower frequency of W and NREMS episodes, compared with the control group (W, − 50.0 %, t 22 0 4.45, p < 0.001; NREMS, −14.8 %, t22 0 3.02, p <0.01), whereas only the

Table 2 Frequency band power densities in the control and RF-EMF-exposed groups at ambient temperatures of 24 and 31 °C Control group 24 °C W Delta (0.75–4.0 Hz) Theta (5.5–9.0 Hz) Sigma (11.0–16.0 Hz) Delta (0.75–4.0 Hz) Theta (5.5–9.0 Hz) Sigma (11.0–16.0 Hz) Delta (0.75–4.0 Hz) Theta (5.5–9.0 Hz) Sigma (11.0–16.0 Hz) 44.8±4.2 21.8±0.9 5.9±0.3 82.9±8.8 32.0±1.0 9.1±0.4 29.4±3.2 52.3±5.3 5.2±0.4 31 °C 39.5±2.5 22.4±1.2 6.5±0.3 70.5±5.7 32.5±1.3 10.0±0.5 35.9±5.7 53.9±4.5 6.7±0.4 Exposed group 24 °C 43.2±2.8 27.1±2.6 6.4±0.5 65.5±5.2 34.9±2.5 9.1±0.6 30.3±3.1 66.3±7.7 6.3±0.7 31 °C 43.5±3.2 27.1±2.6 7.0±0.5 66.3±6.0 32.8±2.3 9.9±0.7 28.5±2.8 65.3±7.9 6.8±0.8 NS NS F1,22 0 10.00, p <0.01 NS NS F1,22 0 15.40, p <0.001 NS NS F1,22 0 14.21, p <0.01 Ta effect

NREMS

REMS

Power densities in the delta, theta and sigma bands for the EEG signals in the control group (n 0 11) and the RF-EMF-exposed group (n 0 13) during W, NREMS and REMS. Values are expressed as means±SE. The right-hand columns indicate the effects due to the ambient temperature (“Ta effect”). There were no significant effects due to RF-EMF exposure and no significant interactions between RF-EMF exposure and Ta NS non-significant

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Fig. 5 Total quantity of food intake per hour for the control group (no exposure to RF-EMF, white columns, n 0 11) and the RF-EMF-exposed group (black columns, n 0 13) at ambient temperatures of 24 and 31 °C. Values are expressed as mean ± SEM. Statistically significant (p <0.001) differences are indicated by *** for inter-group comparisons and by ††† for inter-temperature comparisons

Fig. 4 Cortical temperatures (a) and tail temperatures (b) in the control group (open columns, n 0 9) and the RF-EMF-exposed group (filled columns, n 0 11) at ambient temperatures of 24 and 31 °C. Values are means±SE. Statistical significance (p <0.001) is indicated by *** for inter-group comparisons and by ††† for inter-temperature comparisons

duration of W episodes was lower at 24 °C (−27.8 %, t22 0 4.64, p <0.001). As far as the sleep state frequencies were concerned, in the RF-EMF-exposed group, the frequency of W and NREMS episodes was lower at 31 °C than at 24 °C (W, −30.8 %, t12 0 3.58, p <0.01; NREMS, −13.6 %, t12 0

4.29, p <0.001). Only the duration of NREMS episodes in the RF-EMF-exposed group was higher at 31 °C than at 24 ° C (+23.1 %, t12 0 6.70, p <0.001). In the control group, the duration of W episodes was lower at 31 °C than at 24 °C (−44.4 %, t10 0 5.48, p <0.001). Exposure to RF-EMF did not appear to have a significant effect on the spectral power density of the EEG signals. The effects of RF-EMF exposure on body temperatures were Ta dependent (F1,34 0 10.28). At 31 °C, Ttail was lower in the RF-EMF-exposed group than in the control group (−1.21 °C, t55 0 5.66, p <0.001; Fig. 4b). The differences for individual sleep stages (Table 3) (W, −1.17 °C, t17 0 3.22; NREMS, −1.20 °C, t17 0 3.09; REMS, −1.25 °C, t17 0 3.17, p <0.01) did not differ significantly (F2,34 0 1.32, p 0 0.28). Exposure to RF-EMF did not appear to have a significant effect on Tcor (F1,17 0 0.24, p 0 0.63). Figure 6 shows the time-response pattern for Ttail before and after the injection of prazosin. As reported above, Ttail was lower in the RF-EMF-exposed group than in the control group before the injection at a Ta of 31 °C (midday–1 pm:

Table 3 Cortical and tail temperatures during wakefulness and sleep stages in the control and the RF-EMF-exposed groups at ambient temperatures of 24 and 31 °C Control group 24 °C Tcor W NREMS REMS W NREMS REMS 36.71±0.09 36.68±0.09 36.77±0.08 32.62±0.32 32.64±0.32 32.47±0.33 31 °C 36.91±0.08 36.89±0.08 36.87±0.08 35.08±0.26 35.10±0.26 35.03±0.27 Exposed group 24 °C 36.68±0.05 36.64±0.05 36.70±0.04 32.53±0.32 32.53±0.33 32.45±0.34 31 °C 36.83±0.07 36.80±0.07 36.78±0.06 33.91±0.25* 33.90±0.28* 33.78±0.28* F1,17 0 15.57, p <0.001 F1,17 0 16.00, p <0.001 NS F1,17 0 120.21, p <0.001 F1,17 0 107.70, p <0.001 F1,17 0 119.92, p <0.001 NS NS NS F1, 17 0 9.44, p <0.01 F1, 17 0 8.80, p <0.01 F1, 17 0 11.98, p <0.01 Ta effect RF × Ta interaction

Ttail

Cortical (Tcor) and tail (Ttail) temperatures (in degrees Celsius) during W, NREMS and REMS episodes in the control group (n 0 9) and the RF-EMFexposed group (n 0 11). Values are expressed as mean±SEM. The right-hand columns indicate effects due to the ambient temperature (“Ta effect”) and those due to interaction between RF-EMF exposure and the ambient temperature (“RF × Ta interaction”) NS non-significant
*

p <0.01, statistically significant differences for comparisons of the control and exposed groups

Environ Sci Pollut Res Fig. 6 Tail temperatures in the control group (open circles, n 0 5) and the RF-EMF-exposed group (filled circles, n 0 5) in the presence (right panel) or absence of prazosin (left panel) at ambient temperatures of 24 °C (top panel) and of 31 °C (bottom panel). Values are expressed as mean±SEM. Statistically significant differences between the control and the RF-EMFexposed groups are indicated by * for p <0.05 and ** for p <0.01

z 0 1.98, p <0.05; 1 pm–2 pm: z 0 2.61, p <0.01; 2 pm–3 pm: z 0 2.40, p <0.05). The drug dampened the specific effect of RF-EMF exposure, since Ttail of the exposed animals increased after the injection (by an average of 0.46 °C over 6 h, z 0 2.16, p <0.05), whereas that of the control group did not change (p 0 0.99). At 24 °C, prazosin's local, vasodilatory effect on the thermoregulatory effector organs (i.e. Ttail) was observed in the two groups of animals over the 6-h period (+1.20 °C for the control group, on average, z 0 3.28, p <0.01; +1.35 °C for the RF-EMF-exposed group, on average, z 0 3.88, p <0.001). The effects of RF-EMF exposure on the quantity of food intake were Ta dependent (F1,22 0 5.10, Fig. 5). At 31 °C, the exposed animals ate more and thus gained more energy from their nutrients than those of the control group (+0.22 gh−1, i.e. +3.09 kJh−1, t22 0 4.52, p <0.001). The difference between the two groups at 24 °C was not statistically significant.

Discussion To the best of our knowledge, this is the first study to have analysed the simultaneous effects of prolonged exposure to low-intensity RF-EMF on three main functions (sleep, thermoregulation and energy intake) that control the body heat balance in a growing organism. In the present study, we assess the specific effects of RF-EMF by exposing rats to an intensity below the threshold SAR of 4 Wkg−1, above which

body tissues are heated (ICNIRP 1998). The absence of body heating in our experiment is confirmed by the fact that the control and the exposed groups did not differ significantly in terms of Tcor for a given environmental temperature. Since Tcor is closely related to the temperature of the hypothalamic structures (Gao et al. 1995) known to be the main central controllers of sleep, thermoregulation and feeding behaviour, one can reasonably conclude that disturbances reported here did not result from the heating of central structures. After just over 5 weeks of exposure at 24 °C, the animals were exposed at 31 °C. This temperature has been previously described as corresponding to a thermoneutral environment in which the external thermal load is minimal for sleeping Wistar rats (Kumar et al. 2009; Szymusiak and Satinoff 1981). Our results confirm this observation; the amount and duration of REMS (a state in which thermoregulatory processes are impaired in animals) (Parmeggiani 1968) were higher at 31 °C than at 24 °C, whereas the amount of wakefulness (during which these processes are fully efficient) was lower at 31 °C than at 24 °C. At 31 °C, the power density in the sigma bands (11.0–16.0 Hz) was higher at 31 °C than at 24 °C. This finding was particularly true in NREMS and appears to be a specific feature of sleep in the rat (Bjorvatn et al. 1998). This change could be related to an increase in subcortical activity, which is often observed in a state of alertness (Cajochen et al. 2001). These findings clearly indicate that for sleeping Wistar rats, 24 °C corresponds to the lower limit of the thermoneutral zone and

Environ Sci Pollut Res

31 °C can be used as a standard ambient temperature to study sleep (Romanovsky et al. 2002; Szymusiak and Satinoff 1981). These values are quoted for sleeping rats because sleep pressure is more important during the light period than during the dark period (Cajochen et al. 2001; Vyazovskiy et al. 2007). Our finding that the food intake (corresponding to the energy requirements to produce metabolic heat) was lower at 31 °C (with only basal metabolism) than at 24 °C confirmed previous work (Larson et al. 1996; Strubbe and van Dijk 2002). Likewise, oxygen consumption (VO 2 ) data in the literature (Gautier 2000; Szymusiak and Satinoff 1981) suggest that 31 °C corresponds to thermoneutrality (oxygen consumption is minimal). In the present experiment, we chose not to make VO2 measurements because they preclude feeding and would thus have biassed our food intake measurements. We observed a specific effect of RF-EMF exposure on REMS episodes with an increase in frequency. It is noteworthy that changes during REMS correspond to sleep stage instability. This may correspond to an increase in alertness of the exposed animals, since it has often been reported that reactivity decreases in REMS, during which the animal is particularly vulnerable to environmental stress (Horne 1988). Our study demonstrated that various effects of RFEMF on sleep are temperature dependent. The combined effect of RF-EMF exposure and the ambient temperature might partly account for the divergent results reported in the literature. Moreover, the thermoneutral zone and thus the external thermal load (which changes homeothermia) differ from one breed to another (Romanovsky et al. 2002). For all these reasons, it is particularly relevant to indicate the ambient thermal conditions when studying the effects of RFEMF exposure studies. In the present investigation, RF-EMF exposure was found not to modify the power density in the various EEG frequency bands. Similar observations have been reported by Mitchell et al. (1989), who did not find any changes in the root mean square power densities for bands ranging between 1.5 and 70.0 Hz in rats exposed to 2.45-GHz microwave radiations (SAR 0 2.7 Wkg−1) continuously for 7 h. Similar results were found in animals exposed to microwave radiations at frequencies of 945 MHz (1.0–2.0 Wm−2, bands from 0.5 to 26.5 Hz), 1.8 GHz (0.048–0.36 Wkg−1, bands from 0.5 to 14.0 Hz) and 2.45 GHz (0.31–1.58 Wkg−1, bands from 0.5 to 14.0 Hz) (Crouzier et al. 2007a, b; Vorobyov et al. 1997). It is difficult to compare when these environmental factors (as stated by the authors) differed. One of the most remarkable findings in the present work was that Ttail was lower in exposed animals than in the control group at Ta of 31 °C; this shows that peripheral vasomotor tone is dampened by RF-EMF exposure, since vasoconstrictor tone disappeared following injection of the vasodilatory drug in exposed animals only. At 31 °C,

peripheral vasodilatation is at its peak since prazosin had no effect on the control group of animals. The control mechanism for peripheral vasomotor tone involves internal influences from the preoptic area and the anterior hypothalamus (Ishiwata et al. 2001), both of which are also involved in sleep control (John et al. 1994). Peripheral blood flow (the main thermoregulatory effector) is thus sleep stage dependent (Parmeggiani 1977). In our experiment, the lower Ttail found in exposed rats did not significantly differ when comparing values in W, NREMS and REMS. This observation suggests that RF-EMF exposure acts directly at a peripheral level, without any central involvement. This observation was reinforced by the fact that (1) the RF-EMF-exposed and control groups did not differ in terms of Tcor (a guide to the central temperature) and (2) prazosin acts predominantly on the postganglionic adrenergic receptors involved in the control of peripheral vasomotor tone. Thus, the tail's vasoconstriction induced by RF-EMF exposure reduces radiative and convective heat losses from the body to the environment by decreasing the difference between the skin temperature and the environmental temperature. Moreover, at 31 °C, food intake was higher in the RF-EMF-exposed group than in the control group. This difference would promote heat production—heat that would be stored due to vasoconstriction. These results strongly suggest that the exposure to RF-EMF triggers mechanisms for energy saving. An increase in food intake could be initiated by signals from the skin cooling prompted by vasoconstriction, since it is generally acknowledged that food intake regulation operates through negative feedback from the periphery on hypothalamic structures (Strubbe et al. 1986). Indeed, the observed increase in the frequency of REMS episodes could also have been initiated by peripheral signals (skin cooling), prompting a return to wakefulness or NREMS. It is also possible that a higher frequency of REMS episodes could facilitate metabolic processes (such as lipolysis) that are induced by an increase in energy input during the light phase (Danguir and Nicolaidis 1980). These results concerned young animals that have been exposed during their development, a period particularly sensitive to environmental agents. As mechanisms of energy saving are the same in young rats as in adults, it could be interesting to look whether such a disturbance could similarly or at a lesser degree also affect the adult, to know if the observed effects were specific to young rats. The use of implanted metallic electrodes and probes and the associated leads can be a problem when interpreting the results. When conductive leads are exposed to RF-EMF, induced currents are generated along the leads which could affect dosimetry and EEG measurements (Schmid et al. 2007; Valentini et al. 2007). The observed effects might result from the locally amplified fields in the brain or elsewhere where well-conducting material is present (Angelone

Environ Sci Pollut Res

et al. 2004). The amount of induced current depends on the frequency and the specific geometry of the leads with respect to the source (Angelone et al. 2010; Hamblin et al. 2007). In particular, the vertical alignment of the wires coming out from the head of the animals should minimize the coupling of the field, as the polarisation close from the antennas is still parallel to the plane of the antennas. Only the lead of the skin temperature in the tail could significantly be coupled to the horizontal electric field. However, the main hypothesis that RF-EMF triggers mechanisms for energy saving comes from the results and can be confirmed in further studies that can be done without any implanted metallic parts. Exposure of growing rats to chronic, low-level RF-EMF (900 MHz, 1 Vm−1) at thermoneutrality does not modify the overall sleep pattern but slightly increases REMS fragmentation. An increase in peripheral vasoconstriction promotes heat storage. When combined with an increase in food intake, this thermal response attests to an energy-saving state.
Acknowledgments This study was funded by grants from the French Ministry of Research and a “Post-Grenelle” from the French Ministry of Ecology, as part of the “Pôle applicatif en Toxicologie et Ecotoxicologie” programme coordinated by the French National Institute of Environment and Industrial Risks (INERIS). We thank Patrice Cagnon for his technical assistance with RF-EMF dosimetry.

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