Environmental Research 135 (2014) 236–246

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Exposure to extremely low frequency electromagnetic fields alters the calcium dynamics of cultured entorhinal cortex neurons Fen-Lan Luo a,1, Nian Yang a,1, Chao He a, Hong-Li Li b, Chao Li a, Fang Chen a, Jia-Xiang Xiong a, Zhi-An Hu a,n, Jun Zhang a,n a b

Department of Physiology, Third Military Medical University, Chongqing 400038, PR China Department of Histology and Embryology, Third Military Medical University, Chongqing 400038, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 14 July 2014 Received in revised form 25 August 2014 Accepted 13 September 2014

Previous studies have revealed that extremely low frequency electromagnetic field (ELF-EMF) exposure affects neuronal dendritic spine density and NMDAR and AMPAR subunit expressions in the entorhinal cortex (EC). Although calcium signaling has a critical role in control of EC neuronal functions, however, it is still unclear whether the ELF-EMF exposure affects the EC neuronal calcium homeostasis. In the present study, using whole-cell recording and calcium imaging, we record the whole-cell inward currents that contain the voltage-gated calcium currents and show that ELF-EMF (50 Hz, 1 mT or 3 mT, lasting 24 h) exposure does not influence these currents. Next, we specifically isolate the high-voltage activated (HVA) and low-voltage activated (LVA) calcium channels-induced currents. Similarly, the activation and inactivation characteristics of these membrane calcium channels are also not influenced by ELF-EMF. Importantly, ELF-EMF exposure reduces the maximum amplitude of the high-K þ -evoked calcium elevation in EC neurons, which is abolished by thapsigargin, a Ca2 þ ATPase inhibitor, to empty the intracellular calcium stores of EC neurons. Together, these findings indicate that ELF-EMF exposure specifically influences the intracellular calcium dynamics of cultural EC neurons via a calcium channelindependent mechanism. & 2014 Elsevier Inc. All rights reserved.

Keywords: Extremely low frequency electromagnetic fields Entorhinal cortex Calcium channel Calcium dynamics

1. Introduction The extremely low-frequency electromagnetic field (ELF-EMF), primarily produced by the electrical wires and equipments, is one of the prominent types of pollutions in modern industrial society (Lacy-Hulbert et al., 1998). Given that exposure to ELF-EMF occurs throughout a person's entire life, concerns about the health risks associated with exposure are accumulating (Hardell and Sage, 2008; Kheifets et al., 2006). Indeed, it has been reported that the ELF-EMF exposure affects the neuronal functions in the hippocampus and prefrontal cortex, both of which are closely related to the learning and memory. In the hippocampus, ELF-EMF (1–60 Hz, 0.05–0.56 mT) disrupted neuronal rhythmic slow activity (Bawin et al., 1996) and altered the neuronal Ca2 þ signaling events, leading to the aberrant NMDA receptor activities (Manikonda et al., 2007). In the prefrontal cortex, ELF-EMF (50 Hz, 0.1–1 mT) exposure induced the increase of 5-HT2A receptor density (Janac et al., 2009) and affected neurotrophic signaling (Di Loreto et al., n

Corresponding author. E-mail addresses: [email protected] (Z.-A. Hu), [email protected] (J. Zhang). 1 Co-first author. http://dx.doi.org/10.1016/j.envres.2014.09.023 0013-9351/& 2014 Elsevier Inc. All rights reserved.

2009) and anti-oxidative enzymatic activity (Falone et al., 2008). The entorhinal cortex (EC) has been recognized as the major relay station between the neocortex and hippocampus (Van Cauter et al., 2013; van Strien et al., 2009). It contains spatial information-related functional cells and plays an essential role in the spatial learning and memory (Burak, 2014; Si and Treves, 2013; Yartsev et al., 2011). Previous studies in our laboratory have found that EC neurons are also vulnerable to the ELF-EMF. ELF-EMF (50 Hz, 0.5 mT) exposure reduced the dendritic spine density of the EC neurons and affected their NMDAR and AMPAR subunit expressions, although it did not affect the rat spatial learning ability (Li et al., 2014; Xiong et al., 2013). Calcium signaling plays an important role in controlling a variety of EC neuronal functions, including neurotransmitter release, membrane excitability and gene expression (Berridge, 1998). Because of the key role of Ca2 þ signaling in cellular functions, the cytosolic Ca2 þ is rigorously and largely controlled by the membrane calcium channels and intracellular calcium dynamics in normal condition (Mills, 1991; Ross, 1989). Dysfunctions of calcium channels or intracellular calcium dynamics can disrupt the calcium homeostasis, which is involved in the pathogenesis of cognitive-related diseases (Small, 2009; Supnet and Bezprozvanny, 2010). Interestingly, the regulatory machineries

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that control the calcium homeostasis are affected by a number of environmental factors. Among these factors, ionizing and microwave radiation have attracted great scientific interest. In rat whole-brain synaptosomes, gamma irradiation reduced KCl-stimulated voltage-dependent uptake of Ca2 þ via the inhibition of protein kinase C activity (Kandasamy and Harris, 1992). In stem cell-derived neuronal cells and dorsal root ganglion neurons, microwave radiation significantly increased the cytosolic Ca2 þ concentration through its influence on N-type calcium channels and TRPV1 channels, respectively (Ghazizadeh and Naziroglu, 2014; Titushkin et al., 2009). Moreover, it has been shown that radiofrequency radiation and microwave affected the Ca2 þ efflux from human neuroblastoma cells (Dutta et al., 1989, 1984). Although the calcium dynamics is a principal mediator of many vital neuronal activities and sensitive to environmental factors (Berridge, 1998), it is still unclear whether the ELF-EMF exposure affects the EC neuronal calcium homeostasis. In the present study, using whole-cell patch clamp recordings and calcium imaging, we investigate the effects of ELF-EMF (50 Hz, 1 mT or 3 mT) with exposure duration of 24 h on the EC neuronal membrane ion channels, especially calcium channels, and the intracellular calcium dynamics. We found ELF-EMF exposure specifically influenced the intracellular calcium dynamics, but not the membrane calcium channel activities. These results reveal a novel interaction mechanism between ELF-EMF and the EC, which might partially interpret the ELF-EMF-induced changes in cellular functions.

2. Materials and methods 2.1. Primary EC neuron culture All experimental procedures involving animals were in accordance with the guidelines for the care and use of laboratory animals in the Third Military Medical University. Postnatal 0-day-old Sprague–Dawley rats obtained from the Center of Animal Laboratory of the Third Military Medical University were soaked in alcohol for acute disinfection and then decapitated to remove out the brain. After isolating the cerebellum and removing the meninges, the rostral parts of the EC were cut away. Each pair of EC (left and right) from a rat was used for a single culture. The dissected tissue pieces were then collected in 1 ml ice-cold Hank's balanced salt solution (composition in g/L: 8 NaCl, 0.4 KCl, 0.06 NaH2PO4, 0.0475 NaH2PO4, 1 D-glucose, pH 7.2, Hyclone). Next, trypsin (750 μl, 0.25%, Sigma) was added to the solution. After incubation at 37 °C for 20 min, the trypsin was inactivated with 750 μl cold fetal calf serum. Then, the mixed solution was centrifuged at 1800 rpm for 5 min. After removing the supernatant, fresh neurobasal medium (1 ml, Gibco), which can help to suppress glial cells, was added to resuspend the cells. Dissociation of tissue fragments was achieved by gentle trituration using a firepolished Pasteur pipette. Next, cells from the each pair of EC were seeded on a single culture dish, respectively, and all cultures were maintained for 6 hours at 37 °C in 5% CO2 in neurobasal medium (1 ml, Gibco) supplemented with 2.5 mg/ml B27 (2%), 2 mM Lglutamine, 160 U/ml penicillin, and 200 U/ml streptomycin. The medium was changed 6 h after cell dissection and subsequently every third day for 12 days. 2.2. The exposure procedure to ELF-EMF After the 12 day culture period, cultured EC neurons were divided into sham exposure or ELF-EMF exposure (sinusoidal waveform, 50 Hz, 1 or 3 mT) groups and subjected to a 24 h exposure procedure, in which sham or ELF-EMF exposure was applied alternately, 5 min on and 10 min off. After the completion

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of the exposure at the thirteenth day (DIV13), the cultural neurons were immediately used for the whole-cell patch clamp recording or the calcium imaging experiments. As described previously, this kind of intermittent exposure pattern but not continuous may not only mimic real life exposure conditions to the time-varying magnetic fields that are the primary sources of ELF-EMF, but also achieve the possibly greatest effects of ELF-EMF (Focke et al., 2010). During the exposure procedure, each one or two cultures exposed to ELF-EMF was always accompanied by a sham-exposed culture through an sXcELF system (IT'IS Foundation, Switzerland) at the same time. In brief, the exposure system consisted of two four-coil setups (2 coils with 56 windings, 2 coils with 50 windings), each of which was placed inside a Mu-metal box. The currents in the bifilar coils could be switched between parallel for field exposure and non-parallel for sham control. Thus the field exposure and sham exposure can be achieved at the same time. Two fans were mounted per box to guarantee enough atmospheric exchange within the exposure chambers. Both setups were placed inside a commercial incubator (Heracell 240i, Thermo scientific) to ensure constant environmental conditions (37 °C, 5% CO2, 95% humidity). In addition, the temperature was monitored at the location of the culture dishes with Pt100 probes and maintained at 37.0–37.5 °C during exposure. The temperature difference between the chambers did not exceed 0.1 °C. Thus the possible thermal effects could be ruled out. A current source was developed based on four audio amplifiers (Agilent Technologies, Zurich, Switzerland) and applied to the bifilar coils to allow magnetic fields up to 3.5 mT. The field could be arbitrarily varied in the frequency range from 3 to 1250 Hz by a computer controlled arbitrary function generator. The entire system has been optimized for homogeneous field distribution, maximum field strength, minimum temperature increase and minimum vibrations. Non-uniformity of the magnetic field is o1% for all possible petri dish locations. And the fields and all sensors were continuously monitored. 2.3. Whole-cell patch clamp recording With the aid of an upright microscope (BX51WI, Olympus) equipped with a 40  water immersion objective and a video imaging camera, visualized whole-cell patch clamp recordings in voltage clamp mode were made on cultured EC neurons immediately and up to 4 h after finishing the 24 h sham or ELF-EMF exposure procedure at room temperature (22–25 °C). After gigaohm seal formation and patch rupture, the membrane potential of neurons before and after the voltage pulse stimulus were held at  70 or  80 mV for whole-cell current or calcium channel activity recordings, respectively. The current signal was amplified using an Axopatch 200B amplifier (Molecular Device) with a low cutoff frequency of 1 or 2 kHz and stored in a computer with a sampling frequency of 10 or 20 kHz for off-line analysis. Throughout the entire experiment, membrane capacitance and series resistance were continually monitored. Although the series resistance was not compensated, the recorded neuron was included in the final analysis only if the series resistance did not exceed 20 MΩ and did not change over 15%. In the off-line analysis session, the scaled P/N leak subtraction was applied routinely to correct for passive membrane current (N ¼4 or 5). For whole-cell current recordings, the culture medium was replaced by artificial cerebrospinal fluid (ACSF, composition in mM: 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1.3 MgSO4, 26 NaHCO3, 2 CaCl2, 10 Dglucose) before the recording sessions. The patch pipette (2.5–5 MΩ) was filled with normal K þ -based internal solution (composition in mM: 130 K-gluconate, 5 KCl, 2 MgCl2, 10 HEPES, 0.1 EGTA, 2 Na-ATP, 0.3 Na2-GTP, 4 Na2-phosphocreatine, adjusted to pH 7.25 with 1 mM KOH). After finishing the recordings, tetrodotoxin (TTX, 1 μM)þtetraethylammonium (TEA, 10 mM)þ4-aminopyridine (4-AP, 5 mM) were

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applied to the external recording solution on several occasions. In this situation, the whole-cell current was largely suppressed, suggesting that the whole-cell current mainly consists of sodium and potassium currents. While recording the activity of calcium channels, the culture medium was replaced by modified ACSF (composition in mM: 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1.3 MgCl2, 26 NaHCO3, 5 BaCl2, 10 D-glucose) in order to change the charge carrier from calcium to barium. Then, TTX (1 μM) was added to the modified ACSF to block sodium channels. The pipette was filled with Cs-based internal solution (composition in mM: 122 CsCl, 2 MgCl2, 10 HEPES, 0.1 EGTA, 2 Na-ATP, 0.3 Na2-GTP, 4 Na2-phosphocreatine, 10 TEA-Cl, adjusted to pH 7.25 with 1 mM CsOH) to block potassium channels. After finishing the recordings, Cd2þ (2 mM) was applied to the external recording solution to confirm the recorded calcium activity on several occasions, and indeed, all activity was totally blocked in these conditions. 2.4. Calcium imaging Once the sham or ELF-EMF exposure procedure was finished, the EC neurons in the culture dish were immediately loaded with 2 μM fluo-4/AM (Molecular Probes) and 0.02% pluronic F127 (Sigma) in HEPES-buffered ACSF (composition in mM: 126 NaCl, 3 KCl, 2 MgSO4, 2 CaCl2, 10 D-glucose, 10 HEPES, pH 7.4) at 37 °C for 30 min in darkness. Cells were then rinsed three times in fluo-4/ AM-free HEPES-buffered ACSF at room temperature. After the

culture dish was mounted in the imaging chamber (containing 2 ml fluo-4/AM-free HEPES-buffered ACSF) of an inverted confocal microscope (Olympus), the dye in the selected cytoplasmic part of the cells was excited by a wavelength of 488 nm, and the fluorescence images were captured at 2.0 μs per pixel by an Olympus fluoview ver.3.1a controlled by a computer. In this session, the fluorescence intensity was directly used to describe the intracellular calcium concentration. Thus, in order to compare the fluorescence intensity among different repetitive trials, the strength of the exciting light and the parameters used for capture were kept constant. The calcium response was evoked by local application (15 s) of 10 mM K þ -modified HEPES-buffered ACSF near the recorded cells only once when the recorded fluorescence intensity was stable for at least 5 min, and thus the amplitude of the calcium response in each culture was analyzed from only one single randomly selected observation field.

2.5. Statistical analysis All data are presented as the mean 7s.e.m. Analysis of variance (ANOVA), Fisher's protected least significant difference (LSD) post hoc testing, and Student's t-tests were employed for statistical analysis. Significant differences were accepted when P o0.05.

Fig. 1. Whole-cell currents/current densities of cultured EC neurons are not influenced by the exposure to ELF-EMF. (A1–A3) Representative traces of the activation of wholecell currents from a cultured EC neuron after the sham (A1), 1 mT (A2), or 3 mT (A3) ELF-EMF exposures, respectively. Group data of the inward (B1), transient outward (C1), and sustained outward (D1) components of whole-cell currents between the sham (black rectangle), 1 mT (blue rectangle), or 3 mT (red rectangle) ELF-EMF exposure groups shows no significant differences. Group data of the inward (B2), transient outward (C2), and sustained outward (D2) components of whole-cell current densities between the sham (black rectangle), 1 mT (blue rectangle), or 3 mT (red rectangle) ELF-EMF exposure groups also shows no significant differences. Error bars represent s.e.m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Table 1 Summary of statistical methods and results used in the present study. Item

Sub-item

Statistical method

F value

P value

Cm Whole-cell current

Inward component

One-way ANOVA Repeated measures two-way ANOVA

Transient outward component

Repeated measures two-way ANOVA

Sustained outward component

Repeated measures two-way ANOVA

Inward component

Repeated measures two-way ANOVA

Transient outward component

Repeated measures two-way ANOVA

Sustained outward component

Repeated measures two-way ANOVA

Transient component

Repeated measures two-way ANOVA

Sustained component

Repeated measures two-way ANOVA

Transient component

Repeated measures two-way ANOVA

Sustained component

Repeated measures two-way ANOVA

F(2,69) ¼ 1.464 group: F(2,19) ¼ 0.733 holding level: F(14,266) ¼ 24.643 interaction: F(28,266) ¼ 0.787 group: F(2,19) ¼ 2.192 holding level: F(14,266) ¼ 64.654 interaction: F(28,266) ¼ 2.018 group: F(2,19) ¼ 1.506 holding level: F(14,266) ¼ 48.344 interaction: F(28,266) ¼ 1.476 group: F(2,19) ¼ 0.198 holding level: F(14,266) ¼ 31.702 interaction: F(28,266) ¼ 0.236 group: F(2,19) ¼ 0.195 holding level: F(14,266) ¼ 98.599 interaction: F(28,266) ¼ 0.200 group: F(2,19) ¼ 0.077 holding level: F(14,266) ¼ 88.428 interaction: F(28,266) ¼ 0.106 group: F(2,26) ¼ 0.346 holding level: F(8,208) ¼52.337 interaction: F(16,208) ¼0.583 group: F(2,26) ¼ 0.175 holding level: F(8,208) ¼51.392 interaction: F(16,208) ¼0.821 group: F(2,26) ¼ 0.037 holding level: F(8,208) ¼68.474 interaction: F(16,208) ¼0.900 group: F(2,26) ¼ 0.190 holding level: F(8,208) ¼86.339 interaction: F(16,208) ¼1.936 group: F(2,26) ¼ 0.287 holding level: F(8,208) ¼80.683 interaction: F(16,208) ¼0.138 group: F(2,26) ¼ 1.219 holding level: F(8,208) ¼63.577 interaction: F(16,208) ¼0.835 F(2,19) ¼ 1.012 F(2,19) ¼ 1.106 F(2,15) ¼ 0.277 F(2,19) ¼ 0.231 F(2,123) ¼ 0.903 F(2,116) ¼ 1.230 F(2,123) ¼ 27.020 F(2,116) ¼ 0.748

0.238 0.494 o0.001 0.505 0.139 o0.001 0.155 0.247 o0.001 0.253 0.822 o0.001 0.904 0.824 o0.001 0.851 0.927 o0.001 0.917 0.711 o0.001 0.719 0.84 o0.001 0.534 0.964 o0.001 0.502 0.828 o0.001 0.075 0.753 o0.001 0.978 0.312 o0.001 0.511 0.382 0.351 0.762 0.796 0.408 0.296 o0.01 0.476

Whole-cell current density

HVA current

HVA current density

LVA current

Repeated measures two-way ANOVA

LVA current density

Repeated measures two-way ANOVA

Inactivation of HVA current Inactivation of LVA current Intracellular basal calcium level Maximum amplitude of calcium elevation

Half maximal inactivation voltage Slope factor Half maximal inactivation voltage Slope factor Normal ACSF In thapsigargin Normal ACSF In thapsigargin

3. Results 3.1. ELF-EMF exposure does not alter the whole-cell inward current of cultured EC neurons The activation of membrane voltage-gated calcium channels generates inward currents. To investigate the effect of ELF-EMF exposure on their activity, we firstly recorded the whole-cell inward currents of the EC neurons by a series of voltage steps (holding at –100  þ40 mV for 500 ms, increment: þ10 mV). As shown in Fig. 1B1, in the sham, 1 mT, and 3 mT exposure groups, the inward component of the whole-cell current (n ¼8 from 6 cultures, 9 from 4 cultures, and 7 from 4 cultures, respectively) exhibited a similar voltage dependence. Further statistical analysis revealed that there was no significant difference in the inward component among the three groups (Fig. 1B1; Table 1). Considering that the amount of whole-cell current may be highly influenced by cell membrane size, cell surface area-based whole-cell current density, which is usually calculated by dividing whole-cell current by membrane capacitance, may be more suitable for assessing the effects of ELF-EMF on the membrane ion channels of cultured EC neurons. After calculating the whole-cell current

One-way One-way One-way One-way One-way One-way One-way One-way

ANOVA ANOVA ANOVA ANOVA ANOVA ANOVA ANOVA ANOVA

densities, we found that there was no significant difference in the inward component of the density between the sham, 1 mT, and 3 mT exposure groups as well (Fig. 1B2, C2, and D2; Table 1). We also analyzed the whole-cell transient outward (positive peak) (Fig. 1C1) and the sustained outward current (mean during the last 50 ms period of the voltage pulse) (Fig. 1D1). As did the inward component, the amplitude and current density of transient outward and the sustained outward currents was not significantly different among the sham and exposure groups as well (Fig. 1C and D; Table 1). Together, these results indicate that the ELF-EMF exposure might have little impact on the membrane voltage-gated channels. 3.2. The activation of the voltage-gated calcium channels was not affected by ELF-EMF exposure in cultured EC neurons In order to further prove the negative effect of ELF-EMF on membrane calcium channels, we specifically isolated the highvoltage activated calcium (HVA) and low-voltage activated calcium (LVA) currents after blockade of the voltage-gated sodium and potassium channels. HVA currents were evoked by a series of voltage steps (holding at  40  þ40 mV for 200 ms, increment:

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Fig. 2. HVA currents/current densities of EC neurons are not influenced by the exposure to ELF-EMF. (A1–A3) Representative traces of the activation of HVA currents from a cultured EC neuron after the sham (A1), 1 mT (A2), or 3 mT (A3) ELF-EMF exposures, respectively. Note that for clarity, only the current traces during odd voltage steps are shown. Group data of the transient component of HVA currents (B1) or current densities (B2) between the sham (black rectangle), 1 mT (blue rectangle), or 3 mT (red rectangle) ELF-EMF exposure groups shows no significant differences. Group data of the sustained component of HVA currents (C1) or current densities (C2) between the sham (black rectangle), 1 mT (blue rectangle), or 3 mT (red rectangle) ELF-EMF exposure groups also shows no significant differences. Error bars represent s.e.m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

þ10 mV) with a pre-depolarization holding at  50 mV for 3 s, and the transient and sustained components of the currents were analyzed (Fig. 2A1–A3). As shown in Fig. 2B1, in the sham, 1 mT, and 3 mT exposure groups, the transient component (negative peak) of the HVA current (n ¼10 from 5 cultures, 10 from 5 cultures, and 10 from 6 cultures, respectively) exhibited a similar voltage dependence, as did the sustained component (negative plateau in the last 50 ms, Fig. 2C1). Further statistical analysis revealed that there was no significant difference in the transient or the sustained component between the three groups (Figs. 2B1 and 2 C1; Table 1). After calculating HVA current densities (n ¼10, 10, and 10, respectively), no significant difference was found in the transient or the sustained component of the density between the three groups as well (Figs. 2B2 and 2 C2; Table 1). LVA currents were evoked by a series of voltage steps (holding at  70  30 mV for 200 ms, increment: þ10 mV) with a prehyperpolarization holding at  120 mV for 3 s (Fig. 3A1–A3). The

range of voltage steps did not exceed  30 mV in order to avoid a large activation of HVA current. As shown in Fig. 3B, the LVA current (n ¼7 from 5 cultures, 11 from 5 cultures, and 13 from 6 cultures, respectively) in the sham, 1 mT, and 3 mT exposure groups exhibited similar voltage dependences in this voltage range. Further statistical analysis revealed that there was no significant different in the LVA current or the LVA current density between the three groups (Figs. 3B and C; Table 1). 3.3. ELF-EMF exposure does not influence the inactivation of the voltage-gated calcium channels in cultured EC neurons To thoroughly investigate the effects of ELF-EMF exposure on the calcium channels of cultured EC neurons, we further analyzed the inactivation characteristic of HVA and LVA currents after the 24 h sham or ELF-EMF exposure procedure. As shown in Fig. 4A1, the voltage dependence of steady-state inactivation of the HVA

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Fig. 3. LVA currents/current densities of EC neurons are not influenced by the exposure to ELF-EMF. (A1–A3) Representative traces of the activation of LVA currents from a cultured EC neuron after the sham (A1), 1 mT (A2), or 3 mT (A3) ELF-EMF exposures, respectively. Group data of the LVA currents (B) or current densities (C) between the sham (black rectangle), 1 mT (blue rectangle), or 3 mT (red rectangle) ELF-EMF exposure groups shows no significant differences. Error bars represent s.e.m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

current was determined by a test pulse of þ10 mV for 100 ms, preceded by conditioning voltage steps ranging from  105 to þ15 mV for 3 s (increment: þ15 mV) in a sample cultured EC neuron. Then, the normalized test pulse evoked currents were plotted as a function of conditioning voltage (Fig. 4A2), and the relation between them was well described by the following Boltzmann equation:

I (V )/Imax = 1/(1 + exp[(V − Vh)/Vc]) where Vh is the half maximal inactivation voltage, Vc is the slope factor, and Imax is the maximal evoked current. Therefore, we calculated the half maximal inactivation voltage and the slope factor to describe the inactivation characteristics of the HVA current. Through further statistical analysis, we found that there was no significant difference between the sham, 1 mT, and 3 mT exposure groups in half maximal inactivation voltage (57.272.4 mV,  62.773.3 mV, and  56.173.5 mV, respectively) or slope factor (16.270.9 mV, 14.170.6 mV, and 14.571.0 mV, respectively) for the inactivation of HVA current (n¼6 from 4 cultures, 6 from 3 cultures, and 10 from 6 cultures, respectively, Figs. 4B1 and B2; Table 1). Similarly, the voltage dependence of the steady-state inactivation of the LVA current in a sample cultured EC neuron, which was determined by a test pulse of  40 mV for 100 ms, preceded by conditioning voltage steps ranging from 120 to  45 mV for 3 s (increment: þ15 mV), and the corresponding Boltzmann fitting are shown in Figs. 4C1 and C2, respectively. Analysis of group data revealed that there was no significant difference between the sham, 1 mT, and 3 mT exposure groups in the half maximal inactivation voltage ( 88.873.0 mV, 86.472.2 mV, and  88.472.0 mV, respectively) or the slope factor (9.971.4 mV, 11.070.6 mV, and 10.371.4 mV, respectively) for the inactivation of LVA current as well (n¼ 6 from 6 cultures, 6, from 3 cultures and 6 from 5 cultures, respectively, Figs. 4D1 and D2; Table 1).

3.4. ELF-EMF exposure attenuates the high-K þ -evoked intracellular calcium elevation via a membrane calcium channel-independent mechanism in cultured EC neurons Although the unchanged membrane calcium channel characteristic, the intracellular calcium dynamics may also be influenced by changed calcium influx-induced secondary processes, including calcium release from intracellular stores and subsequent uptake. Therefore, it is necessary to directly investigate the change in the EC neurons′ calcium dynamics induced by ELF-EMF exposure. Through employing calcium imaging, we first found that the cultured EC neurons (n¼ 59 from 4 cultures, 36 from 3 cultures, and 31 from 3 cultures, respectively) in the sham, 1 mT, and 3 mT exposure groups had the same intracellular basal calcium level (1277.6 784.6, 1129.7 762.4, and 1130.9 7135.3, respectively, Figs. 5A1, B1, C1, and D1; Table 1) and exhibited a similar dynamic response with an increase in intracellular calcium concentration following a high-K þ stimulus (Figs. 5A2, A3, B2, B3, C2, and C3). Intriguingly, through carefully analyzing the dynamic response, we further found that ELF-EMF exposure reduced the maximum amplitude of calcium elevation (sham: 144.5 7 2.1%, 1 mT: 129.3 72.3%, 3 mT: 121.47 2.5% of baseline, respectively) in a dose-dependent manner (Fig. 5D2; Table 1), suggesting that ELFEMF exposure indeed changes calcium dynamics in cultured EC neurons. These results combining our electrophysiological results showing that calcium channel activity is not influenced by ELF-EMF exposure raise the strong possibility that ELF-EMF exposure changes the calcium dynamics in cultured EC neurons through a calcium channel-independent mechanism. To confirm this possibility, we next pre-treated the cultured EC neurons with the Ca2 þ ATPase inhibitor thapsigargin (5 μM, 15 min) to empty the intracellular calcium stores (Treiman et al., 1998). We first noted that the pre-incubation of thapsigargin may increase the intracellular calcium levels of cultured EC neurons. Therefore, elevated

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from 3 cultures, and 30 from 3 cultures, respectively) in the sham, 1 mT, and 3 mT exposure groups also had the same intracellular basal calcium level (1547.6795.5, 1752.4 7138.8, and 1460.6 7158.2, respectively, Fig. 6B; Table 1), although the overall mean level (1584.27 71.6, n ¼119) was increased compared to the (1199.2 754.7, n ¼126) previous conditions (t-test: Po 0.01). Moreover, the cultured EC neurons in the sham, 1 mT, and 3 mT exposure groups also exhibited a similar dynamic response with an increase in intracellular calcium concentration following a high-K þ stimulus (Figs. 6A1, A2, and A3). However, the maximum amplitude of calcium elevation (sham: 114.0 7 1.0%, 1 mT: 112.1 71.5%, 3 mT: 114.3 7 1.4% of baseline, respectively) was not significantly different between the three groups (Fig. 6C; Table 1), providing substantial evidence that ELF-EMF exposure changes the calcium dynamics in cultured EC neurons independent of calcium channels.

4. Discussion

Fig. 4. Inactivation of HVA and LVA currents are not influenced by the exposure to ELF-EMF. (A1) Representative traces of the steady-state inactivation of HVA currents from a cultured EC neuron. (A2) Boltzmann fit of the steady-state inactivation of HVA currents from the cultured EC neuron shown in (A1). Group data of half maximal inactivation voltage (B1) and slope factor (B2) for the inactivation of HVA current between the sham, 1 mT, or 3 mT ELF-EMF exposure groups shows no significant differences. (C1) Representative traces of the steadystate inactivation of LVA currents from a cultured EC neuron. (C2) Boltzmann fit of the steady-state inactivation of LVA currents from the cultured EC neuron shown in (C1). Group data of half maximal inactivation voltage (D1) and slope factor (D2) for the inactivation of LVA current between the sham, 1 mT, or 3 mT ELF-EMF exposure groups shows no significant differences. Error bars represent s.e.m.

extracellular calcium, from 2 mM to 5 mM, was employed in the following experiments. In these conditions, calcium imaging revealed that the cultured EC neurons (n¼ 55 from 5 cultures, 34

There are a great number of studies concerning about the cellular effects ELF-EMF exposure in several cognitive brain regions, including the hippocampus and the prefrontal cortex. However, the influence of ELF-EMF exposure on the entorhinal neurons, which mediates the majority connections between hippocampus and cortex, remains largely unknown. In the present study, we focused on the neuronal calcium homeostasis of EC neurons and found the ELF-EMF exposure altered the intracellular calcium dynamics, independent of the membrane calcium channels. These results indicate the regulatory machineries of EC neuronal calcium homeostasis are sensitive to the environmental ELF-EMF, and the observed dysfunctions of EC neurons caused by ELF exposure may involve altered Ca2 þ signaling events. There is one viewpoint that the cell membrane is a major site of interaction for ELF-EMF, and thus, the permeability of ions through the membrane is usually assumed to be altered by exposure to ELF-EMF (Moghadam et al., 2011). Indeed, it has been shown that the expression and activity of several types of ion channels are changed by ELF-EMF exposure (Grassi et al., 2004; He et al., 2013; Piacentini et al., 2008). However, in the present study, ELF-EMF did not influence the mixture inward currents (mainly reflecting the fast voltage-gated sodium currents), transient and sustained outward currents (mainly reflecting the potassium currents) evoked by voltage steps. Given that the membrane calcium current may contribute largely to the activities and functions of the neurons, we investigated the effects of ELF-EMF exposure on their activity in cultured EC neurons. Notably, through replacing extracellular Ca2 þ with Ba2 þ , the possible calcium influx-induced cellular responses and the influence of the original intracellular basal calcium concentration of cultured EC neurons are excluded in order to avoid influencing the analysis of calcium channel activity. In these conditions, we found that the activity and inactivation characteristics of calcium channels, including HVA and LVA channels, are not influenced by 24 h exposure to ELF-EMF, suggesting that neither the activity characteristics nor the expression of membrane calcium channels are altered by ELF-EMF. Consistent with our results, in pheochromocytoma cells, the ELF-EMF exposure has no effect on the activity of calcium channels (Obo et al., 2002). However, the activity of membrane calcium channels in diverse other types of cells has been found to be increased by both the short (r 24 h) and long (3 days) duration of ELF-EMF exposure (Grassi et al., 2004; Marchionni et al., 2006; Piacentini et al., 2008).Furthermore, several studies indicate that the ELFEMF exposure especially target L-type (Cav1) channels (Cuccurazzu et al., 2010; Leone et al., 2014; Morgado-Valle et al., 1998). Overall, the effects of ELF-EMF exposure on the calcium channels are

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Fig. 5. High-K þ evoked calcium elevation in cultured EC neurons is reduced by the exposure to ELF-EMF. Representative calcium imaging from a cultured EC neuron at rest after the sham (A1), 1 mT (B1), or 3 mT (C1) ELF-EMF exposure are shown, respectively. After the stimulation of high-K þ , the calcium fluorescence intensity is increased in the three sham (A2), 1 mT (B2), or 3 mT (C2) ELF-EMF exposed cultured EC neurons, and the corresponding time-response curves are plotted in (A3), (B3), and (C3), respectively. Note that the arrowhead represents the application of high-K þ (10 mM) and the gray curve indicates the relative calcium fluorescence intensity changes. Group data of intracellular basal calcium fluorescence intensity (D1) between the sham, 1 mT, or 3 mT ELF-EMF exposure groups shows no significant differences, but the group data of maximum amplitude of high-K þ evoked calcium elevation (D2) between the sham, 1 mT, or 3 mT ELF-EMF exposure groups shows a dose-dependent decrease. Scale bar: 5 μm. Error bars represent s.e.m. n indicates Po 0.05 and nn P o0.01.

inconsistent. One of the reasons for the complex effects might be attributed to the different expression patterns of the calcium channels in these experimental models. A previous study has found that L-type calcium channels are expressed in acutely isolated neurons from EC and contribute to approximately 52– 55% of total HVA calcium currents (Castelli and Magistretti, 2006). But it is still unclear the expression pattern of calcium channels in the cultural EC neurons. One possibility for negative effects observed in the present study might due to the L-type calcium channels are poorly expressed in cultural EC neurons. On the other hand, it should be noted that the distinct exposure parameters and

radiosensitivities of the cells might also contribute the diverse results. In the present study, we just administrated ELF-EMF with short exposure duration. Considering the positive effects of short exposure duration on the calcium channel activity in several other cell types (Grassi et al., 2004; Marchionni et al., 2006), thus the calcium channels in the cultural EC neurons might be less sensitive to the ELF-EMF. Nevertheless, we still cannot rule out the possibility that the membrane calcium channels can be affected by the chronic ELF-EMF exposure and further studies are needed to address this issue.

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Fig. 6. Thapsigargin abolishes the reduction effect of ELF-EMF on high-K þ evoked calcium elevation in EC neurons. Representative time-response curves of high-K þ -evoked relative calcium fluorescence intensity changes from a culture of EC neurons after the sham (A1), 1 mT (A2), or 3 mT (A3) ELF-EMF exposure are shown, respectively. (B) Group data of intracellular basal calcium fluorescence intensity between the sham, 1 mT, or 3 mT ELF-EMF exposure groups shows no significant differences in the presence of thapsigargin. (C) Group data of maximum amplitude of high-K þ evoked calcium elevation between the sham, 1 mT, or 3 mT ELF-EMF exposure groups shows no significant differences in the presence of thapsigargin. Error bars represent s.e.m.

Although the activity of membrane calcium channels largely influences intracellular calcium dynamics and several reports have mentioned that altered activity of calcium channels would subsequently influence intracellular calcium (Lisi et al., 2006; Yu-Hong et al., 2007), the characteristics of intracellular calcium dynamics are not determined exclusively by the activity of membrane calcium channels and thus may be influenced by ELF-EMF independent of the membrane ion channel pathways. Indeed, in several studies in which intracellular calcium was reportedly altered by ELF-EMF, the underlying mechanism was stated to be independent of membrane ion channels (Bernabo et al., 2007; Morabito et al., 2010a, 2010b). Therefore, we next employed calcium imaging to directly investigate the effects of ELF-EMF exposure on the intracellular basal calcium level and the high-K þ -evoked calcium dynamics in cultured EC neurons. In previous reports, authors usually first investigated the changes in the intracellular basal calcium level induced by ELF-EMF to indirectly reflect calcium dynamics, and diverse results in different types of cells have been demonstrated, including increases (Lisi et al., 2006; Manikonda et al., 2007; Morabito et al., 2010a, 2010b; Sert et al., 2011; Tonini et al., 2001; Zhang et al., 2010), decreases (Bernabo et al., 2007; McCreary et al., 2002), and no change (Madec et al., 2003; Pilger et al., 2004; Ramstad et al., 2000; Yamaguchi et al., 2002). However, in cultured EC neurons, we found that ELF-EMF exposure does not influence the intracellular basal calcium level. We next investigated the high-K þ -evoked calcium dynamics in cultured EC neurons to directly observe the effects of ELF-EMF exposure. Among the sham, 1 mT, and 3 mT exposure groups, the cultured EC neurons exhibited similar dynamic responses with an increase in intracellular calcium concentration following a high-K þ stimulus. The maximum amplitude of the calcium elevation was reduced by ELF-EMF exposure in a dose-dependent manner. This means that ELF-EMF exposure indeed alters calcium dynamics in cultured EC neurons. Intriguingly, similar results have been reported previously in diverse types of cells (Gaetani et al., 2009; McCreary et al., 2006; Morabito et al., 2010b; Zhao et al., 2008), and the underlying mechanism has been proven to include membrane calcium channels (Zhao et al.,

2008) and intracellular calcium stores (Gaetani et al., 2009; Morabito et al., 2010b). Regardless, in our present study, considering that membrane calcium channels of cultured EC neurons are not influenced by ELF-EMF exposure, the mechanism for the altered calcium dynamics must be dependent on calcium influxinduced secondary processes rather than on membrane calcium channel activity. Consistently, after application of thapsigargin, a Ca2 þ ATPase inhibitor, to empty the intracellular calcium stores of cultured EC neurons, our results reveal that the high-K þ -evoked increase in intracellular calcium concentration was not affected by ELF-EMF exposure, confirming that a mechanism independent of calcium channels is involved. Overall, our results suggest that ELFEMF affect the calcium dynamics via an intracellular calcium stores-dependent process. Indeed, the calcium dynamics can be tightly controlled by intracellular calcium stores primarily via two secondary processes of calcium release from intracellular stores and/or the subsequent uptake (Small, 2009; Supnet and Bezprozvanny, 2010). However, the question of which types of secondary processes involved is still unclear. In the future study, the administration of specific agonists and antagonists of calcium release and uptake from intracellular stores would be very helpful. The intracellular calcium dynamics play an essential role in initiation and maintenance of EC neuronal functions. Intriguingly, the release of calcium from intracellular stores is an important modulator of dendritic spine structure. In cultured hippocampal neurons, calcium released from the ryanodine-sensitive stores induced a transient rise of Ca2 þ in dendrites and spines, and caused a fast and significant increase in the size of existing dendritic spines. Moreover, it could leads to formation of new dendritic spines (Harris, 1999; Korkotian and Segal, 1999). Previous study in our laboratory found that chronic ELF-EMF exposure significantly reduced the spine density in the EC (Xiong et al., 2013). It is reasonable to speculate that this phenomenon might be partially resulted from the aberrant activity-dependent intracellular calcium dynamics after the ELF-EMF exposure. Importantly, long-term synaptic potentiation induction requires the large increases in postsynaptic calcium, which activates multiple downstream signaling enzymes, including the kinases calcium/

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calmodulin-dependent protein kinase II and protein kinase C (Ho et al., 2011). The ELF-EMF exposure-induced reduction in the activity-evoked calcium dynamics, combined with its effects on the AMPAR and NMDAR subunit expressions (Li et al., 2014), thus might substantially affect the synaptic plasticity in the EC. There are several theories suggesting that only ELF-EMF with the proper frequency would significantly interact with biosystems (Gaetani et al., 2009; Obo et al., 2002). However, this may not be the case. ELF-EMF of diverse frequency has been used in previous research, and positive interactions are often reported. In the present study, we chose the following ELF-EMF exposure parameters: 50 Hz, 1 or 3 mT, 24 h duration, and a 5 min on and 10 min off cycling pattern. We used this type of exposure to mimic the daily exposure that a human being might encounter in normal life, although we have clearly acknowledged that the cultured preparations and other aspects of this work might not very suitable for accomplishing this task. A previous report revealed that when the frequency of ELF-EMF is fixed, high-intensity/short-duration exposure may produce an effect similar to that produced by lowerintensity/longer-duration exposure (Lai and Carino, 1999). Therefore, we may conclude that our present study used exposure parameters that at least satisfy our original research aim, which was to investigate the correlation between the daily ELF-EMF exposure encountered by an average human being and EC neurons. In summary, the ELF-EMF exposure used in the present study does not alter membrane calcium channel activities, but it does influence the intracellular calcium dynamic of EC neurons and thus might affect normal functions of entorhinal neurons. It should also be noted, however, that in previous studies about the correlation between ELF-EMF and calcium intracellular dynamics in cognition-related brain regions, diverse types of exposure parameters were used, including different frequencies, densities, durations, and patterns, and diverse results were observed, as described above. We cannot rule out the possibility that this is the reason that our present study reveals discrepant results compared to previous observations. Another possibility is that different characteristics among investigated tissues may substantially influence their responses to ELF-EMF exposure. On the other hand, other possible influences of ELF-EMF on the EC, especially on the neurotransmitter/receptor systems in the EC, need more detailed investigation, as mentioned above. Another important aspect is that the sXcELF system used for the ELF-EMF exposure procedure effectively excluded the thermal effects of the exposure on EC neurons. Therefore, the pure effects of ELF-EMF exposure on EC neuronal calcium homeostasis are demonstrated in the present study.

Acknowledgments This work was supported by the National Basic Research Program of China 2011CB503700 (to Hu ZA) and the Youth Project of Third Military Medical University 2011XQN63 (to Zhang J).

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