- Email: [email protected]
Effects of exposure to extremely low frequency electromagnetic fields on hippocampal long-term potentiation in hippocampal CA1 region
Biochemical and Biophysical Research Communications 517 (2019) 513e519
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Effects of exposure to extremely low frequency electromagnetic fields on hippocampal long-term potentiation in hippocampal CA1 region Yu Zheng a, *, Jianhao Cheng a, Lei Dong b, Xiaoxu Ma a, Qingyao Kong c a
School of Electronics and Information Engineering, Tianjin Polytechnic University, Tianjin, 300387, China State Key Laboratory of Precision Measurement Technology and Instruments, Tianjin University, Tianjin, 300072, China c Department of Anesthesia and Critical Care, University of Chicago, Chicago, IL, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2019 Accepted 22 July 2019 Available online 31 July 2019
Exposure to environmental electromagnetic fields, especially to the extremely low-frequency (ELF < 300 Hz) electromagnetic fields (EMFs) might produce modulation effects on neuronal activity. Long-term changes in synaptic plasticity such as long-term potentiation (LTP) involved in learning and memory may have contributions to a number of neurological diseases. However, the modulation effects of ELF-EMFs on LTP are not yet fully understood. In our present study, we aimed to evaluate the effects of exposure to ELF-EMFs on LTP in hippocampal CA1 region in rats. Hippocampal slices were exposed to magnetic fields generated by sXcELF system with different frequencies (15, 50, and 100 Hz [Hz]), intensities (0.5, 1, and 2 mT [mT]), and duration (10 s [s], 20 s, 40 s, 60 s, and 5 min), then the baseline signal recordings for 20 min and the evoked field excitatory postsynaptic potentials (fEPSPs) were recorded. We found that the LTP amplitudes decreased after magnetic field exposure, and the LTP amplitudes decreased in proportion to exposure doses and durations, suggesting ELF-EMFs may have dose and duration-dependent inhibition effects. Among multiple exposure duration and doses combinations, upon 5 min magnetic field exposure, 15 Hz/2 mT maximally inhibited LTP. Under 15 Hz/2 mT ELF-EMFs, LTP amplitude decreases in proportion to the length of exposure durations within 5 min time frame. Our findings illustrated the potential effects of ELF-EMFs on synaptic plasticity and will lead to better understanding of the influence on learning and memory. © 2019 Elsevier Inc. All rights reserved.
Keywords: Extremely low frequency electromagnetic fields Short-term effects of ELF-EMF exposure Hippocampal CA1 area Synaptic plasticity Long-term potentiation
1. Introduction Extremely low frequency electromagnetic fields (ELF-EMFs) affect several types of neuronal activities including memory [1,2]. Previous study [3] has shown that the widespread use of electricity increases the potential sources of radiation resulting in continuous or intermittent exposure of living beings to extremely low frequency electromagnetic fields. Because ELF-EMFs stimulate neuronal cells by the depolarization of voltage-gated ion channels [4,5], which may evoke pre- and postsynaptic depolarization that is required for generation of LTP [6], we hypothesize that ELF-EMFs may affect the induction or the expression of LTP. To test this hypothesis, we previously tested whether ELF-EMFs induce LTP in vitro. Interestingly, we found that exposure to ELF-EMFs itself doesn't induce LTP but modulates LTP at Schaffer collateral-CA1
* Corresponding author. E-mail address: [email protected] (Y. Zheng). https://doi.org/10.1016/j.bbrc.2019.07.085 0006-291X/© 2019 Elsevier Inc. All rights reserved.
synapses on rat hippocampal slices [7]. Consistent findings demonstrate that exposure to ELF-EMFs produces a marked change in LTP at hippocampal perforant pathway to dentate gyrus specifically in postsynapses [8], which supports our argument that ELFEMFs modulates LTP in rat hippocampus. Studying the effects of ELF-EMFs exposure on LTP in brain tissues or living animals is important because accumulation evidence indicates that ELF-EMFs exposure affects spatial learning and memory and is associated with neurodegenerative including Parkinson's disease (PD) and Alzheimer's disease (AD) [9]. [10e12]. Therefore, understanding the neurobiological mechanisms that underlie the modulation effects of ELF-EMFs on LTP would be highly desirable. Since LTP is a key component in spatial learning and alterations in LTP may contribute to PD and AD ewhether and how exposure to ELF-EMFs affects LTP will be a key question in tackling the biological mechanisms involved in the ELF-EMFs’ effects on animal behaviors and diseases. We are interested in synaptic plasticity in hippocampus because the hippocampus in the deep brain plays an important
514
Y. Zheng et al. / Biochemical and Biophysical Research Communications 517 (2019) 513e519
role in short-term learning and memory including spatial learning [13e15] that reportedly can be affected by exposure to ELF-EMFs [1]. Thus, understanding the effects of ELF-EMFs on vital hippocampal pathways may shed light on potential therapeutic strategies for prevention and treatment of learning related diseases. Considering hippocampus LTP enhances synaptic efficiency and is essential for learning and memory processes [16e18], and LTP at schaffer collaterals-CA1 is a well-established model in studying synaptic plasticity [19], we chose LTP at schaffer collaterals-CA1 as a model in exploring the effects of ELF-EMFs on synaptic plasticity. Previous studies indicate that utilizing ELF-EMFs at different strengths may have divergent effects on synaptic plasticity at CA1 region in rats [8,20]. However, whether dose-response relationships exist in the effects of ELF-EMFs on synaptic plasticity has never been studied before [21]. In the present study, we ask how exposure to ELF-EMFs at different frequencies or intensities in different exposure durations affects LTP in CA1, what potential dose-response relationships are involved. Answers to these questions are essential to understanding of the neurobiological mechanisms underlying the effects of ELF-EMFs. To investigate how exposure to ELF-EMFs modulates LTP, we utilized a high-precision magnetic exposure device sXcELF (IT'IS Foundation, Zurich, Switzerland) that can generate uniform magnetic fields at wide frequency and intensity ranges (frequencies between 3 and 1250 Hz [Hz] and intensities ranging from 0.04 to 3.5 mT) at high accuracy [22]. In recent years, sXcELF device has been widely used in studies on the principle, characteristics and biomedical application of ELF-EMFs [20,22e25], however we did not find any application of sXcELF in studies on synaptic plasticity. In our research, sXcELF device enables us to study the effect of the ELF-EMFs in wide ranges of frequency/intensity. In our study, rat hippocampal slices were stimulated using the external exposure device sXcELF, followed by a recording system of field excitatory postsynaptic potentials (fEPSPs), which contains a stimulation electrode and a recording electrode that measures the amplitude of hippocampal fEPSPs. We investigated the impact of ELF-EMFs at different frequencies (15, 50, and 100 Hz), intensities (0.5, 1, and, 2 mT), and duration (10 s [s], 20s, 40s, 60s, and, 5 min [min]) on LTP amplitudes at hippocampal schaffer-CA1 region in rats. Our results showed the LTP amplitudes decreased after magnetic field exposure, and the LTP amplitudes decreased in proportion to exposure doses and durations, indicating ELFEMFs exposure inhibits LTP amplitude in dose and durationdependent patterns. Among multiple exposure duration and doses combinations, upon 5 min magnetic field exposure, 15 Hz/ 2 mT maximally inhibited LTP. Under 15 Hz/2 mT ELF-EMFs, LTP amplitude decreases in proportion to the length of exposure durations.
2. Materials and methods 2.1. Animals A total of 18 male Sprague-Dawley rats (14e18 days old at the time of surgery) were obtained from the Academy of Military Medical Sciences (Tianjin, China), certification number SCXK-(PLA) 2014e0001. Each rat was kept in a separate cage in a clean room maintained under a 12-h light/dark cycle at a constant temperature (25 ± 2 C), and with access to food and water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee protocol of Tianjin Polytechnic University, Tianjin, China. The experiments were designed to minimize any animal suffering as well as the number of animals used.
2.2. Tissue preparation SpragueeDawley rats were anesthetized with chloral hydrate [23,25], at a concentration of 0.1 ml/20 g (10%), and their brains were rapidly removed and stored in 4 C cutting solution containing: 90 mM of sucrose, 87.2 mM of NaCl, 2.5 mM of KCl, 7 mM of MgCl2, 0.5 of mM CaCl2, 1.25 mM of NaH2PO4, 25 mM of NaHCO3, and 16.7 mM of glucose. The solution was continuously bubbled with 95% O2/5% CO2. Subsequently, 400 mm thick horizontal slices were cut using the vibrating tissue slicer, VF-200 (Precisionary Instruments New Jersey, USA) in ice-cold cutting solution. Slices were then incubated at 33 C in artificial cerebrospinal fluid (ACSF) for 1 h and an individual slice was moved to a perfusion chamber for recording. The ACSF consisted of: 120 mM of NaCl, 2.5 mM of KCl, 2 mM of MgSO4$7H2O, 2 mM of CaCl2, 1.25 mM of NaH2PO4$2H2O, 26 mM of NaHCO3, and 10 mM of glucose, continuously bubbled with 95% O2/5% CO2 at a pH of 7.4. Moreover, when fEPSPs were being recorded, pipettes were filled with 3 mol/L of NaCl. All other reagents were of analytical grade made in China (Tianjin Fengchuan Chemical Reagent Technologies Co., Ltd, Tianjin). All brain slices were randomly assigned either to the control or experimental group. 2.3. Magnetic stimulator and electrophysiological recordings The magnetic exposure device sXcELF used in this study (Fig. 1a) consisted of four parts: two four-coil systems, a commercial incubator, a monitor, and a data acquisition unit (PC). The two four-coil systems were placed into two porous metal boxes labeled “Chamber 100 and “Chamber 200 , in order to enable the generation of magnetic fields with a frequency range of 3e1250 Hz and a maximum magnetic flux density of 3.5 mT root-mean-square amplitude [22]. The commercial incubator BSC-PC (Harvard Apparatus, Horiston, MA) was placed above the chambers to maintain the chamber temperature at 33 C. Both the dish holder and mumetal box were placed on elastically damped feet in order to minimize vibration. A software-controlled arbitrary function generator was used as the signal source for the two current sources. The current source converted the voltage signal to the desired current and sent it to the coil inside the incubator. The temperature of the flasks was then monitored during exposure with Pt100 probes in order to control the output of the desired signal as a voltage. The slices were random placed in either chamber, which an EMFs exposure apparatus (experimental group) and a similar device without EMFs (control group), during this procedure, the solution was continuously bubbled with 95% O2/5% CO2. The EMF produced by experimental group was set at an intensity of 0.5, 1, or 2 mT and the frequency was set at 15, 50, or 100 Hz. The experimental group slice exposure duration was set to 10 s, 20s, 40 s, 60 s, and 5 min. The stratum radiatum of CA1 was viewed under the upright microscope, BX51-WI (Olympus, Tokyo, Japan) equipped with a long-range water immersion objective (40 ) and an infrared video camera (IR1000, DAGE-MTI, Richmond Inner Harbor) (Fig. 1b) of the electrophysiological recording system. The bipolar concentric stimulation, CBARC75 (FHC, Bowdoin, ME), was placed on the stratum radiatum of CA1. The recording electrodes (resistance 3e8 mega-ohms [MU]) were also placed in either the stratum radiatum, approximately 250 mm from the stimulation electrode in CA1 to record the amplitude of fEPSPs [26,27]. The distribution of the magnetic field around the center of the perfusion chamber, where brain slices were placed during the experiments, was measured using the mill-Tesla device, HT108 (Htmagnet, Shanghai China) to ensure the accuracy of the exposure. Although, the above-mentioned Tesla-meter was able to measure
Y. Zheng et al. / Biochemical and Biophysical Research Communications 517 (2019) 513e519
515
Fig. 1. Experimental setup of the magnetic exposure device and the field excitatory postsynaptic potential (fEPSP) recording system. (a) The exposure device sXcELF produces shortterm extremely low frequency electromagnetic field (ELF-EMF) stimulation on brain slices. (b) The fEPSPs recording system: brain slices are first stimulated using the external exposure device sXcELF, followed by recordings of fEPSP amplitudes using respective electrodes.
the magnetic field strength in brain slices, the test results were inaccurate due to the large size of the Tesla-meter probe, when compared with the brain slices. In order to estimate the distribution of the magnetic field in the brain slices, we also conducted a simulation experiment (COMSOL Multiphysics 5.2a, COMSOL Inc, Stockholm, Sweden). Subsequently, Auto CAD 2017 (Autodesk, California, USA) was used to construct a brain tissue model with a width of 1 cm and a thickness of 400 mm. This model was then imported into the COMSOL software; the characteristics of the materials used in the simulation are shown in Supplementary file (Table S1). Results from the simulation experiments showed that the simulated magnetic field intensity was similar to the experimental magnetic field strength as seen in Supplementary file (Fig. 1Sa). The induced electric field intensity in the radiation layer is shown in Supplementary file (Fig. 1Sb). Notably, the difference in the distribution of induced electric field intensity was negligible in the radiation layer. 2.4. Stimulation protocol In this paper, the preparation was stimulated electrically for 20 min at a test of the stimulus frequency of 0.06 Hz. The baseline served as a reference to evaluate changes in the slope of fEPSPs. Stimulation strength was adjusted to obtain approximately 40% of the maximal response. The duration of single pulses was 0.1 ms. Stable baseline fEPSPs were recorded each minute for at least 20 min before any plasticity induction was applied. fEPSPs were then recorded again every minute for 60 min after plasticity was induced. We first determined whether priming uninterrupted sine, ELF-EMFs interfered with the electrically induced LTP. The stimulus protocol used in the experiments is depicted in Supplementary file (Fig. 2S). We first conducted the control experiments (without any priming of ELF-EMFs, only electrical high-frequency electric stimulation [HFS] induced LTP). For this, a stable baseline fEPSPs was recorded for 20 min before any plasticity induction was applied. The fEPSPs were then recorded again for 60 min, after plasticity induction. The electrical HFS paradigm consisted of 4 sweeps of 100 pulses (1s apart) with a 20 s interval. Then, we applied priming offline sine ELF-EMFs, as an uninterrupted sinusoidal signal with frequencies of 15 Hz, 50 Hz, and 100 Hz, to hippocampal brain slices, with a duration of 10s, 20s, 40s, 60s, and 5 min, according to the experimental process shown above. 2.5. Statistical analysis The raw data was processed using the Origin 8.0 software platform (Origin Lab, Northampton, MA) and the statistical analysis
was performed using the GraphPad Prism 7 (GraphPad Software Incorporation, San Diego, CA). The mean values of the fEPSPs baseline amplitudes in the control group and experimental group were normalized to a 100 value. All data were analyzed using oneway analyses of variance (ANOVA) on ranks with Tukey's post hoc test and a two-way ANOVA on Tukey's multiple comparisons test. The results were expressed as the mean ± standard deviation (SD). The differences observed were considered to be statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001. 3. Results 3.1. The effect of ELF-EMFs on LTP is frequency and intensitydependent Hippocampal slices were treated using the stimulation protocol described in Supplementary file (Fig. 2S), with each experimental group containing results obtained from 5 hippocampal brain slices. Baseline and electrical HFS-induced LTP are recorded following ELF-EMF exposure. The effects of the 0.5 mT, 1 mT, and 2 mT magnetic fields of 15 Hz, 50 Hz, and 100 Hz on the fEPSPs amplitude were analyzed separately as shown in Fig. 2. The magnetic field exposure duration was fixed to 5 min. We observed the fEPSPs amplitudes decreased after exposure to a series of priming magnetic fields with increasing intensities, and the electrical stimulation-induced LTP decreased in proportion to the magnetic field intensity. In addition, we performed statistical analysis accordingly. The baseline signal recordings for the first 20 min in each group were normalized, and the induced EPSP amplitudes were treated according to the corresponding normalized coefficients, with the average fEPSP amplitude obtained being 99.28 ± 1.43 mV (n ¼ 5). Without applying the magnetic field, the fEPSP amplitudes corresponding to the different frequencies were 100.55 ± 1.69 mV, 100.34 ± 1.72 mV, and 99.08 ± 4.14 mV (n ¼ 5). According to our statistical analysis, we concluded that there were no significant differences between these datasets. After a 100 Hz high frequency stimulation, the fEPSP was 202.71 ± 9.29 mV (n ¼ 5). The fEPSP amplitudes of slices exposed to ELF-EMFs are significantly lower than controls (Fig. 2a). For magnetic fields with a fixed intensity of 0.5 mT, the inhibition ratio was as follows: 15 Hz, 33.79%; 50 Hz, 25.97%; 100 Hz, 21.36%; (n ¼ 5). In contrast, when the intensity was increased to 1 mT, the inhibition rate was as follows: 15 Hz, 39.67%; 50 Hz, 31.81%; 100 Hz, 31.54% (n ¼ 5) (Fig. 2b). Correspondingly, the inhibition rates of the 2 mT magnetic fields were: 40.82%, 33.39%, and 35.48% (n ¼ 5) in three frequencies, respectively (Fig. 2c). These results indicated that fEPSP amplitudes reduced significantly after
516
Y. Zheng et al. / Biochemical and Biophysical Research Communications 517 (2019) 513e519
Fig. 2. Schaffer collateral pathway LTP responses to different sine ELF-EMFs intensities and frequencies. (a-c) The influence of 0.5 mT, 1 mT, and 2 mT priming ELF-EMFs on LTP (the light-colored line represents the baseline and the dark line represents the induced LTP.). (d-f) Statistical analysis and effect comparison of magnetic fields of different frequencies and intensities on LTP induction. Compared with the control group, the 0.5 mT, 1 mT, and 2 mT priming uninterrupted sine magnetic field significantly inhibits LTP induction. (g) Average fEPSP amplitudes at different stimulation intensities (0.5, 1, and 2 mT) for each frequency. (h) Average fEPSP amplitudes at different stimulation frequencies (15, 50, and 100 Hz) for each intensity, n ¼ 5. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.
magnetic exposure. The two-way ANOVA test results are shown in Table 1. The fEPSP amplitudes upon exposure to magnetic fields with a series of frequencies at 15, 50, and 100 Hz with a fixed intensity of 0.5 mT, were 134.20 ± 8.38 mV, 150.07 ± 9.69 mV, and
159.40 ± 12.40 mV (n ¼ 5), respectively. We concluded that 15 Hz magnetic field induced greatest inhibition in fEPSPs (Fig. 2g). In order to determine the influence of the magnetic intensity on the fEPSP amplitude, we stimulated brain slices with 15 Hz/0.5 mT, 15 Hz/1 mT, and 15 Hz/2 mT magnetic fields. The corresponding
Table 1 The two-way ANOVA results of magnetic field intensity and frequency. Magnetic stimulation frequency 15 Hz (b) Control (a) 0.5 mT
1 mT
2 mT
Intensity (F, P) Intensity *Frequency (F, P)
202.71 ± 9.29 134.20 ± 8.38 Pab < 0.001, Pac < 0.001, Pad < 0.001 Pbc ¼ 0.099, Pbd ¼ 0.006, Pcd ¼ 0.479 122.30 ± 12.19 Pab < 0.001, Pac < 0.001, Pad < 0.001 Pbc ¼ 0.072, Pbd ¼ 0.006, Pcd ¼ 0.479 119.97 ± 8.59 Pab < 0.001, Pac < 0.001, Pad < 0.001 Pbc ¼ 0.033, Pbd ¼ 0.164, Pcd ¼ 0.823 F(3,16) ¼ 80.70 P < 0.001 F (4, 36) ¼ 1.012, P ¼ 0.4144
50 Hz (c)
100 Hz (d)
Frequency (F, P)
150.07 ± 9.69
159.40 ± 12.40
F (3,16) ¼ 42.53, P < 0.001
138.231 ± 6.36
138.78 ± 8.96
F (3,16) ¼ 71.36, P < 0.001
135.03 ± 6.21
130.79 ± 6.48
F (3,16) ¼ 117.40, P < 0.001
F (3,16) ¼ 76.37 P < 0.001
F (3,16) ¼ 80.70 P < 0.001
Y. Zheng et al. / Biochemical and Biophysical Research Communications 517 (2019) 513e519
517
Fig. 3. Analysis of the effect of ELF-EMFs stimulation on the threshold effect of LTP of Schaffer-CA1. (a) The effect of 15 Hz/0.1 mT on the amplitude of LTP. (B) The fitting curve of the influence of magnetic field dose on LTP.
fEPSP amplitudes were 134.20 ± 8.38 mV, 122.30 ± 12.19 mV, and 119.97 ± 8.59 mV (n ¼ 5), respectively. These results indicated that magnetic field intensity of 2 mT produced the greatest inhibition effect in fEPSPs, and the fEPSP amplitude decreased in proportion to intensity levels. We then performed statistical analysis and plotted our data on the graph depicted in Fig. 2h. The influence of the 15 Hz priming magnetic field on LTP induction was as follows: 0.5 mT versus 1 mT, P ¼ 0.11; 0.5 mT versus 2 mT, P ¼ 0.046; and 1 mT versus 2 mT, P ¼ 0.913. Similarly, the influence of the 50 Hz priming magnetic field on LTP induction was as follows: 0.5 mT versus 1 mT, P ¼ 0.112; 0.5 mT versus 2 mT, P ¼ 0.034; and 1 mT versus 2 mT, P ¼ 0.843. Finally, the influence of the 100 Hz priming magnetic field on LTP induction was as follows: 0.5 mT versus 1 mT, P ¼ 0.003; 0.5 mT versus 2 mT, P < 0.001; and 1 mT versus 2 mT, P ¼ 0.356. To further determine the threshold analysis of the LTP on the magnetic field, we tested the effect of extremely low intensity of 0.1 mT/15 Hz magnetic field on LTP, because it can be seen from Fig. 2 that the 15 Hz EMF stimulation has the most significant effect on LTP. We found that after 0.1 mT/15 Hz magnetic field exposure, the amplitudes of evoked-LTP were significantly lower than controls, suggesting the extremely low intensity of 0.1 mT magnetic field can still inhibit LTP (Fig. 3a). And the inhibition rate is 15.90% compare with the control group. Meanwhile, the inhibition rate of 0.5 mT, 1 mT and 2 mT is 27.10%, 28.80%, 32.40%, respectively. We have
curve fitting the above data (Fig. 3b), the result of curve fitting for the inhibition rate at different intensities (0, 0.1, 0.5, 1, 2 mT) revealed a logarithmical increase in the inhibition rates. Thus, we can conclude that the inhibitory effect of magnetic field dose on LTP is regulatory effect, and the inhibition is gradually enhanced with the change of dose. The inhibition effect changes rapidly between 0 and 0.1 mT, but reach to 0.5 mT, the inhibition tends to be gentle.
3.2. The effect of the ELF-EMF on LTP is related to exposure duration In the next experiment, we used the 15 Hz/2 mT magnetic field to study the effect of different magnetic field exposure durations on LTP. Control experiment was conducted without any magnetic exposure, and LTP was recorded after magnetic exposure of 10s, 20s, 40s, 60s, and 5 min (Fig. 4a). We observed the fEPSP amplitudes in the magnetic exposure groups are lower than that in control group. We found 10s and 20s of exposure duration induced similar inhibition effects, and 40s, 60s, and 5 min induced much greater inhibition. We conducted statistical analysis on the resulting data (Fig. 4b). The one-way ANOVA test results are shown in Table 2. Therefore, we can conclude that the fEPSP amplitude decreased with an increase in the magnetic exposure duration with constant frequency and intensity.
Fig. 4. LTP on the Schaffer collateral pathway using different sine ELF-EMFs duration. (a) Influence of 0s, 10s, 20s, 40s, 60s, and 5 min priming ELF-EMFs on LTP induction. (b) Statistical analysis and comparisons of the effect of magnetic fields of different duration on LTP induction. Compared with the control group, each group significantly inhibits LTP induction. n ¼ 5. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.
518
Y. Zheng et al. / Biochemical and Biophysical Research Communications 517 (2019) 513e519
Table 2 The one-way ANOVA results of magnetic field duration. Magnetic stimulation duration Control (a) 15 Hz 2 mT P values
Duration (F, P)
10s (b)
20s (c)
40s (d)
60s (e)
5min (f)
169.463± 145.021± 143.976± 114.130± 113.854± 115.465 ± 6.34752 6.19931 4.27713 8.19402 4.68080 5.79459 Pab < 0.001, Pac < 0.001, Pad < 0.001, Pae < 0.001, Paf < 0.001, Pbc ¼ 0.986, Pbd<0.001, Pbe<0.001, Pbf<0.001, Pcd<0.001,Pce<0.001, Pcf<0.001, Pde>0.999, Pdf ¼ 0.957, Pef ¼ 0.950 F (5, 24) ¼ 364.2, P < 0.001
4. Discussion LTP in the schaffer collaterals of the hippocampal CA1 region is an important model in studying neural plasticity and contributes to learning and memory processing [28]. In our research, we utilized a high-precision quantitative magnetic exposure device, sXcELF, which enabled us to generate short-term ELF-EMFs with certain parameters that can stimulate brain tissues quantitatively. In addition, we proved the feasibility of this device in electrophysiological studies. We observed that the HFS induced-LTP in the hippocampal CA1 radiation layer significantly decreased under wide ranges of magnetic field strengths or magnetic field frequencies, indicating that magnetic fields have an inhibitory effect on the amplitude of LTP. Most studies have shown that priming magnetic stimulation inhibits the fEPSP slope induced by TMS and tonic stimulation [29], and the priming magnetic stimulation do not change the synaptic plasticity increases the threshold for LTP [30]. Therefore, we guess there are three possible mechanisms explaining the effects of electromagnetic field on LTP: 1. the electromagnetic field affects the opening and closing of the presynaptic membrane voltage-gated channels, thus affecting the entry of calcium ions into the presynaptic membrane, thereby affecting the release of neurotransmitters mediating LTP [31,32]. 2. The electromagnetic field could potentially block neural signal transmission [20]. 3. The electromagnetic field may affect the removal of magnesium ions (Mg2þ) blocking Nmethyl-D-aspartic acid (NMDA) receptors, thereby affecting calcium (Ca2þ) and sodium (Naþ) ion neural levels in the postsynaptic regions, as well as intracellularly, thus decreasing the binding capacity of NMDA receptor-linked channels and permeability to Ca2þ [33]. Meanwhile, Ca2þ can in turn activate the calcium/calmodulindependent protein kinase (CaMKII) and protein kinase C (PKC), as well as a series of other signaling pathways that can induce the production of LTP [34]. The use of PKC inhibitors can therefore be able to block the production of LTP. Similarly, CaMKII inhibitors or a knockout CaMKII mouse model can also inhibit LTP. Despite of these possible molecular biological understandings, further experimental confirmation is still needed. Furthermore, it has been reported that magnetite is present in human hippocampal brain tissue [35], implying ELF-EMFs may induce magnetite rotating, which in turn lead to transient changes of ion channel gating state at excitable synapses and affect synaptic plasticity. Meanwhile, possibly via acting on magnetite, magnetic fields induce electric fields and currents in human body, stimulate hippocampal [33], central nervous system and muscles, and change the excitability of excitatory neurons [36,37]. Though the magnetite's function in the brain is still unknown, it is a possible target of external magnetic fields, which renders dose-dependent inhibition of LTP, and further studies is needed in understanding of the underlying mechanisms. Our present study has three advantages in studying the effect of ELF-EMFs on neuronal activity. First, the sXcELF system utilized is able to shield external low intensity and low frequency magnetic
fields existing in the experiment space, which guarantees the precision and the accuracy of ELF-EMFs treatment. Second, the thermal controlling system in our sXcELF system is able to keep ELF-EMFs exposure space at a specific temperature that is most suitable for the brain slice viability, which is superior to other similar magnetic field exposure studies [38]; Petra et al., 2010). At last, the large exposure capacity of our sXcELF system enables us to treat experimental and control groups simultaneously, ensuring unwanted variables are factored out, which a unique advantage in ELF-EMFs studies [39]. Nevertheless, our study has some limitations. First, ELF-EMFs exposure and electrophysiological recording has to be performed in separate setups, thus, it takes short period of time to transfer the brain slices to the recording site, which may bring small variance to LTP recordings. Besides, though a dose-dependent inhibition effect on LTP is characterized, we are still unable to identify a threshold intensity that induces minimal changes in LTP, because extremely low magnetic field intensities (<0.04 mT) cannot be generated in our setup. We expect further studies on extremely low intensities can be achieved by using micro-magnetic in the future [40]. In conclusion, ELF-EMFs could alter the synaptic plasticity in the hippocampal CA1 brain region in Sprague-Dawley rats. Based on our results, we assume that the LTP can be induced by HFS regardless of the intensities and frequencies of the ELF-EMFs applied to brain slices. Meanwhile, we investigated the mutual relationship between ELF-EMF stimulation and HFS on inducing LTP. In a previous study, we conducted sinusoidal ELF-EMFs and HFS induction LTP experiments separately and found that a 100 Hz sinusoidal ELF-EMFs induction alone does not induce LTP. We then hypothesized that sinusoidal ELF-EMFs function as modulators, rather than inducers of LTP [7]. From an experimental point of view, in our current study we illustrated the possible effects of ELF-EMFs on synaptic plasticity. These results may provide a theoretical and experimental basis for future research focused on the understanding of LTP induction in learning and memory formation. Significance statement Comparing to previous in vitro brain devices developed for magnetic exposure, the sXcELF exposure system can flexibly generate high-precision quantitatively uninterrupted sinusoidal magnetic fields. On this basis, we investigated the effects of shortterm ELF-EMFs on synaptic plasticity in the hippocampal area CA1 using the sXcELF exposure system. Our results indicate that shortterm ELF-EMFs exposure dose-dependently inhibit LTP, which can serve as a reference in understanding the influence of ELF-EMFs on learning and memory. Funding Support or grant information: This paper was supported by grants from the National Natural Science Foundation of China (61871288).
Y. Zheng et al. / Biochemical and Biophysical Research Communications 517 (2019) 513e519
Conflicts of interest None. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.07.085. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.07.085. References [1] T. Liu, S. Wang, L. He, K. Ye, Chronic exposure to low-intensity magnetic field improves acquisition and maintenance of memory, Neuroreport 19 (2008) 549e552. [2] S. Atsushi, T. Masayuki, M. Kei, S. Yukihisa, J. Yasuhiko, N. Satoshi, Response of cultured neuronal network activity after high-intensity power frequency magnetic field exposure, Front. Physiol. 9 (2018) 189. [3] A. Boland, D. Delapierre, D. Mossay, A. Dresse, V. Seutin, Effect of intermittent and continuous exposure to electromagnetic fields on cultured hippocampal cells, Bioelectromagnetics 23 (2) (2010) 97e105. [4] E.Kennard L. Mathie A, L. Veale E, Neuronal ion channels and their sensitivity to extremely low frequency weak electric field effects, Radiat. Prot. Dosim. 106 (4) (2003) 311e315. [5] R.D. Saunders, J.G. Jefferys, A NEUROBIOLOGICAL BASIS FOR ELF GUIDELINES, Health Phys. 92 (6) (2007) 596e603. €m, B. Gustafsson, Postsynaptic control of hippocampal long-term [6] H. Wigstro potentiation, J. Physiol. (Paris) 81 (4) (1986) 228e236. [7] L. Dong, Y. Zheng, Z.Y. Li, G. Li, L. Lin, Modulating effects of on-line low frequency electromagnetic fields on hippocampal long-term potentiation in young male Sprague-Dawley rat, J. Nurs. Res. 96 (11) (2018) 1775e1785. https://doi.org/10.1002/jnr.24276. [8] A. Komakin, A. Khalili, I. Salehi, S. Shahidi, A. Sarihi, Effects of exposure to an extremely low frequency electromagnetic field on hippocampal long-term potentiation in rat, Brain Res. 1564 (2014) 1e8. [9] J. Huang, T. Tang, G. Hu, J. Zheng, Y. Wang, Q. Wang, J. Su, Y. Zou, X. Peng, Association between exposure to electromagnetic fields from high voltage transmission lines and neurobehavioral function in children, PLoS One 8 (2013), e67284. [10] H. Lai, Spatial learning deficit in the rat after exposure to 60 Hz magnetic field, Bioelectromagnetics 17 (1996) 494e496. [11] R.M. Mostafa, Y.M. Mostafa, A. Ennaceur, Effects of exposure to extremely low-frequency magnetic field of 2G intensity on memory and corticosterone level in rats, Physiol. Behav. 76 (2002) 589e595. zquezgarcía, D. Elíasvin ~ as, G. Reyesguerrero, A. Domínguezgonz [12] M. Va alez, n, Exposure to extremely low-frequency L. Verdugodíaz, R. Guevaraguzma electromagnetic fields improves social recognition in male rats, Physiol. Behav. 82 (2004) 685e690. [13] L. Nadel, The hippocampus and space revisited, Hippocampus 1 (1991) 221e229. [14] H. Eichenbaum, The hippocampal system and declarative memory in animals, J. Cogn. Neurosci. 4 (1992) 217. [15] Z. Ahmed, A. Wieraszko, The mechanism of magnetic field-induced increase of excitability in hippocampal neurons, Brain Res. 1221 (2008) 30e40. [16] T.J. Teyler, P. DiScenna, Long-term potentiation, Annu. Rev. Neurosci. 249 (1990) 131e161. [17] T.V. Bliss, G.L. Collingridge, A synaptic model of memory: long-term potentiation in the hippocampus, Nature 361 (1993) 31e39. [18] S.F. Cooke, T.V. Bliss, Plasticity in the human central nervous system, Brain 129
519
(2006) 1659. [19] R.C. Malenka, M.F. Bear, LTP and LTD: an embarrassment of riches, Neuron 44 (2004) 5e21. gi, [20] T. Balassa, P. Varro, S. Elek, O. Drozdoversuszky, R. Szemerszky, I. Vila G. Bardos, Changes in synaptic efficacy in rat brain slices following extremely low-frequency magnetic field exposure at embryonic and early postnatal age, Int. J. Dev. Neurosci. 31 (2013) 724e730. [21] X. Liu, H. Zuo, D. Wang, R. Peng, T. Song, S. Wang, X. Xu, Y. Gao, Y. Li, S.X. Wang, L. Wang, L. Zhao, Improvement of spatial memory disorder and hippocampal damage by exposure to electromagnetic fields in an alzheimer's disease rat model, PLoS One 10 (2015), e0126963. [22] J. Schuderer, W. Oesch, N. Felber, D. Spat, N. Kuster, In vitro exposure apparatus for ELF magnetic fields, Bioelectromagnetics 25 (2004) 582. , R. Szemerszky, G. B gi, Changes in synaptic efficacy and [23] P. Varro ardos, I. Vila seizure susceptibility in rat brain slices following extremely low-frequency electromagnetic field exposure, Bioelectromagnetics 30 (2009) 631. [24] Y. Zheng, W. Ma, L. Dong, J.R. Dou, Y. Gao, Influence of the on-line ELF-EMFs stimulation on the electrophysiological properties of the rat hippocampal CA1 neurons in vitro, Rev. Sci. Instrum. 88 (2017a) 105106. [25] Y. Zheng, J.R. Dou, Y. Gao, L. Dong, G. Li, Effects of 15 Hz square wave magnetic fields on the voltage-gated sodium and potassium channels in prefrontal cortex pyramidal neurons, Int. J. Radiat. Oncol. 93 (2017b) 449e455. [26] S. Nabavi, H.W. Kessels, S. Alfonso, J. Aow, R. Fox, R. Malinow, Metabotropic NMDA receptor function is required for NMDA receptor-dependent long-term depression, Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 4027e4032. [27] N. Zhang, M. Xing, Y. Wang, , et al.H. Tao, Y. Cheng, Repetitive transcranial magnetic stimulation enhances spatial learning and synaptic plasticity via the VEGF and BDNF-NMDAR pathways in a rat model of vascular dementia, Neuroscience 311 (2015) 284e291. [28] S. Komai, T. Matsuyama, K. Matsumoto, K. Kato, M. Kobayashi, K. Imamura, S. Yoshida, S. Ugawa, S. Schiosaka, Neuropsin regulates an early phase of Schaffer-collateral long-term potentiation in the murine hippocampus, Eur. J. Neurosci. 12 (2000) 1479e1486. € hling, NMDA receptor[29] T. Tokay, T. Kirschstein, M. Rohde, V. Zschorlich, R. Ko dependent metaplasticity by high-frequency magnetic stimulation, Neural Plast. (2014) (2014) 1e8. [30] H.J. Park, H.K. Kang, M. Wang, J. Jo, E. Chunq, S. Kim, A pilot study of planar coil based magnetic stimulation using acute hippocampal slice in mice, Conf. Proc. IEEE Eng. Med. Biol. Soc. (2017) (2017) 1118e1121. [31] T. Taghian, D.A. Narmoneva, A.B. Kogan, Modulation of cell function by electric field: a high-resolution analysis, J. R. Soc. Interface 12 (107) (2015). [32] V. Vajrala, J.R. Claycomb, H. Sanabria, J.H. Miller Jr., Effects of oscillatory electric fields on internal membranes: an analytical model, Biophys. J. 94 (6) (2008) 2043e2052. [33] G. Kronberg, M. Bridi, T. Abel, M. Bikson, L.C. Parra, Direct current stimulation modulates LTP and LTD: activity dependence and dendritic effects, Brain Stimul. 10 (1) (2016) 51e58. S1935861X16302595. [34] H. Schulman, Protein phosphorylation in neuronal plasticity and gene expression, Curr. Opin. Neurobiol. 5 (1995) 375e381. [35] J. Dobson, P. Grassi, Magnetic properties of human hippocampal tissue– evaluation of artefact and contamination sources, Brain Res. Bull. 39 (4) (1996) 255e259. € ttner, P. Lehofer, U. Schmidpeter, M. Rampetsreiter, [36] N. Leitgeb, R. Cech, J. Schro Magnetic emissions of electric appliances, Int. J. Hyg Environ. Health 211 (1e2) (2008) 69e73. [37] F. Ranieri, M.V. Podda, E. Riccardi, M. Frisullo, P. Dileone, F. Profice, V. Pilato, Di Lazzaro, C. Grassi, Modulation of LTP at rat hippocampal CA3-CA1 synapses by direct current stimulation, J. Neurophysiol. 107 (7) (2012) 1868e1880. [38] M. Jadidi, M.S. Khatami, F. Mohammad-Pour, A. Bandavi, A. Rashidy-Pour, A.A. Vafaei, A.A. Taherian, H. Miladi-Gorji, Effects of extremely low frequency magnetic field on the development of tolerance to the analgesic effect of morphine in rats, Bioelectromagnetics 38 (8) (2017) 618e625. €hling, High-frequency [39] T. Tokay, N. Holl, T. Kirschstein, V. Zschorlich, R. Ko magnetic stimulation induces long-term potentiation in rat hippocampal slices, Neurosci. Lett. 461 (2) (2009) 150e154. [40] G. Bonmassar, S.W. Lee, D.K. Freeman, M. Polasek, S.I. Fried, J.T. Gale, Microscopic magnetic stimulation of neural tissue, Nat. Commun. 3 (2012).
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Effects of exposure to extremely low frequency electromagnetic fields on hippocampal long-term potentiation in hippocampal CA1 region Yu Zheng a, *, Jianhao Cheng a, Lei Dong b, Xiaoxu Ma a, Qingyao Kong c a
School of Electronics and Information Engineering, Tianjin Polytechnic University, Tianjin, 300387, China State Key Laboratory of Precision Measurement Technology and Instruments, Tianjin University, Tianjin, 300072, China c Department of Anesthesia and Critical Care, University of Chicago, Chicago, IL, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2019 Accepted 22 July 2019 Available online 31 July 2019
Exposure to environmental electromagnetic fields, especially to the extremely low-frequency (ELF < 300 Hz) electromagnetic fields (EMFs) might produce modulation effects on neuronal activity. Long-term changes in synaptic plasticity such as long-term potentiation (LTP) involved in learning and memory may have contributions to a number of neurological diseases. However, the modulation effects of ELF-EMFs on LTP are not yet fully understood. In our present study, we aimed to evaluate the effects of exposure to ELF-EMFs on LTP in hippocampal CA1 region in rats. Hippocampal slices were exposed to magnetic fields generated by sXcELF system with different frequencies (15, 50, and 100 Hz [Hz]), intensities (0.5, 1, and 2 mT [mT]), and duration (10 s [s], 20 s, 40 s, 60 s, and 5 min), then the baseline signal recordings for 20 min and the evoked field excitatory postsynaptic potentials (fEPSPs) were recorded. We found that the LTP amplitudes decreased after magnetic field exposure, and the LTP amplitudes decreased in proportion to exposure doses and durations, suggesting ELF-EMFs may have dose and duration-dependent inhibition effects. Among multiple exposure duration and doses combinations, upon 5 min magnetic field exposure, 15 Hz/2 mT maximally inhibited LTP. Under 15 Hz/2 mT ELF-EMFs, LTP amplitude decreases in proportion to the length of exposure durations within 5 min time frame. Our findings illustrated the potential effects of ELF-EMFs on synaptic plasticity and will lead to better understanding of the influence on learning and memory. © 2019 Elsevier Inc. All rights reserved.
Keywords: Extremely low frequency electromagnetic fields Short-term effects of ELF-EMF exposure Hippocampal CA1 area Synaptic plasticity Long-term potentiation
1. Introduction Extremely low frequency electromagnetic fields (ELF-EMFs) affect several types of neuronal activities including memory [1,2]. Previous study [3] has shown that the widespread use of electricity increases the potential sources of radiation resulting in continuous or intermittent exposure of living beings to extremely low frequency electromagnetic fields. Because ELF-EMFs stimulate neuronal cells by the depolarization of voltage-gated ion channels [4,5], which may evoke pre- and postsynaptic depolarization that is required for generation of LTP [6], we hypothesize that ELF-EMFs may affect the induction or the expression of LTP. To test this hypothesis, we previously tested whether ELF-EMFs induce LTP in vitro. Interestingly, we found that exposure to ELF-EMFs itself doesn't induce LTP but modulates LTP at Schaffer collateral-CA1
* Corresponding author. E-mail address: [email protected] (Y. Zheng). https://doi.org/10.1016/j.bbrc.2019.07.085 0006-291X/© 2019 Elsevier Inc. All rights reserved.
synapses on rat hippocampal slices [7]. Consistent findings demonstrate that exposure to ELF-EMFs produces a marked change in LTP at hippocampal perforant pathway to dentate gyrus specifically in postsynapses [8], which supports our argument that ELFEMFs modulates LTP in rat hippocampus. Studying the effects of ELF-EMFs exposure on LTP in brain tissues or living animals is important because accumulation evidence indicates that ELF-EMFs exposure affects spatial learning and memory and is associated with neurodegenerative including Parkinson's disease (PD) and Alzheimer's disease (AD) [9]. [10e12]. Therefore, understanding the neurobiological mechanisms that underlie the modulation effects of ELF-EMFs on LTP would be highly desirable. Since LTP is a key component in spatial learning and alterations in LTP may contribute to PD and AD ewhether and how exposure to ELF-EMFs affects LTP will be a key question in tackling the biological mechanisms involved in the ELF-EMFs’ effects on animal behaviors and diseases. We are interested in synaptic plasticity in hippocampus because the hippocampus in the deep brain plays an important
514
Y. Zheng et al. / Biochemical and Biophysical Research Communications 517 (2019) 513e519
role in short-term learning and memory including spatial learning [13e15] that reportedly can be affected by exposure to ELF-EMFs [1]. Thus, understanding the effects of ELF-EMFs on vital hippocampal pathways may shed light on potential therapeutic strategies for prevention and treatment of learning related diseases. Considering hippocampus LTP enhances synaptic efficiency and is essential for learning and memory processes [16e18], and LTP at schaffer collaterals-CA1 is a well-established model in studying synaptic plasticity [19], we chose LTP at schaffer collaterals-CA1 as a model in exploring the effects of ELF-EMFs on synaptic plasticity. Previous studies indicate that utilizing ELF-EMFs at different strengths may have divergent effects on synaptic plasticity at CA1 region in rats [8,20]. However, whether dose-response relationships exist in the effects of ELF-EMFs on synaptic plasticity has never been studied before [21]. In the present study, we ask how exposure to ELF-EMFs at different frequencies or intensities in different exposure durations affects LTP in CA1, what potential dose-response relationships are involved. Answers to these questions are essential to understanding of the neurobiological mechanisms underlying the effects of ELF-EMFs. To investigate how exposure to ELF-EMFs modulates LTP, we utilized a high-precision magnetic exposure device sXcELF (IT'IS Foundation, Zurich, Switzerland) that can generate uniform magnetic fields at wide frequency and intensity ranges (frequencies between 3 and 1250 Hz [Hz] and intensities ranging from 0.04 to 3.5 mT) at high accuracy [22]. In recent years, sXcELF device has been widely used in studies on the principle, characteristics and biomedical application of ELF-EMFs [20,22e25], however we did not find any application of sXcELF in studies on synaptic plasticity. In our research, sXcELF device enables us to study the effect of the ELF-EMFs in wide ranges of frequency/intensity. In our study, rat hippocampal slices were stimulated using the external exposure device sXcELF, followed by a recording system of field excitatory postsynaptic potentials (fEPSPs), which contains a stimulation electrode and a recording electrode that measures the amplitude of hippocampal fEPSPs. We investigated the impact of ELF-EMFs at different frequencies (15, 50, and 100 Hz), intensities (0.5, 1, and, 2 mT), and duration (10 s [s], 20s, 40s, 60s, and, 5 min [min]) on LTP amplitudes at hippocampal schaffer-CA1 region in rats. Our results showed the LTP amplitudes decreased after magnetic field exposure, and the LTP amplitudes decreased in proportion to exposure doses and durations, indicating ELFEMFs exposure inhibits LTP amplitude in dose and durationdependent patterns. Among multiple exposure duration and doses combinations, upon 5 min magnetic field exposure, 15 Hz/ 2 mT maximally inhibited LTP. Under 15 Hz/2 mT ELF-EMFs, LTP amplitude decreases in proportion to the length of exposure durations.
2. Materials and methods 2.1. Animals A total of 18 male Sprague-Dawley rats (14e18 days old at the time of surgery) were obtained from the Academy of Military Medical Sciences (Tianjin, China), certification number SCXK-(PLA) 2014e0001. Each rat was kept in a separate cage in a clean room maintained under a 12-h light/dark cycle at a constant temperature (25 ± 2 C), and with access to food and water ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee protocol of Tianjin Polytechnic University, Tianjin, China. The experiments were designed to minimize any animal suffering as well as the number of animals used.
2.2. Tissue preparation SpragueeDawley rats were anesthetized with chloral hydrate [23,25], at a concentration of 0.1 ml/20 g (10%), and their brains were rapidly removed and stored in 4 C cutting solution containing: 90 mM of sucrose, 87.2 mM of NaCl, 2.5 mM of KCl, 7 mM of MgCl2, 0.5 of mM CaCl2, 1.25 mM of NaH2PO4, 25 mM of NaHCO3, and 16.7 mM of glucose. The solution was continuously bubbled with 95% O2/5% CO2. Subsequently, 400 mm thick horizontal slices were cut using the vibrating tissue slicer, VF-200 (Precisionary Instruments New Jersey, USA) in ice-cold cutting solution. Slices were then incubated at 33 C in artificial cerebrospinal fluid (ACSF) for 1 h and an individual slice was moved to a perfusion chamber for recording. The ACSF consisted of: 120 mM of NaCl, 2.5 mM of KCl, 2 mM of MgSO4$7H2O, 2 mM of CaCl2, 1.25 mM of NaH2PO4$2H2O, 26 mM of NaHCO3, and 10 mM of glucose, continuously bubbled with 95% O2/5% CO2 at a pH of 7.4. Moreover, when fEPSPs were being recorded, pipettes were filled with 3 mol/L of NaCl. All other reagents were of analytical grade made in China (Tianjin Fengchuan Chemical Reagent Technologies Co., Ltd, Tianjin). All brain slices were randomly assigned either to the control or experimental group. 2.3. Magnetic stimulator and electrophysiological recordings The magnetic exposure device sXcELF used in this study (Fig. 1a) consisted of four parts: two four-coil systems, a commercial incubator, a monitor, and a data acquisition unit (PC). The two four-coil systems were placed into two porous metal boxes labeled “Chamber 100 and “Chamber 200 , in order to enable the generation of magnetic fields with a frequency range of 3e1250 Hz and a maximum magnetic flux density of 3.5 mT root-mean-square amplitude [22]. The commercial incubator BSC-PC (Harvard Apparatus, Horiston, MA) was placed above the chambers to maintain the chamber temperature at 33 C. Both the dish holder and mumetal box were placed on elastically damped feet in order to minimize vibration. A software-controlled arbitrary function generator was used as the signal source for the two current sources. The current source converted the voltage signal to the desired current and sent it to the coil inside the incubator. The temperature of the flasks was then monitored during exposure with Pt100 probes in order to control the output of the desired signal as a voltage. The slices were random placed in either chamber, which an EMFs exposure apparatus (experimental group) and a similar device without EMFs (control group), during this procedure, the solution was continuously bubbled with 95% O2/5% CO2. The EMF produced by experimental group was set at an intensity of 0.5, 1, or 2 mT and the frequency was set at 15, 50, or 100 Hz. The experimental group slice exposure duration was set to 10 s, 20s, 40 s, 60 s, and 5 min. The stratum radiatum of CA1 was viewed under the upright microscope, BX51-WI (Olympus, Tokyo, Japan) equipped with a long-range water immersion objective (40 ) and an infrared video camera (IR1000, DAGE-MTI, Richmond Inner Harbor) (Fig. 1b) of the electrophysiological recording system. The bipolar concentric stimulation, CBARC75 (FHC, Bowdoin, ME), was placed on the stratum radiatum of CA1. The recording electrodes (resistance 3e8 mega-ohms [MU]) were also placed in either the stratum radiatum, approximately 250 mm from the stimulation electrode in CA1 to record the amplitude of fEPSPs [26,27]. The distribution of the magnetic field around the center of the perfusion chamber, where brain slices were placed during the experiments, was measured using the mill-Tesla device, HT108 (Htmagnet, Shanghai China) to ensure the accuracy of the exposure. Although, the above-mentioned Tesla-meter was able to measure
Y. Zheng et al. / Biochemical and Biophysical Research Communications 517 (2019) 513e519
515
Fig. 1. Experimental setup of the magnetic exposure device and the field excitatory postsynaptic potential (fEPSP) recording system. (a) The exposure device sXcELF produces shortterm extremely low frequency electromagnetic field (ELF-EMF) stimulation on brain slices. (b) The fEPSPs recording system: brain slices are first stimulated using the external exposure device sXcELF, followed by recordings of fEPSP amplitudes using respective electrodes.
the magnetic field strength in brain slices, the test results were inaccurate due to the large size of the Tesla-meter probe, when compared with the brain slices. In order to estimate the distribution of the magnetic field in the brain slices, we also conducted a simulation experiment (COMSOL Multiphysics 5.2a, COMSOL Inc, Stockholm, Sweden). Subsequently, Auto CAD 2017 (Autodesk, California, USA) was used to construct a brain tissue model with a width of 1 cm and a thickness of 400 mm. This model was then imported into the COMSOL software; the characteristics of the materials used in the simulation are shown in Supplementary file (Table S1). Results from the simulation experiments showed that the simulated magnetic field intensity was similar to the experimental magnetic field strength as seen in Supplementary file (Fig. 1Sa). The induced electric field intensity in the radiation layer is shown in Supplementary file (Fig. 1Sb). Notably, the difference in the distribution of induced electric field intensity was negligible in the radiation layer. 2.4. Stimulation protocol In this paper, the preparation was stimulated electrically for 20 min at a test of the stimulus frequency of 0.06 Hz. The baseline served as a reference to evaluate changes in the slope of fEPSPs. Stimulation strength was adjusted to obtain approximately 40% of the maximal response. The duration of single pulses was 0.1 ms. Stable baseline fEPSPs were recorded each minute for at least 20 min before any plasticity induction was applied. fEPSPs were then recorded again every minute for 60 min after plasticity was induced. We first determined whether priming uninterrupted sine, ELF-EMFs interfered with the electrically induced LTP. The stimulus protocol used in the experiments is depicted in Supplementary file (Fig. 2S). We first conducted the control experiments (without any priming of ELF-EMFs, only electrical high-frequency electric stimulation [HFS] induced LTP). For this, a stable baseline fEPSPs was recorded for 20 min before any plasticity induction was applied. The fEPSPs were then recorded again for 60 min, after plasticity induction. The electrical HFS paradigm consisted of 4 sweeps of 100 pulses (1s apart) with a 20 s interval. Then, we applied priming offline sine ELF-EMFs, as an uninterrupted sinusoidal signal with frequencies of 15 Hz, 50 Hz, and 100 Hz, to hippocampal brain slices, with a duration of 10s, 20s, 40s, 60s, and 5 min, according to the experimental process shown above. 2.5. Statistical analysis The raw data was processed using the Origin 8.0 software platform (Origin Lab, Northampton, MA) and the statistical analysis
was performed using the GraphPad Prism 7 (GraphPad Software Incorporation, San Diego, CA). The mean values of the fEPSPs baseline amplitudes in the control group and experimental group were normalized to a 100 value. All data were analyzed using oneway analyses of variance (ANOVA) on ranks with Tukey's post hoc test and a two-way ANOVA on Tukey's multiple comparisons test. The results were expressed as the mean ± standard deviation (SD). The differences observed were considered to be statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001. 3. Results 3.1. The effect of ELF-EMFs on LTP is frequency and intensitydependent Hippocampal slices were treated using the stimulation protocol described in Supplementary file (Fig. 2S), with each experimental group containing results obtained from 5 hippocampal brain slices. Baseline and electrical HFS-induced LTP are recorded following ELF-EMF exposure. The effects of the 0.5 mT, 1 mT, and 2 mT magnetic fields of 15 Hz, 50 Hz, and 100 Hz on the fEPSPs amplitude were analyzed separately as shown in Fig. 2. The magnetic field exposure duration was fixed to 5 min. We observed the fEPSPs amplitudes decreased after exposure to a series of priming magnetic fields with increasing intensities, and the electrical stimulation-induced LTP decreased in proportion to the magnetic field intensity. In addition, we performed statistical analysis accordingly. The baseline signal recordings for the first 20 min in each group were normalized, and the induced EPSP amplitudes were treated according to the corresponding normalized coefficients, with the average fEPSP amplitude obtained being 99.28 ± 1.43 mV (n ¼ 5). Without applying the magnetic field, the fEPSP amplitudes corresponding to the different frequencies were 100.55 ± 1.69 mV, 100.34 ± 1.72 mV, and 99.08 ± 4.14 mV (n ¼ 5). According to our statistical analysis, we concluded that there were no significant differences between these datasets. After a 100 Hz high frequency stimulation, the fEPSP was 202.71 ± 9.29 mV (n ¼ 5). The fEPSP amplitudes of slices exposed to ELF-EMFs are significantly lower than controls (Fig. 2a). For magnetic fields with a fixed intensity of 0.5 mT, the inhibition ratio was as follows: 15 Hz, 33.79%; 50 Hz, 25.97%; 100 Hz, 21.36%; (n ¼ 5). In contrast, when the intensity was increased to 1 mT, the inhibition rate was as follows: 15 Hz, 39.67%; 50 Hz, 31.81%; 100 Hz, 31.54% (n ¼ 5) (Fig. 2b). Correspondingly, the inhibition rates of the 2 mT magnetic fields were: 40.82%, 33.39%, and 35.48% (n ¼ 5) in three frequencies, respectively (Fig. 2c). These results indicated that fEPSP amplitudes reduced significantly after
516
Y. Zheng et al. / Biochemical and Biophysical Research Communications 517 (2019) 513e519
Fig. 2. Schaffer collateral pathway LTP responses to different sine ELF-EMFs intensities and frequencies. (a-c) The influence of 0.5 mT, 1 mT, and 2 mT priming ELF-EMFs on LTP (the light-colored line represents the baseline and the dark line represents the induced LTP.). (d-f) Statistical analysis and effect comparison of magnetic fields of different frequencies and intensities on LTP induction. Compared with the control group, the 0.5 mT, 1 mT, and 2 mT priming uninterrupted sine magnetic field significantly inhibits LTP induction. (g) Average fEPSP amplitudes at different stimulation intensities (0.5, 1, and 2 mT) for each frequency. (h) Average fEPSP amplitudes at different stimulation frequencies (15, 50, and 100 Hz) for each intensity, n ¼ 5. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.
magnetic exposure. The two-way ANOVA test results are shown in Table 1. The fEPSP amplitudes upon exposure to magnetic fields with a series of frequencies at 15, 50, and 100 Hz with a fixed intensity of 0.5 mT, were 134.20 ± 8.38 mV, 150.07 ± 9.69 mV, and
159.40 ± 12.40 mV (n ¼ 5), respectively. We concluded that 15 Hz magnetic field induced greatest inhibition in fEPSPs (Fig. 2g). In order to determine the influence of the magnetic intensity on the fEPSP amplitude, we stimulated brain slices with 15 Hz/0.5 mT, 15 Hz/1 mT, and 15 Hz/2 mT magnetic fields. The corresponding
Table 1 The two-way ANOVA results of magnetic field intensity and frequency. Magnetic stimulation frequency 15 Hz (b) Control (a) 0.5 mT
1 mT
2 mT
Intensity (F, P) Intensity *Frequency (F, P)
202.71 ± 9.29 134.20 ± 8.38 Pab < 0.001, Pac < 0.001, Pad < 0.001 Pbc ¼ 0.099, Pbd ¼ 0.006, Pcd ¼ 0.479 122.30 ± 12.19 Pab < 0.001, Pac < 0.001, Pad < 0.001 Pbc ¼ 0.072, Pbd ¼ 0.006, Pcd ¼ 0.479 119.97 ± 8.59 Pab < 0.001, Pac < 0.001, Pad < 0.001 Pbc ¼ 0.033, Pbd ¼ 0.164, Pcd ¼ 0.823 F(3,16) ¼ 80.70 P < 0.001 F (4, 36) ¼ 1.012, P ¼ 0.4144
50 Hz (c)
100 Hz (d)
Frequency (F, P)
150.07 ± 9.69
159.40 ± 12.40
F (3,16) ¼ 42.53, P < 0.001
138.231 ± 6.36
138.78 ± 8.96
F (3,16) ¼ 71.36, P < 0.001
135.03 ± 6.21
130.79 ± 6.48
F (3,16) ¼ 117.40, P < 0.001
F (3,16) ¼ 76.37 P < 0.001
F (3,16) ¼ 80.70 P < 0.001
Y. Zheng et al. / Biochemical and Biophysical Research Communications 517 (2019) 513e519
517
Fig. 3. Analysis of the effect of ELF-EMFs stimulation on the threshold effect of LTP of Schaffer-CA1. (a) The effect of 15 Hz/0.1 mT on the amplitude of LTP. (B) The fitting curve of the influence of magnetic field dose on LTP.
fEPSP amplitudes were 134.20 ± 8.38 mV, 122.30 ± 12.19 mV, and 119.97 ± 8.59 mV (n ¼ 5), respectively. These results indicated that magnetic field intensity of 2 mT produced the greatest inhibition effect in fEPSPs, and the fEPSP amplitude decreased in proportion to intensity levels. We then performed statistical analysis and plotted our data on the graph depicted in Fig. 2h. The influence of the 15 Hz priming magnetic field on LTP induction was as follows: 0.5 mT versus 1 mT, P ¼ 0.11; 0.5 mT versus 2 mT, P ¼ 0.046; and 1 mT versus 2 mT, P ¼ 0.913. Similarly, the influence of the 50 Hz priming magnetic field on LTP induction was as follows: 0.5 mT versus 1 mT, P ¼ 0.112; 0.5 mT versus 2 mT, P ¼ 0.034; and 1 mT versus 2 mT, P ¼ 0.843. Finally, the influence of the 100 Hz priming magnetic field on LTP induction was as follows: 0.5 mT versus 1 mT, P ¼ 0.003; 0.5 mT versus 2 mT, P < 0.001; and 1 mT versus 2 mT, P ¼ 0.356. To further determine the threshold analysis of the LTP on the magnetic field, we tested the effect of extremely low intensity of 0.1 mT/15 Hz magnetic field on LTP, because it can be seen from Fig. 2 that the 15 Hz EMF stimulation has the most significant effect on LTP. We found that after 0.1 mT/15 Hz magnetic field exposure, the amplitudes of evoked-LTP were significantly lower than controls, suggesting the extremely low intensity of 0.1 mT magnetic field can still inhibit LTP (Fig. 3a). And the inhibition rate is 15.90% compare with the control group. Meanwhile, the inhibition rate of 0.5 mT, 1 mT and 2 mT is 27.10%, 28.80%, 32.40%, respectively. We have
curve fitting the above data (Fig. 3b), the result of curve fitting for the inhibition rate at different intensities (0, 0.1, 0.5, 1, 2 mT) revealed a logarithmical increase in the inhibition rates. Thus, we can conclude that the inhibitory effect of magnetic field dose on LTP is regulatory effect, and the inhibition is gradually enhanced with the change of dose. The inhibition effect changes rapidly between 0 and 0.1 mT, but reach to 0.5 mT, the inhibition tends to be gentle.
3.2. The effect of the ELF-EMF on LTP is related to exposure duration In the next experiment, we used the 15 Hz/2 mT magnetic field to study the effect of different magnetic field exposure durations on LTP. Control experiment was conducted without any magnetic exposure, and LTP was recorded after magnetic exposure of 10s, 20s, 40s, 60s, and 5 min (Fig. 4a). We observed the fEPSP amplitudes in the magnetic exposure groups are lower than that in control group. We found 10s and 20s of exposure duration induced similar inhibition effects, and 40s, 60s, and 5 min induced much greater inhibition. We conducted statistical analysis on the resulting data (Fig. 4b). The one-way ANOVA test results are shown in Table 2. Therefore, we can conclude that the fEPSP amplitude decreased with an increase in the magnetic exposure duration with constant frequency and intensity.
Fig. 4. LTP on the Schaffer collateral pathway using different sine ELF-EMFs duration. (a) Influence of 0s, 10s, 20s, 40s, 60s, and 5 min priming ELF-EMFs on LTP induction. (b) Statistical analysis and comparisons of the effect of magnetic fields of different duration on LTP induction. Compared with the control group, each group significantly inhibits LTP induction. n ¼ 5. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.
518
Y. Zheng et al. / Biochemical and Biophysical Research Communications 517 (2019) 513e519
Table 2 The one-way ANOVA results of magnetic field duration. Magnetic stimulation duration Control (a) 15 Hz 2 mT P values
Duration (F, P)
10s (b)
20s (c)
40s (d)
60s (e)
5min (f)
169.463± 145.021± 143.976± 114.130± 113.854± 115.465 ± 6.34752 6.19931 4.27713 8.19402 4.68080 5.79459 Pab < 0.001, Pac < 0.001, Pad < 0.001, Pae < 0.001, Paf < 0.001, Pbc ¼ 0.986, Pbd<0.001, Pbe<0.001, Pbf<0.001, Pcd<0.001,Pce<0.001, Pcf<0.001, Pde>0.999, Pdf ¼ 0.957, Pef ¼ 0.950 F (5, 24) ¼ 364.2, P < 0.001
4. Discussion LTP in the schaffer collaterals of the hippocampal CA1 region is an important model in studying neural plasticity and contributes to learning and memory processing [28]. In our research, we utilized a high-precision quantitative magnetic exposure device, sXcELF, which enabled us to generate short-term ELF-EMFs with certain parameters that can stimulate brain tissues quantitatively. In addition, we proved the feasibility of this device in electrophysiological studies. We observed that the HFS induced-LTP in the hippocampal CA1 radiation layer significantly decreased under wide ranges of magnetic field strengths or magnetic field frequencies, indicating that magnetic fields have an inhibitory effect on the amplitude of LTP. Most studies have shown that priming magnetic stimulation inhibits the fEPSP slope induced by TMS and tonic stimulation [29], and the priming magnetic stimulation do not change the synaptic plasticity increases the threshold for LTP [30]. Therefore, we guess there are three possible mechanisms explaining the effects of electromagnetic field on LTP: 1. the electromagnetic field affects the opening and closing of the presynaptic membrane voltage-gated channels, thus affecting the entry of calcium ions into the presynaptic membrane, thereby affecting the release of neurotransmitters mediating LTP [31,32]. 2. The electromagnetic field could potentially block neural signal transmission [20]. 3. The electromagnetic field may affect the removal of magnesium ions (Mg2þ) blocking Nmethyl-D-aspartic acid (NMDA) receptors, thereby affecting calcium (Ca2þ) and sodium (Naþ) ion neural levels in the postsynaptic regions, as well as intracellularly, thus decreasing the binding capacity of NMDA receptor-linked channels and permeability to Ca2þ [33]. Meanwhile, Ca2þ can in turn activate the calcium/calmodulindependent protein kinase (CaMKII) and protein kinase C (PKC), as well as a series of other signaling pathways that can induce the production of LTP [34]. The use of PKC inhibitors can therefore be able to block the production of LTP. Similarly, CaMKII inhibitors or a knockout CaMKII mouse model can also inhibit LTP. Despite of these possible molecular biological understandings, further experimental confirmation is still needed. Furthermore, it has been reported that magnetite is present in human hippocampal brain tissue [35], implying ELF-EMFs may induce magnetite rotating, which in turn lead to transient changes of ion channel gating state at excitable synapses and affect synaptic plasticity. Meanwhile, possibly via acting on magnetite, magnetic fields induce electric fields and currents in human body, stimulate hippocampal [33], central nervous system and muscles, and change the excitability of excitatory neurons [36,37]. Though the magnetite's function in the brain is still unknown, it is a possible target of external magnetic fields, which renders dose-dependent inhibition of LTP, and further studies is needed in understanding of the underlying mechanisms. Our present study has three advantages in studying the effect of ELF-EMFs on neuronal activity. First, the sXcELF system utilized is able to shield external low intensity and low frequency magnetic
fields existing in the experiment space, which guarantees the precision and the accuracy of ELF-EMFs treatment. Second, the thermal controlling system in our sXcELF system is able to keep ELF-EMFs exposure space at a specific temperature that is most suitable for the brain slice viability, which is superior to other similar magnetic field exposure studies [38]; Petra et al., 2010). At last, the large exposure capacity of our sXcELF system enables us to treat experimental and control groups simultaneously, ensuring unwanted variables are factored out, which a unique advantage in ELF-EMFs studies [39]. Nevertheless, our study has some limitations. First, ELF-EMFs exposure and electrophysiological recording has to be performed in separate setups, thus, it takes short period of time to transfer the brain slices to the recording site, which may bring small variance to LTP recordings. Besides, though a dose-dependent inhibition effect on LTP is characterized, we are still unable to identify a threshold intensity that induces minimal changes in LTP, because extremely low magnetic field intensities (<0.04 mT) cannot be generated in our setup. We expect further studies on extremely low intensities can be achieved by using micro-magnetic in the future [40]. In conclusion, ELF-EMFs could alter the synaptic plasticity in the hippocampal CA1 brain region in Sprague-Dawley rats. Based on our results, we assume that the LTP can be induced by HFS regardless of the intensities and frequencies of the ELF-EMFs applied to brain slices. Meanwhile, we investigated the mutual relationship between ELF-EMF stimulation and HFS on inducing LTP. In a previous study, we conducted sinusoidal ELF-EMFs and HFS induction LTP experiments separately and found that a 100 Hz sinusoidal ELF-EMFs induction alone does not induce LTP. We then hypothesized that sinusoidal ELF-EMFs function as modulators, rather than inducers of LTP [7]. From an experimental point of view, in our current study we illustrated the possible effects of ELF-EMFs on synaptic plasticity. These results may provide a theoretical and experimental basis for future research focused on the understanding of LTP induction in learning and memory formation. Significance statement Comparing to previous in vitro brain devices developed for magnetic exposure, the sXcELF exposure system can flexibly generate high-precision quantitatively uninterrupted sinusoidal magnetic fields. On this basis, we investigated the effects of shortterm ELF-EMFs on synaptic plasticity in the hippocampal area CA1 using the sXcELF exposure system. Our results indicate that shortterm ELF-EMFs exposure dose-dependently inhibit LTP, which can serve as a reference in understanding the influence of ELF-EMFs on learning and memory. Funding Support or grant information: This paper was supported by grants from the National Natural Science Foundation of China (61871288).
Y. Zheng et al. / Biochemical and Biophysical Research Communications 517 (2019) 513e519
Conflicts of interest None. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.07.085. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.07.085. References [1] T. Liu, S. Wang, L. He, K. Ye, Chronic exposure to low-intensity magnetic field improves acquisition and maintenance of memory, Neuroreport 19 (2008) 549e552. [2] S. Atsushi, T. Masayuki, M. Kei, S. Yukihisa, J. Yasuhiko, N. Satoshi, Response of cultured neuronal network activity after high-intensity power frequency magnetic field exposure, Front. Physiol. 9 (2018) 189. [3] A. Boland, D. Delapierre, D. Mossay, A. Dresse, V. Seutin, Effect of intermittent and continuous exposure to electromagnetic fields on cultured hippocampal cells, Bioelectromagnetics 23 (2) (2010) 97e105. [4] E.Kennard L. Mathie A, L. Veale E, Neuronal ion channels and their sensitivity to extremely low frequency weak electric field effects, Radiat. Prot. Dosim. 106 (4) (2003) 311e315. [5] R.D. Saunders, J.G. Jefferys, A NEUROBIOLOGICAL BASIS FOR ELF GUIDELINES, Health Phys. 92 (6) (2007) 596e603. €m, B. Gustafsson, Postsynaptic control of hippocampal long-term [6] H. Wigstro potentiation, J. Physiol. (Paris) 81 (4) (1986) 228e236. [7] L. Dong, Y. Zheng, Z.Y. Li, G. Li, L. Lin, Modulating effects of on-line low frequency electromagnetic fields on hippocampal long-term potentiation in young male Sprague-Dawley rat, J. Nurs. Res. 96 (11) (2018) 1775e1785. https://doi.org/10.1002/jnr.24276. [8] A. Komakin, A. Khalili, I. Salehi, S. Shahidi, A. Sarihi, Effects of exposure to an extremely low frequency electromagnetic field on hippocampal long-term potentiation in rat, Brain Res. 1564 (2014) 1e8. [9] J. Huang, T. Tang, G. Hu, J. Zheng, Y. Wang, Q. Wang, J. Su, Y. Zou, X. Peng, Association between exposure to electromagnetic fields from high voltage transmission lines and neurobehavioral function in children, PLoS One 8 (2013), e67284. [10] H. Lai, Spatial learning deficit in the rat after exposure to 60 Hz magnetic field, Bioelectromagnetics 17 (1996) 494e496. [11] R.M. Mostafa, Y.M. Mostafa, A. Ennaceur, Effects of exposure to extremely low-frequency magnetic field of 2G intensity on memory and corticosterone level in rats, Physiol. Behav. 76 (2002) 589e595. zquezgarcía, D. Elíasvin ~ as, G. Reyesguerrero, A. Domínguezgonz [12] M. Va alez, n, Exposure to extremely low-frequency L. Verdugodíaz, R. Guevaraguzma electromagnetic fields improves social recognition in male rats, Physiol. Behav. 82 (2004) 685e690. [13] L. Nadel, The hippocampus and space revisited, Hippocampus 1 (1991) 221e229. [14] H. Eichenbaum, The hippocampal system and declarative memory in animals, J. Cogn. Neurosci. 4 (1992) 217. [15] Z. Ahmed, A. Wieraszko, The mechanism of magnetic field-induced increase of excitability in hippocampal neurons, Brain Res. 1221 (2008) 30e40. [16] T.J. Teyler, P. DiScenna, Long-term potentiation, Annu. Rev. Neurosci. 249 (1990) 131e161. [17] T.V. Bliss, G.L. Collingridge, A synaptic model of memory: long-term potentiation in the hippocampus, Nature 361 (1993) 31e39. [18] S.F. Cooke, T.V. Bliss, Plasticity in the human central nervous system, Brain 129
519
(2006) 1659. [19] R.C. Malenka, M.F. Bear, LTP and LTD: an embarrassment of riches, Neuron 44 (2004) 5e21. gi, [20] T. Balassa, P. Varro, S. Elek, O. Drozdoversuszky, R. Szemerszky, I. Vila G. Bardos, Changes in synaptic efficacy in rat brain slices following extremely low-frequency magnetic field exposure at embryonic and early postnatal age, Int. J. Dev. Neurosci. 31 (2013) 724e730. [21] X. Liu, H. Zuo, D. Wang, R. Peng, T. Song, S. Wang, X. Xu, Y. Gao, Y. Li, S.X. Wang, L. Wang, L. Zhao, Improvement of spatial memory disorder and hippocampal damage by exposure to electromagnetic fields in an alzheimer's disease rat model, PLoS One 10 (2015), e0126963. [22] J. Schuderer, W. Oesch, N. Felber, D. Spat, N. Kuster, In vitro exposure apparatus for ELF magnetic fields, Bioelectromagnetics 25 (2004) 582. , R. Szemerszky, G. B gi, Changes in synaptic efficacy and [23] P. Varro ardos, I. Vila seizure susceptibility in rat brain slices following extremely low-frequency electromagnetic field exposure, Bioelectromagnetics 30 (2009) 631. [24] Y. Zheng, W. Ma, L. Dong, J.R. Dou, Y. Gao, Influence of the on-line ELF-EMFs stimulation on the electrophysiological properties of the rat hippocampal CA1 neurons in vitro, Rev. Sci. Instrum. 88 (2017a) 105106. [25] Y. Zheng, J.R. Dou, Y. Gao, L. Dong, G. Li, Effects of 15 Hz square wave magnetic fields on the voltage-gated sodium and potassium channels in prefrontal cortex pyramidal neurons, Int. J. Radiat. Oncol. 93 (2017b) 449e455. [26] S. Nabavi, H.W. Kessels, S. Alfonso, J. Aow, R. Fox, R. Malinow, Metabotropic NMDA receptor function is required for NMDA receptor-dependent long-term depression, Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 4027e4032. [27] N. Zhang, M. Xing, Y. Wang, , et al.H. Tao, Y. Cheng, Repetitive transcranial magnetic stimulation enhances spatial learning and synaptic plasticity via the VEGF and BDNF-NMDAR pathways in a rat model of vascular dementia, Neuroscience 311 (2015) 284e291. [28] S. Komai, T. Matsuyama, K. Matsumoto, K. Kato, M. Kobayashi, K. Imamura, S. Yoshida, S. Ugawa, S. Schiosaka, Neuropsin regulates an early phase of Schaffer-collateral long-term potentiation in the murine hippocampus, Eur. J. Neurosci. 12 (2000) 1479e1486. € hling, NMDA receptor[29] T. Tokay, T. Kirschstein, M. Rohde, V. Zschorlich, R. Ko dependent metaplasticity by high-frequency magnetic stimulation, Neural Plast. (2014) (2014) 1e8. [30] H.J. Park, H.K. Kang, M. Wang, J. Jo, E. Chunq, S. Kim, A pilot study of planar coil based magnetic stimulation using acute hippocampal slice in mice, Conf. Proc. IEEE Eng. Med. Biol. Soc. (2017) (2017) 1118e1121. [31] T. Taghian, D.A. Narmoneva, A.B. Kogan, Modulation of cell function by electric field: a high-resolution analysis, J. R. Soc. Interface 12 (107) (2015). [32] V. Vajrala, J.R. Claycomb, H. Sanabria, J.H. Miller Jr., Effects of oscillatory electric fields on internal membranes: an analytical model, Biophys. J. 94 (6) (2008) 2043e2052. [33] G. Kronberg, M. Bridi, T. Abel, M. Bikson, L.C. Parra, Direct current stimulation modulates LTP and LTD: activity dependence and dendritic effects, Brain Stimul. 10 (1) (2016) 51e58. S1935861X16302595. [34] H. Schulman, Protein phosphorylation in neuronal plasticity and gene expression, Curr. Opin. Neurobiol. 5 (1995) 375e381. [35] J. Dobson, P. Grassi, Magnetic properties of human hippocampal tissue– evaluation of artefact and contamination sources, Brain Res. Bull. 39 (4) (1996) 255e259. € ttner, P. Lehofer, U. Schmidpeter, M. Rampetsreiter, [36] N. Leitgeb, R. Cech, J. Schro Magnetic emissions of electric appliances, Int. J. Hyg Environ. Health 211 (1e2) (2008) 69e73. [37] F. Ranieri, M.V. Podda, E. Riccardi, M. Frisullo, P. Dileone, F. Profice, V. Pilato, Di Lazzaro, C. Grassi, Modulation of LTP at rat hippocampal CA3-CA1 synapses by direct current stimulation, J. Neurophysiol. 107 (7) (2012) 1868e1880. [38] M. Jadidi, M.S. Khatami, F. Mohammad-Pour, A. Bandavi, A. Rashidy-Pour, A.A. Vafaei, A.A. Taherian, H. Miladi-Gorji, Effects of extremely low frequency magnetic field on the development of tolerance to the analgesic effect of morphine in rats, Bioelectromagnetics 38 (8) (2017) 618e625. €hling, High-frequency [39] T. Tokay, N. Holl, T. Kirschstein, V. Zschorlich, R. Ko magnetic stimulation induces long-term potentiation in rat hippocampal slices, Neurosci. Lett. 461 (2) (2009) 150e154. [40] G. Bonmassar, S.W. Lee, D.K. Freeman, M. Polasek, S.I. Fried, J.T. Gale, Microscopic magnetic stimulation of neural tissue, Nat. Commun. 3 (2012).