Bioelectromagnetics 30:631^640 (2009)

Changes in Synaptic Efficacy and SeizureSusceptibility in Rat Brain Slices FollowingExtremely Low-Frequency Electromagnetic

Field Exposure

Petra Varro¤ ,1,2 Rena¤ ta Szemerszky,1 Gyo« rgy Ba¤ rdos,1 and Ildiko¤ Vila¤ gi1*1Department of Physiology and Neurobiology, Eo« tvo« s Lora¤ nd University,

Budapest, Hungary2AnimalBreeding andAnimal Hygiene Research Group of the HungarianAcademy of

Sciences andKaposva¤ r University, Kaposva¤ r, Hungary

The effects of electromagnetic fields (EMFs) on living organisms are recently a focus of scientificinterest, as they may influence everyday life in several ways. Although the neural effects of EMFs havebeen subject to a considerable number of investigations, the results are difficult to compare sincedissimilar exposure protocols have been applied on different preparations or animals. In the presentseries of experiments, whole rats or excised rat brain slices were exposed to a reference level-intensity(250–500 mT, 50 Hz) EMF in order to examine the effects on the synaptic efficacy in the centralnervous system. Electrophysiological investigation was carried out ex vivo, on neocortical andhippocampal slices; basic synaptic functions, short- and long-term plasticity and seizure susceptibilitywere tested. The most pronounced effect was a decrease in basic synaptic activity in slices treateddirectly ex vivo observed as a diminution in amplitude of evoked potentials. On the other hand,following whole-body exposure an enhanced short- and long-term synaptic facilitation inhippocampal slices and increased seizure susceptibility in neocortical slices was also observed.However, these effects seem to be transient. We can conclude that ELF-EMF exposure exertssignificant effects on synaptic activity, but the overall changes may strongly depend on the synapticstructure and neuronal network of the affected region together with the specific spatial parameters andconstancy of EMF. Bioelectromagnetics 30:631–640, 2009. � 2009 Wiley-Liss, Inc.

Key words: ELF-EMF; rat; hippocampus; neocortex; slices

INTRODUCTION

Electromagnetic fields (EMFs) have recently beena focus of scientific interest, as they are present ineveryday life and may have an influence on livingorganisms. The effects of low-frequency (non-ionising)radiation are rather controversial. Certain EMFs havetherapeutic applications, for example, transcranialmagnetic stimulation is utilised for the treatment ofepilepsy [Joo et al., 2007] and the relief of pain[Summers et al., 2004]. It is also a powerful techniquefor non-invasive brain imaging investigations [Walshand Cowey, 2000]. Extremely low-frequency electro-magnetic field (ELF-EMF) application has beenproposed for the treatment of osteoporosis and bonefractures [Otter et al., 1998; Sert et al., 2002]. It also hasa chondroprotective effect in osteoarthritis [Fini et al.,2008]. On the other hand, ELF-EMFs are presumed tohave adverse effects also. Such radiation is generated byelectrical devices, power lines and transformer stations,with a frequency of 50 or 60 Hz, and individuals may

be occupationally or residentially exposed to it intheir everyday life. Reference levels for exposure in theEuropean Union are prescribed as 100mT at homefor 24 h and 500mT at work for 8 h [InternationalCommission on Non-Ionizing Radiation Protection(ICNIRP), 1998]. Epidemiological studies suggest a

92009Wiley-Liss, Inc.

——————Grant sponsors: OTKA grants to G.B. (T047170 and K 76880);OMFB grant to I.V. (01609/2006); KVVM grant to P.V. (K 324/2008).

*Correspondence to: Ildiko Vilagi, Department of Physiology andNeurobiology, Eotvos Lorand University, Pazmany Peter setany1/C, Budapest H-1117, Hungary. E-mail: vilagildi@ludens.elte.hu

Received for review 7 February 2008; Final revision received22 April 2008

DOI 10.1002/bem.20517Published online 1 July 2009 in Wiley InterScience(www.interscience.wiley.com).

connection between ELF-EMF exposure and certaindiseases. Such exposure may play a role in thedevelopment of childhood leukaemia [Li et al., 1998]or in the progress of Alzheimer’s disease [Harmanciet al., 2003]. ELF-EMF exposure may stimulatethe secretion of b-amyloid peptide in exposed culturedneuroglioma cells [Del Giudice et al., 2007]. It also hasbeen shown that ELF-EMFs may promote the develop-ment of breast cancer in female rats [Loscher andMevissen, 1995]. However, these results might becontroversial, and highly dependent on species andstrains investigated [Anderson et al., 1999; Fedrowitzet al., 2004]. The above-mentioned disorders may beclosely connected with the enhanced production of freeradicals [Jelenkovic et al., 2006].

Subjects exposed to ELF-EMF often complain ofheadaches, nausea, paraesthesias and other non-specifichealth problems [Bergdahl, 1995; Frick et al., 2002;Stenberg et al., 2002; Roosli et al., 2004; Rubinet al., 2005]. In the background there might be thehigh vulnerability of the central nervous system to theinfluences of electric and magnetic fields, due to itshigh electrical sensitivity and capacity of integration[Saunders and Jefferys, 2007]. Behavioural studies onrats and mice indicated reductions in locomotor activityand anxiety behaviour [Choleris et al., 2001], and animpaired spatial learning capacity [Lai et al., 1998;Sienkiewicz et al., 1998]. On the other hand, somestudies reported the improvement of spatial memoryacquisition [Kavaliers et al., 1996; Liu et al., 2008].Some results could not be replicated when attempt-ed [Stern et al., 1996]. Thus, reported behaviouraleffects are sometimes contradictory, in most cases mild,transient and reversible [Sienkiewicz et al., 1996, 1998;Choleris et al., 2001; Prolic et al., 2005].

Although there is a considerable body of evidencethat weak magnetic fields have neuronal effects, theresults are still controversial [Valberg et al., 1997].The comparison and interpretation of the outcome ofdifferent exposure protocols and experimental techni-ques are rather difficult. The aim of our present studywas to determine the effects of different types of ELF-EMF exposure on synaptic functions in the brain usingdifferent treatments and the same recording technique.In one part of the experiment, the whole body of livingrats was exposed to an ELF-EMF and brain slicesprepared from the previously treated animals wereinvestigated. In the other part of the investigation, brainslices were directly exposed to ELF-EMF following thedissection. The EMF intensities (250–500 mT, 50 Hz)were chosen on the basis of the literature, according tothe reference level for occupational exposure [ICNIRP,1998]. Experiments were performed on the neocorticaland hippocampal area, and alterations in basic synaptic

functions, synaptic plasticity and seizure sensitivitywere analysed.

MATERIALS AND METHODS

The experiments were performed on young adult,male Wistar rats (100–200 g, Charles River, Budapest,Hungary). The experimental design was approved byEotvos Lorand University Animal Care Committeeand by the Hungarian National Animal Health CareAuthority. The rats were kept under a constant 12-hlight/dark cycle at controlled temperature (22� 2 8C).Standard pellet food and tap water were available adlibitum.

Treatment of Animals and Slices

In the present series of investigations, differenttypes of ELF-EMF treatments were applied. Formodelling exposure of human beings in their home,whole-body (WhB) exposure of rats (50 Hz, 500mT)was applied for a single 15 h period between 18.00and 9.00 h (WhB15 group), whereas direct effect of theELF-EMF field on the neuronal activity was studiedin brain slices exposed to a 250–320mT EMF for 1 himmediately preceding the recording session (Slicegroup). To study the long-term effect of a whole-bodyexposure, rats were exposed to a 500mT EMF throughfour consecutive days between 18.00 and 9.00 h, for atotal of 60 h (WhB60 group).

For the WhB irradiation, the animals were placedinto a standard Helmholtz-coil apparatus in a plasticcage. The device consisted of two solenoids (diameter:42 cm) placed on a common axis spaced apart at adistance equal to their radii (21 cm) with equal currentsflowing in the same direction (Fig. 1A). Helmholtz coilsproduced a vertical and homogenous field in the spacebetween the coils (Fig. 1C). The coils were constructedof glaze-insulated copper wire (d¼ 1.4 mm) andhad 240 turns (DC resistance was 2.9O). Fifty hertzEMF frequency was generated by sinusoidal current(1.6 A in each coil) at the output of the circuit, driven bya 230 V, 180 VA adjustable thoroid-transformer. EMFwas measured by a hand-held Electric and MagneticField Meter (Maschek-ESM-100, Bad Worishofen,Germany) and the value of the EMF was fixed at500� 25 mT. The electric gradient was between 525 and575 V/m. The ambient background level of the EMFwas <10 mT.

For the WhB irradiation, after 3 days of habitua-tion to the laboratory environment, the animals were putin either an EMF exposure apparatus or in a similardevice without EMF (control). Subjects were placed inpairs into the centre of the Helmholtz coils in opaqueplastic boxes (35 cm� 35 cm� 17 cm (height)) with a

632 Varro¤ et al.

Bioelectromagnetics

perforated plexiglass cover and wood chip bedding.Rats were removed from their boxes on a daily basis forcleaning the boxes and replacing food and water.Experiments were carried out at an ambient roomtemperature (24� 0.5 8C) and no significant tem-perature change was detected between the twoactivated Helmholtz coils. Rats of WhB60 group(n¼ 11) were sacrificed 3–10 days after the end ofthe exposure, while recordings on the WhB15 group(n¼ 10) were performed immediately following theexposure. Control slices came from sham-exposedanimals (n¼ 10).

For the ex vivo slice treatment, a glass dish(diameter: 9 cm) on a plastic tray containing brain slicesfrom untreated rats (n¼ 11) was placed for 1 h on thetop of two ring-shaped solenoids tightly bound ona common axis with equal currents flowing in thesame direction (Fig. 1B). The coils (diameter: 12 cm)were constructed of glaze-insulated copper wire(d¼ 1.4 mm) and had 35 turns. The EMF producedby this device was not homogenous; it ranged between

250 and 320 mT on the level of the glass dish containingthe brain slices (approximately 0.5 cm above theplane of the upper coil) (Fig. 1D,E). The slices werepositioned above the centre of the coils, parallel withthe plane of the coils. During the procedure thesolution was continuously bubbled with carbogen(5% CO2–95% O2). Recording started about 30 minfollowing the 1-h incubation/exposure period.

Electrophysiological Recording on Slices

For the electrophysiological recording, rats weredecapitated under deep chloral-hydrate anaesthesia, thebrain was quickly removed and coronal slices (400mmthick) containing the somatosensory cortex and thehippocampus were cut with a vibratome [Gyori et al.,2007]. Slices were incubated at room temperature for1 h in oxygenated artificial cerebrospinal fluid (ACSF)buffered with HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid and its sodium salt, pH 7.1–7.2), the composition of which was (in mM): 120 NaCl;2 KCl; 1.25 KH2PO4; 2 MgSO4; 20 NaHCO3; 2 CaCl2;

Fig. 1. Details of equipment used for irradiation. Standard Helmholtz-coil apparatus for theWhBexposure (A). In the Helmholtz-coil apparatus the fields from the two coils add up to create a netfield that is homogenous in the space between the coils (C). Equipment for the ex vivo brain slicetreatment: ring-shapedsolenoidswith theglassdish containingbrain slices (B).Changeofelectro-magnetic induction along the horizontal top plane of the ring-shaped solenoids. The axis of thesolenoids is in the centre of themeasuring-area (D).Decrease of electromagnetic induction alongtheaxisof thesolenoidsmovingupward fromtheplaneoftheuppersolenoid (E).

ELF-EMFEffects on Rat Brain Slices 633

Bioelectromagnetics

10 glucose; 6.7 HEPES–acid; 3.3 HEPES–Na. Duringex vivo EMF exposition, the slices were kept in thesame type of oxygenated HEPES-buffered ACSFsolution. After the incubation period, a slice wasplaced into an interface type recording chamber (FST,North Vancouver, Canada), through which standardACSF was perfused (2.5 ml/min). The solution wassaturated with carbogen at 33� 1 8C. The compositionof this perfusion solution was (in mM): 126 NaCl;26 NaHCO3; 1.8 KCl; 1.25 KH2PO4; 1.3 MgSO4;2.4 CaCl2; 10 glucose (pH: 7.4).

Evoked field potentials and population spikeswere recorded with extracellular glass microelectrodes(5–10 MO) filled with 1 M NaCl. In the case of theneocortical slices, the recording electrodes werepositioned in the lower part of layer III of the neocortexand bipolar tungsten stimulation electrodes werepositioned immediately below the recording electrodesat the border of the white and grey matter. The durationof the square voltage pulses was 100ms. For thehippocampal slices, the stimulation electrodes wereplaced at the Schaffer collaterals and the recordingelectrodes into the stratum pyramidale of the CA1region. Signals were amplified (Bioamp, Supertech,Pecs, Hungary), A/D converted and recorded withthe SPEL Advanced Intrasys computer program(Experimetria, Budapest, Hungary).

Before the whole procedure, the viability ofthe slices was tested. With the application of singleshock stimulation, the characteristic field responsewas recorded. If the peak-to-peak amplitude of themaximum evoked response was smaller than 1 mV incortical slices, the slice was excluded from the experi-ments. In the hippocampus, the appearance of thepopulation spike was tested. Basic synaptic functionswere tested by determining the voltage thresholdof the evoked field potential (VT), and a stimulusstrength-evoked response amplitude (input–output,I-O) curve was then recorded by gradually increasingthe stimulus intensity from VT to 3VT, in eight steps. Totest short-term plasticity, paired-pulse stimulation wasapplied, using interstimulus intervals of 500, 200, 100,and 50 ms at a stimulus intensity of 2VT. The exact slicenumbers are presented in the tables and figure legends.Altogether 55 neocortical and 31 hippocampal sliceswere tested, and some of the cortical slices were usedfor seizure susceptibility estimation (see below). Inthe remaining slices, initial I-O curve determinationand short-term plasticity test was followed by theinduction of long-term potentiation (LTP) by repetitivestimulation at a stimulus intensity of 2VT, in orderto investigate long-term synaptic plasticity. Thestimulation parameters were as follows: frequency—100 Hz, duration—5 s, four times with 10 s breaks

[Vilagi et al., 2005]. Subsequently, test stimulation wascarried out with 0.1 Hz during 30 min at 2VT. Finally, anI-O curve was again determined.

Seizure susceptibility was analysed in neocorticalslices: the normal perfusion solution was changed toMg2þ-free Ringer-solution (MFR) for 1 h. Thirty and60 min after the solution was switched to MFR, theevoked responses were tested at a stimulus intensity of2VT. Perfusion of Mg2þ-free solution results in theremoval of the Mg2þ from the NMDA-type glutamatereceptor channel, which normally is blocked by thision at resting level, and hence allows its activation[Valenzuela and Benardo, 1995]. The followingdepolarisation contributes to spontaneous seizureevents and also leads to the increase in amplitude andduration of the evoked responses. Spontaneous eventswere registered on a paper chart recorder, and thelatency of the first seizure and the number of seizures inthe second 30 min were determined.

Stored signals were analysed with the SPELAdvanced Intrasys computer program. The peak-to-peak amplitude of the early component of evoked fieldresponses (EPSP) in the neocortex and the populationspike amplitude in the hippocampus were evaluated. Inthe hippocampal slices, the initial slope of the EPSP wasalso measured and its relation to the population spikeamplitude was analysed (E-S curve). The EPSP slope isproportionate to the amount of excitatory synapticinput to the pyramidal cells, while the populationspike amplitude characterises their probability of actionpotential generation, namely, the postsynaptic neuronalexcitability [Wheal et al., 1998].

To compare the control and treated groups, alldata were tested for normal distribution with theKolmogorov–Smirnov test, then Student’s t-testfor independent variables (P< 0.05) was used forstatistical analysis to estimate the differences betweencontrol and each exposed group. Data are presented asmeans� SEM.

RESULTS

Basic Synaptic Functions

Each experimental group afforded resultsobtained on 7–9 slices. The stimulus thresholdsnecessary to evoke field responses were similar in allgroups: they were between 2 and 2.3 V both in thehippocampus and in the neocortex. The amplitudes ofthe population spikes recorded in the hippocampalslices at different stimulus intensities were similar inthe control and the WhB60 and WhB15 slices, while in theSlice group a marked decrease was observed, and thealteration was significant (Fig. 2A). A similar tendency

634 Varro¤ et al.

Bioelectromagnetics

in the effect was observed in the early component ofevoked field potentials recorded in neocortex slices(Fig. 2B). In contrast with the Slice treatment, WhBexposure did not have a significant effect on theamplitudes of evoked responses. The amplitudes for astimulation intensity of 2VT are presented in Table I

(asterisk at the hippocampal Slice group indicates asignificant difference with respect to the control).

In hippocampal slices, the initial slope of theEPSP was also determined. In WhB60 and WhB15 slices,the EPSP slope was significantly steeper than in controlslices. The Slice group did not differ significantly from

Fig. 2. Stimulus intensity-evoked response curves demonstrate ELF-EMF-induced changes inbasic synaptic functionsof the hippocampus (A) and the neocortex (B). Initial inserts showoriginalsample tracesofan EPSP, together with thepopulation spikepotential inahippocampalsliceandatypical fieldpotentialinaneocorticalsliceevokedat astimulusintensityof 2VT. [VT isthestimulationvoltage threshold of the evokedresponse] [Calibration:1mV,5ms (A),0.5 mV,5ms (B)].Thegraphsdepict the changes in amplitude of the population spike amplitude in hippocampal slices (controln¼ 9,WhB60 n¼ 7,WhB15 n¼ 8, Slice n¼ 7) and the field potentials in cortical slices (controln¼13,WhB60 n¼14,WhB15 n¼14, Slice n¼14) as functions of gradually increasing stimulation intensity.Direct ex vivo brain slice exposure to an extremely low-frequency electromagnetic field causedreductions in amplitude of the evoked potentials both in the hippocampus (A) and in the neocortex(B) [*denotessignificantdifferencesatalevelofP< 0.05comparedtotheuntreatedcontrol.Dataarepresentedasmeans�SEM].

TABLE I. Amplitude of Responses Evoked With 2VT Stimulation Intensity in the Slices Treated in Different Ways WithELF-EMF

Control group WhB60 group WhB15 group Ex vivo group

Amplitude of the early component of the evoked responses (mV)Hippocampus 1.65� 0.14 (n¼ 9) 1.79� 0.26 (n� 7) 1.72� 0.24 (n¼ 8) 1.08� 0.15* (n¼ 7)Neocortex 1.06� 0.09 (n¼ 13) 1.07� 0.12 (n¼ 14) 1.12� 0.07 (n¼ 14) 0.816� 0.09 (n¼ 14)

*Significant difference between tretaed and control groups ( p < 0.05).

ELF-EMFEffects on Rat Brain Slices 635

Bioelectromagnetics

the control (Fig. 3). The analysis of the E-S relationshipdid not reveal any marked differences between thegroups (Fig. 4).

Synaptic Plasticity

Short-term synaptic plasticity was examined bymeans of paired-pulse stimulation. In hippocampalslices, typically paired-pulse facilitation (PPF) developsat an interstimulus interval of 50 ms in control cases.In the WhB15 group a significant enhancement of thePPF was observed. In neocortical slices, typically paired-pulse depression (PPD) is detectable in control cases,especially at an interstimulus interval of 50 ms. Neithertype of ELF-EMF exposure had any significant effect onthe cortical short-term synaptic plasticity phenomena(Fig. 5A).

To test long-term synaptic plasticity, LTP wasinduced by repetitive high-frequency stimulationwith an intensity of 2VT. In the hippocampal slices, ahigh increase in amplitude was detected 30 min afterhigh-frequency stimulation: the enhancement at 2VT

stimulation intensity was between 44.8% (control) and86.7% (WhB15). The enhancement was significantlyhigher in the WhB15 group than in the control group. Inthe neocortical slices, the increase in amplitude of theresponses evoked by a stimulation intensity of 2VT wasbetween 16.5% (WhB15) and 31.8% (Slice) (in theControl group, the increase was 26.7%). However, there

were no significant differences between the differentgroups (Fig. 5B).

Seizure Susceptibility

As a consequence of perfusion with Mg2þ-free Ringer-solution, spontaneous seizure-like activitydevelops in neocortical slices. In the Slice group, ELF-EMF exposure inhibited the development of seizure-like activity, but in the WhB groups it was promoted(Table II). In the Slice group, the number of slicesdisplaying seizures and the mean number of seizuresduring the second 30 min of the treatment was lowerthan that in the control group. On the contrary, inthe WhB groups these values were higher than in thecontrol group. Seizure susceptibility changes were alsoobserved in examinations of the latencies of the firstspontaneous event of any type and the first seizure. Inthe WhB15 group, the latencies of appearance of the firstdischarges were shorter than those in the control group.Regarding the evoked potentials measured in MFR,WhB treatments seemed to promote the appearance ofa second component, although the amplitudes of bothcomponents were smaller than those for the controlslices (data not shown). None of the above-mentioneddifferences were statistically significant.

DISCUSSION

This ex vivo study revealed that exposure to a low-intensity ELF-EMF (250–500mT, 50 Hz) has markedeffects on basic neuronal functions that can be detectedin brain slice preparations. Depending on the treatmenttype (ex vivo exposure of slices or whole-body

Fig. 3. The slope of the EPSP measured in hippocampal slices(control n¼ 9,WhB60 n¼ 7,WhB15 n¼ 8, Slice n¼ 7) was plottedagainst the stimulation intensity to characterise the excitatoryinput of CA1 pyramidal cells. The EPSP slope was significantlysteeper in theWhB15 and theWhB60 group than in the control.TheSlice group did not differ significantly from the control [* denotessignificant differences at a level of P< 0.05 compared to theuntreatedcontrol.Dataarepresentedasmeans�SEM].

Fig. 4. E-S relationship analysis of hippocampal (control n¼ 9,WhB60 n¼ 7,WhB15 n¼ 8, Slicen¼ 7) evokedresponseswasper-formedbyplottingtheriseoftheEPSPinitialslopeagainst thepop-ulation spike amplitude. The analysis did not reveal any markeddifferencesbetweenthegroups.

636 Varro¤ et al.

Bioelectromagnetics

exposure), the alterations developed toward oppositedirections. Inhibition of neuronal activity was chieflydetected following direct slice ELF-EMF exposure, butfollowing the whole-body treatments, synaptic activitywas pushed toward excitation.

The molecular basis of ELF-EMF effects maybe explained in terms of Hall-like forces, whichcan generate interactions between cations and their

voltage-gated ion channels. Such interactions arepresumed to produce conformational changes in theproteins and modify the ion flow across the channels[Balcavage et al., 1996]. However, only cation channelsperpendicular to the applied field can be influenced inthis way. We can postulate that the more pronouncedeffect of Slice treatment is caused by the fact that thedissected slices were practically immobile during

Fig. 5. ELF-EMF-induced changes in short-term (A) and long-term (B) synaptic plasticity weretested bymeans of paired-pulse stimulation and repetitive stimulation to induce LTP, respectively.A: Theamplituderatiosofthesecondversusthefirstevokedresponseareshownataninterstimulusinterval of 50ms. In the hippocampal slices (control n¼ 9,WhB60 n¼ 7,WhB15 n¼ 8, Slice n¼ 7),paired-pulse facilitation is characteristic; thiswassignificantlyenhanceddirectlyafter whole-bodyexposure, in theWhB15 group. In the neocortical slices (control n¼13,WhB60 n¼14,WhB15 n¼14,Slice n¼14), a paired-pulse depression is characteristic; this was not changed following electro-magnetic field exposure. B: The long-term change in synaptic efficacy is characterised by theamplitude ratios of the responses evoked before and 30min after the repetitive stimulation at anintensity of 2VT. In the hippocampal slices (control n¼ 9,WhB60 n¼ 7,WhB15 n¼ 8, Slice n¼ 7),whole-body exposure enhanced the efficacy of LTP induction, especially in the WhB15 group. Inthe neocortical slices (control n¼ 9,WhB60 n¼ 9,WhB15 n¼ 9, Slice n¼ 9), however, there was nodifferencebetweenthegroupsinthe increaseofevokedpotentialamplitude [* denotesasignificantdifferenceat alevelofP< 0.05.Dataarepresentedasmeans�SEM].

TABLE II. Effect of ELF-EMF on Seizure Susceptibility in Neocortical Slices: 1 h Perfusion With Magnesium-FreeRinger-Solution (MFR)

Control (n¼ 8) WhB60 (n¼ 8) WhB15 (n¼ 8) Slice (n¼ 8)

Number of slices displaying seizures 4 4 8 2Mean number of seizures 2.125� 1.27 9.125� 5.34 11.75� 4.04 0.25� 0.16Mean latency of the first seizure (min) 29.75� 6.84 25.25� 6.79 21.29� 3.16 26.5� 3.50Mean latency of the first activity (min) 25.125� 3.81 25.875� 3.99 16.037� 3.89 24.125� 5.18Appearance of a 2-day component after30 min (number of slices)

3 3 5 1

Appearance of a 2-day component after60 min (number of slices)

3 7 6 4

ELF-EMFEffects on Rat Brain Slices 637

Bioelectromagnetics

exposure, while in exposed freely moving rats, the ionchannels perpendicular to the field are continuallychanging.

Neuronal activity is greatly influenced by thefunctions of voltage-gated sodium and calciumchannels. Tetrodotoxin-sensitive voltage-gated sodiumchannels have been shown to mediate the activation ofneurons during repetitive transcranial magnetic stim-ulation [Hausmann et al., 2001].

Besides these non-synaptic effects of EMFs whichmay modulate the membrane potential of neurons[Jefferys, 1995], numerous studies have reported thatlow-frequency EMF exposure results in an increase inthe intracellular Ca2þ concentration due to increasedCa2þ influx through voltage-gated Ca2þ channels. Thiseffect has been demonstrated, for example, in ratpituitary cells [Barbier et al., 1996], hippocampus[Manikonda et al., 2007] and dorsal root ganglion cells[Marchionni et al., 2006]. The immediate consequencesare frequently excitatory changes, which may bedemonstrated by c-fos activation [Hausmann et al.,2001]. However, an increased intracellular Ca2þ

concentration alters the Ca2þ-related signalling proc-esses and may lead in the long run, for example, todecreased activity of the NMDA receptors [Manikondaet al., 2007] or alter other Ca2þ-dependent regulatorysignals. It may also activate Ca2þ-dependent Kþ

channels, which reverse the early excitatory effect ofa high intracellular Ca2þ concentration [Marchionniet al., 2006].

Besides effects on voltage gated cation channels-,Ca2þ- and NMDA receptor-dependent processes, theGABAergic system may also be involved in neuronalchanges induced by ELF-EMFs. ELF-EMF exposureleads to a decreased sensitivity in bicuculline-inducedseizures in mice [Sung et al., 2003]. It also increasesclonidine-induced sleep time in chicks and this effectcan be inhibited with GABAA receptor antagonists[Min et al., 2001].

In our experiments, following ex vivo exposure for1 h, we observed significant decreases in the amplitudesof the evoked population spikes in the hippocampus,while no difference was revealed in the E-S relationshipanalysis. An activity decrease could also be seen in theneocortex, though this alteration was not significant. Theeffect of paired-pulse stimulation and LTP inductionwere not affected by this treatment. On the other hand,ELF-EMF exposure of the slices effectively inhibited thedevelopment of spontaneous seizure episodes in Mg2þ-free solution. The reduced occurrence of these phenom-ena in the case of the slices treated ex vivo suggests adecreased activity of the NMDA receptor-dependent orenhanced Kþ channel or GABA receptor-dependentprocesses.

WhB exposure had rather excitatory effects. In thehippocampal slices from the WhB15-treated animals, atendency of increment of the evoked potentials wascharacteristic, but compared to the control slices, thedifference was not significant. In both WhB groups, asignificant increase in the EPSP slope was observed. Itcan be concluded that in these treatment groups, theCA1 pyramidal cells received a stronger excitatory (orweaker inhibitory) input, which may be responsible forthe tendency of increased population spike amplitude.PPF was also enhanced after WhB15 treatment in thehippocampal slices, which may be due to a transientincrease in the probability of transmitter release fromthe presynaptic terminals after synaptic activation. Thisis caused mainly by a residual increase in presynapticintracellular Ca2þ concentration [Castro-Alamancosand Connors, 1997], and the enhancement in PPF ratiocan therefore be explained by Ca2þ-mediated changesin synaptic efficacy. The increases of the evokedpotentials after LTP induction were also enhanced inthe hippocampal slices originating from the WhB15-treated animals. There are a number of pre- andpostsynaptic phenomena that are presumed to underliethe development of LTP. Among others, the activationof NMDA receptors and voltage-gated Ca2þ-channelsplays a crucial role and contributes to an increasedpostsynaptic intracellular Ca2þ level [Malenka, 1995].Regarding the spontaneous activity in MFR, WhB15

treatment had a tendency to increase the occurrence ofseizures, decrease their latency time and promote thedevelopment of a second component of the evokedresponse. These findings likewise indicate the activa-tion of NMDA receptors. The effect may be temporary,as the results obtained with WhB60 treatment were lessexplicit, presumably because of the relatively longand various delays between the end of treatment andthe experimental recording. We did not observe anysignificant difference between WhB60 and controlgroups concerning synaptic transmission or plasticity,or seizure susceptibility.

Although the results of different exposure proto-cols in different test-conditions are difficult to compare,we can conclude that ELF-EMF exposure may resultin a biphasic effect: first an excitatory effect due tothe elevation of intracellular Ca2þ signals, and then aninhibitory effect due to compensatory mechanisms.Both kinds of changes can therefore occur afterdifferent exposure types depending on time andintensity.

Our results suggest that ELF-EMFs have animportant influence on the basic synaptic activity intwo principal brain regions: the hippocampus and theneocortex. Changes observed in basic neuronal activityand susceptibility support the results of other studies

638 Varro¤ et al.

Bioelectromagnetics

which found some effects on a more complex level, likethe behavioural studies or spatial learning experiments[Rudolph et al., 1985; Trzeciak et al., 1993; Smith et al.,1994; Lai et al., 1998; Sienkiewicz et al., 1998; Cobbet al., 2000; Choleris et al., 2001; Del Seppia et al.,2003]. The effects observed on the basic synaptic levelcall for further exploration of the potential health hazardrepresented by ELF-EMF exposure.

On the basis of our experiments we can concludethat ELF-EMF exposure exerts significant effects onthe synaptic activity of different cortical areas, althoughthe alterations are usually transient. However, theevaluation revealed that neuronal effects on synapticlevel may depend on the specific spatial parameters andconstancy of EMF. The effect of the treatment may alsorely on the synaptic structure and neuronal network ofthe affected region. The overall changes may depend onthe connectivity between the affected areas.

REFERENCES

Anderson LE, Boorman GA, Morris JE, Sasser LB, Mann PC,Grumbein SR, Hailey JR, McNally A, Sills RC, HasemannJK. 1999. Effect of 13 week magnetic exposures on DMBA-initiated mammary gland carcinomas in female Sprague-Dawley rats. Carcinogenesis 20:1615–1620.

Balcavage WX, Alvager T, Swez J, Goff CW, Fox MT, AbdullyavaS, King MW. 1996. A mechanism for action of extremely lowfrequency electromagnetic fields on biological systems.Biochem Biophys Res Commun 222:374–378.

Barbier E, Dufy B, Veyret B. 1996. Stimulation of Ca2þ influx in ratpituitary cells under exposure to a 50 Hz magnetic field.Bioelectromagnetics 17:303–311.

Bergdahl J. 1995. Psychologic aspects of patients with symptomspresumed to be caused by electricity or visual display units.Acta Odontol Scand 53:304–310.

Castro-Alamancos MA, Connors BW. 1997. Distinct forms of short-term plasticity at excitatory synapses of hippocampus andneocortex. Proc Natl Acad Sci USA 94:4161–4166.

Choleris E, Thomas AW, Kavaliers M, Prato FS. 2001. A detailedethological analysis of the mouse open field test: Effects ofdiazepam, chlordiazepoxide and an extremely low frequencypulsed magnetic field. Neurosci Biobehav Rev 25:235–260.

Cobb BL, Jauchem JR, Mason PA, Dooley MP, Miller SA, ZiriaxJM, Murphy MR. 2000. Neural and behavioral teratologicalevaluation of rats exposed to ultra-wideband electromagneticfields. Bioelectromagnetics 21:524–537.

Del Giudice E, Facchinetti F, Nofrate V, Boccaccio P, Minelli T,Dam M, Leon A, Moschini G. 2007. Fifty Hertz electro-magnetic field exposure stimulates secretion of beta-amyloidpeptide in cultured human neuroglioma. Neurosci Lett 418:9–12.

Del Seppia C, Mezzasalma L, Choleris E, Luschi P, Ghione S. 2003.Effects of magnetic field exposure on open field behaviourand nociceptive responses in mice. Behav Brain Res 144:1–9.

Fedrowitz M, Kamino K, Loscher W. 2004. Significant differencesin the effects of magnetic field exposure on 7,12-Dimethyl-

benz(a)anthracene-induced mammary carcinogenesis intwo substrains of Sprague-Dawley rats. Cancer Res 64:243–251.

Fini M, Torricelli P, Giavaresi G, Aldini NN, Cavani F, Setti S,Nicolini A, Carpi A, Giardino R. 2008. Effect of pulsedelectromagnetic field stimulation on knee cartilage, sub-chondral and epyphiseal trabecular bone of aged DunkinHartley guinea pigs. Biomed Pharmacother 62:709–715.

Frick U, Rehm J, Eichhammer P. 2002. Risk perception, somatiza-tion, and self report of complaints related to electromagneticfields—A randomized survey study. Int J Hyg Environ Health205:353–360.

Gyori J, Varro P, Zielinska E, Banczerowski-Pelyhe I, Vilagi I. 2007.Bensultap decreases neuronal excitability in molluscan andmammalian central nervous system. Toxicol In Vitro 21:1050–1057.

Harmanci H, Emre M, Gurvit H, Bilgic B, Hanagasi H, Gurol E,Sahin H, Tinaz S. 2003. Risk factors for Alzheimer disease: Apopulation-based case-control study in Istanbul, Turkey.Alzheimer Dis Assoc Disord 17:139–145.

Hausmann A, Marksteiner J, Hinterhuber H, Humpel C. 2001.Magnetic stimulation induces neuronal c-fos via tetrodo-toxin-sensitive sodium channels in organotypic cortex brainslices of the rat. Neurosci Lett 310:105–108.

International Commission on Non-Ionizing Radiation Protection.1998. Guidelines for limiting exposure to time-varyingelectric, magnetic, and electromagnetic fields (up to300 GHz). Health Phys 74:494–522.

Jefferys JG. 1995. Nonsynaptic modulation of neuronal activity inthe brain: Electric currents and extracellular ions. PhysiolRev 75:689–723.

Jelenkovic A, Janac B, Pesic V, Jovanovic DM, Vasiljevic I, Prolic Z.2006. Effects of extremely low-frequency magnetic field inthe brain of rats. Brain Res Bull 68:355–360.

Joo EY, Han SJ, Chung SH, Cho JW, Seo DW, Hong SB. 2007.Antiepileptic effects of low-frequency repetitive transcranialmagnetic stimulation by different stimulation durations andlocations. Clin Neurophysiol 118:702–708.

Kavaliers M, Ossenkopp KP, Prato FS, Innes DG, Galea LA,Kinsella DM, Perrot-Sinal TS. 1996. Spatial learning in deermice: Sex differences and the effects of endogenous opioidsand 60 Hz magnetic fields. J Comp Physiol [A] 179:715–724.

Lai H, Carino MA, Ushijima I. 1998. Acute exposure to a 60 Hzmagnetic field affects rats’ water-maze performance. Bio-electromagnetics 19:117–122.

Li CY, Lee WC, Lin RS. 1998. Risk of leukemia in children livingnear high-voltage transmission lines. J Occup Environ Med40:144–147.

Liu T, Wang S, He L, Ye K. 2008. Chronic exposure to low-intensitymagnetic field improves acquisition and maintenance ofmemory. Neuroreport 19:549–552.

Loscher W, Mevissen M. 1995. Linear relationship between fluxdensity and tumor co-promoting effect of prolongedmagnetic field exposure in a breast cancer model. CancerLett 96:175–180.

Malenka RC. 1995. Synaptic plasticity in hippocampus andneocortex: A comparison. In: Gutnick MJ, Mody I, editors.The cortical neuron. New York: Oxford University Press.pp 98–108.

Manikonda PK, Rajendra P, Devendranath D, Gunasekaran B,Channakeshava, Aradhya RS, Sashidhar RB, SubramanyamC. 2007. Influence of extremely low frequency magneticfields on Ca2þ signaling and NMDA receptor functions in rathippocampus. Neurosci Lett 413:145–149.

ELF-EMFEffects on Rat Brain Slices 639

Bioelectromagnetics

Marchionni I, Paffi A, Pellegrino M, Liberti M, Apollonio F, AbetiR, Fontana F, D’Inzeo G, Mazzanti M. 2006. Comparisonbetween low-level 50 Hz and 900 MHz electromagneticstimulation on single channel ionic currents and on firingfrequency in dorsal root ganglion isolated neurons. BiochimBiophys Acta 1758:597–605.

Min YS, Jeong JH, Choi YM, Lee BC, Huh IH, Lee SY, Sohn UD.2001. The influences of extremely low frequency magneticfields on clonidine-induced sleep in 2-day-old chicks. JAutonPharmacol 21:197–203.

Otter MW, McLeod KJ, Rubin CT. 1998. Effects of electromagneticfields in experimental fracture repair. Clin Orthop Relat Res355(Suppl):S90–S104.

Prolic Z, Janac B, Pesic V, Jelenkovic A. 2005. The effect ofextremely low-frequency magnetic field on motor activityof rats in the open field. Ann NY Acad Sci 1048:381–384.

Roosli M, Moser M, Baldinini Y, Meier M, Braun-Fahrlander C.2004. Symptoms of ill health ascribed to electromagneticfield exposure—A questionnaire survey. Int J Hyg EnvironHealth 207:141–150.

Rubin GJ, Das MJ, Wessely S. 2005. Electromagnetic hyper-sensitivity: A systematic review of provocation studies.Psychosom Med 67:224–232.

Rudolph K, Krauchi K, Wirz-Justice A, Feer H. 1985. Weak 50-Hzelectromagnetic fields activate rat open field behavior.Physiol Behav 35:505–508.

Saunders RD, Jefferys JG. 2007. A neurobiological basis for ELFguidelines. Health Phys 92:596–603.

Sert C, Mustafa D, Duz MZ, Aksen F, Kaya A. 2002. The preventiveeffect on bone loss of 50-Hz, 1-mT electromagnetic fieldin ovariectomized rats. J Bone Miner Metab 20:345–349.

Sienkiewicz ZJ, Haylock RG, Saunders RD. 1996. Acute exposureto power-frequency magnetic fields has no effect on theacquisition of a spatial learning task by adult male mice.Bioelectromagnetics 17:180–186.

Sienkiewicz ZJ, Haylock RG, Saunders RD. 1998. Deficits in spatiallearning after exposure of mice to a 50 Hz magnetic field.Bioelectromagnetics 19:79–84.

Smith RF, Clarke RL, Justesen DR. 1994. Behavioral sensitivity ofrats to extremely-low-frequency magnetic fields. Bioelec-tromagnetics 15:411–426.

Stenberg B, Bergdahl J, Edvardsson B, Eriksson N, Linden G,Widman L. 2002. Medical and social prognosis for patientswith perceived hypersensitivity to electricity and skinsymptoms related to the use of visual display terminals.Scand J Work Environ Health 28:349–357.

Stern S, Laties VG, Nguyen QA, Cox C. 1996. Exposure tocombined static and 60 Hz magnetic fields: Failure toreplicate a reported behavioral effect. Bioelectromagnetics17:279–292.

Summers J, Johnson S, Pridmore S, Oberoi G. 2004. Changes to colddetection and pain thresholds following low and highfrequency transcranial magnetic stimulation of the motorcortex. Neurosci Lett 368:197–200.

Sung JH, Jeong JH, Kim JS, Choi TS, Park JH, Kang HY, Kim YS,Kim DS, Sohn UD. 2003. The influences of extremely lowfrequency magnetic fields on drug-induced convulsion inmouse. Arch Pharm Res 26:487–492.

Trzeciak HI, Grzesik J, Bortel M, Kuska R, Duda D, Michnik J,Malecki A. 1993. Behavioral effects of long-term exposure tomagnetic fields in rats. Bioelectromagnetics 14:287–297.

Valberg PA, Kavet R, Rafferty CN. 1997. Can low-level 50/60 Hzelectric and magnetic fields cause biological effects? RadiatRes 148:2–21.

Valenzuela V, Benardo LS. 1995. An in vitro model of persistentepileptiform activity in neocortex. Epilepsy Res 21:195–204.

Vilagi I, Bardos G, Denes K, Farkas B, Friedrich P. 2005.Enhancement of synaptic strength in the somatosensorycortex following nerve injury does not parallel behaviouralalterations. Brain Res Bull 64:463–469.

Walsh V, Cowey A. 2000. Transcranial magnetic stimulation andcognitive neuroscience. Nat Rev Neurosci 1:73–79.

Wheal HV, Bernard C, Chad JE, Cannon RC. 1998. Pro-epilepticchanges in synaptic function can be accompanied by pro-epileptic changes in neuronal excitability. Trends Neurosci21:167–174.

640 Varro¤ et al.

Bioelectromagnetics