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The action of pulse-modulated gsm radiation increases regional changes in brain activity and c-fos expression in cortical and subcortical areas in a rat model of picrotoxin-induced seizure pronenessJournal of Neuroscience Research 87:1484–1499 (2009) The Action of Pulse-Modulated GSMRadiation Increases Regional Changesin Brain Activity and c-Fos Expressionin Cortical and Subcortical Areas in aRat Model of Picrotoxin-InducedSeizure Proneness E. Lo´pez-Martı´n,1* J. Bregains,2 J.L. Relova-Quinteiro,3 C. Cadarso-Sua´rez,4F.J. Jorge-Barreiro,1 and F.J. Ares-Pena21Morphological Sciences Department, University of Santiago de Compostela, Santiago de Compostela,Spain 2Applied Physics Department, University of Santiago de Compostela, Santiago de Compostela, Spain 3Physiology Department, University of Santiago de Compostela, Santiago de Compostela, Spain 4Biostatistics Unit, University of Santiago de Compostela, Santiago de Compostela, Spain The action of the pulse-modulated GSM radiofrequency that occurs or is observed in relation to low energy, and of mobile phones has been suggested as a physical the results of previous studies have suggested that the phenomenon that might have biological effects on the action pulse-modulated RF ﬁelds such as those emitted mammalian central nervous system. In the present by mobile phones can alter nervous system physiology study, GSM-exposed picrotoxin-pretreated rats showed (Borbe´ly et al., 1999; Huber et al., 2000; Beason and differences in clinical and EEG signs, and in c-Fos Semm, 2002). The general mechanisms proposed for the expression in the brain, with respect to picrotoxin- interaction of pulse-modulated RF signals with the cen- treated rats exposed to an equivalent dose of unmodu- tral nervous system (CNS) are controversial, and include lated radiation. Neither radiation treatment caused tis- alteration of Ca21 efﬂux in vivo (Adey et al., 1982; sue heating, so thermal effects can be ruled out. The Paulraj et al., 1999) but no effect on rat brain tissues most marked effects of GSM radiation on c-Fos exposed in vitro (Shelton and Merritt, 1981; Merritt expression in picrotoxin-treated rats were observed in et al., 1982), low-level exposure-induced increases in limbic structures, olfactory cortex areas and subcortical blood–brain barrier (BBB) permeability and neuronal areas, the dentate gyrus, and the central lateral nucleus damage (Salford et al., 2003) but no changes in the vas- of the thalamic intralaminar nucleus group. Nonpicro- cular barrier (Finne et al., 2002, 2006), and an increase toxin-treated animals exposed to unmodulated radia- in regional cerebral blood ﬂow (Huber et al., 2005). In tion showed the highest levels of neuronal c-Fos this regard, both in vitro experiments (Beason and expression in cortical areas. These results suggest a Semm, 2002; Barteri et al., 2005; Zhao et al., 2007) and speciﬁc effect of the pulse modulation of GSM radiation in vivo experiments in animals (Dubreil et al., 2003; on brain activity of a picrotoxin-induced seizure- Paulraj and Behari, 2004; Lopez-Martı´n et al., 2006) and proneness rat model and indicate that this mobile-phone-type radiation might induce regional changes in Contract grant sponsor: Ministerio de Educacio´n y Ciencia and Secretarı´a Xeral de Investigacio´n e Desenvolvemento of the Xunta de Galicia; C 2008 Wiley-Liss, Inc.
Contract grant number: MTM 2005-00818; Contract grant number:PGIDIT02 BFT 20601 PR; Contract grant number: PGIDIT06 PXIC Key words: seizure; cortex; hippocampus; thalamus; *Correspondence to: Maria Elena Lopez-Martin, Morphological SciencesDepartment, Faculty of Medicine, University of Santiago de Compostela, Research involving evaluation of the cerebral activ- San Francisco s/n Santiago de Compostela La Corunha, 15782 Spain.
ity of humans and laboratory animals during and after E-mail: email@example.com exposure to radiofrequency (RF) radiation has provided Received 6 June 2008; Revised 28 September 2008; Accepted 29 results that do not appear to depend on thermal mecha- nisms. The principal argument for a nonthermal mecha- Published online 29 December 2008 in Wiley InterScience (www.
nism concerns identiﬁcation of an experimental effect interscience.wiley.com). DOI: 10.1002/jnr.21951 ' 2008 Wiley-Liss, Inc.
Pulse-Modulated GSM Radiation Increases c-Fos humans (Maby et al., 2006) have reported biological TABLE I. Description of the experimental groups.
effects after acute GSM-900 exposure. Many of the Picrotoxin-treated (PT) and nonirradiated (no-Rad) responses described have been interpreted as secondary PT and GSM-irradiated (GSM) to a variety of cellular signal transduction pathways and PT and irradiated with unmodulated signal (Unmod) gene expression responses (Belyaev et al., 2006), such as Nonpicrotoxin-treated (no-PT) and no-Rad heat shock responses (Cotgreave, 2005). On the other hand, other authors did not ﬁnd any genotoxic, behav- ioral, or morphological effects on the CNS in in vivo(Kumlin et al., 2007) or in vitro (Chauhan et al., 2006;Joubert et al., 2007) studies.
Pulse-modulated RF ﬁelds can alter electrophysio- gime were used in the study. For treatment with the convul- logical activity of the awake human brain (Eulitz et al., sive agent picrotoxin, rats were injected i.p. with 1 ml of a 1998; Borbe´ly et al., 1999; Hamblin and Wood, 2002; 0.5 mg/ml solution of picrotoxin (Sigma, St. Louis, MO) dis- Krause et al., 2004; Relova et al., 2005) as well as in solved in saline, 5 min before immobilization (see below).
sleep electroencephalograms (Borbe´ly et al., 1999; Huber This dose of picrotoxin has been shown to be subconvulsive et al., 2000, 2002, 2003). Cognitive functions may also (Nutt et al., 1982; Lo´pez-Martı´n et al., 2006).
be affected (Thuroczy et al., 1994; Koivisto et al., 2000;Eldesteyn and Oldershaw, 2002; Mainer et al., 2004).
Experimental Design However, some of these studies have not been replicated Rats were assigned to one of the following groups: by the authors, and there may be some doubts regarding group 1 rats were injected i.p. with picrotoxin and then im- the reliability of the results.
mobilized in methacrylate tubes for 2 hr without exposure to Pulse-modulated RF has been reported to modu- radiation (PT, no-RAD); group 2 rats were injected i.p. with late the effects of drugs (Lai et al., 1990, 1991, 1992a,b), picrotoxin and then immobilized in methacrylate tubes for for example, altering sensitivity to neurotropic drugs 2 hr with exposure to 900-MHz pulse-modulated GSM radia- (Lobanova, 1985) or modulating the action of psychoac- tion (PT, GSM); group 3 rats were injected i.p. with picro- tive drugs (Bruce-Wolfe and Justesen, 1985), and can toxin and then immobilized in methacrylate tubes for 2 hr also affect the function of some neurotransmitters (Gan- with exposure to 900-MHz nonmodulated radiation (PT, dhi and Ross, 1987; Lai et al., 1991; Inaba et al., 1992; UNMOD); and group 4, 5, and 6 rats were injected i.p. with Mausset-Bonnefont et al., 2004).
vehicle only, not picrotoxin, and then immobilized. Rats in The expression of c-Fos has previously been char- group 4 were not exposed to radiation, rats in group 5 were acterized as being elevated in response to various stress exposed to GSM radiation, and rats in group 6 were exposed or neurotoxic stimuli (Graybiel et al., 1990; LaHoste to nonmodulated radiation (no-PT, no-RAD; no-PT, GSM; et al., 1993; Herrera and Robertson, 1996), such as seiz- no-PT, UNMOD; Table I).
ures (Dragunow and Robertson, 1987; Gass et al., 1992)or physical (Morrissey et al., 1999) and nonpathogenic Surgical Procedures environmental conditions, and can be used as a sensitivemarker of neuronal activation. In a previous study, we Electroencephalographic activity was recorded in some found that rats pretreated with a subconvulsive dose of rats from these groups. These rats were anesthetized (30 mg/ the g-aminobutyric acid (GABA) antagonist picrotoxin kg pentobarbital I.P.) and attached to a stereotaxic frame in and then exposed to GSM radiation showed signiﬁcant order to be ﬁtted with stainless-steel screws as electrodes. The alterations in various indicators of brain activity, includ- active leads were placed over the frontal and occipital cortices ing clinical indicators, EEG indicators, and c-Fos levels about 7 mm from midline on both sides, with the reference in the brain (Lo´pez Martı´n et al., 2006). These effects electrode at the vertex. The screws were soldered to a micro- cannot be explained by heating, because the speciﬁc connector. Screws, wires, and connector were embedded to- absorption rate (SAR) was not sufﬁcient to cause bulk gether in acrylic cement. After 1 week, the rats underwent heating of tissue, which suggests that GSM radiation the procedure described below, and electroencephalographic may affect brain activity. The aim of the present study activity was recorded with a 32-channel electroencephalo- was 1) to assess whether the effects on brain activity of pulse-modulated GSM differ from those of nonmodu- LO-RO, LF-LO, and RF-RO, where F indicates frontal, O lated radiation of the same wavelength and, if so, 2) to occipital, L the left side, and R the right side. All experiments study c-Fos expression in seizure-related anatomical cir- were carried out in accordance with the research protocols established by the Animal Care Committee of the Universityof Santiago de Compostela.
MATERIALS AND METHODS Animals and Picrotoxin Treatment Five minutes after i.p. administration of 2 mg/kg picro- Adult male Sprague-Dawley rats weighing 230–250 g toxin (Sigma), with the dose adjusted to individual animal and housed with free access to food and water in individual weights (groups 2, 3, 5, and 6), or vehicle only (groups 1 and cages and maintained at 228C under a 12:12-hr light/dark re- 4), the animals were immobilized in methacrylate tubes, Journal of Neuroscience Research Lo´pez-Martı´n et al.
placed in a 150-346-370-cm radiation cage (incorporating a are commonly related to isolated spiking and were not consid- commercial transmitting antenna) that had previously been ered as seizures. Convulsive seizures were characterized by calibrated to allow measurement of the radiation absorbed by clonic jerks of limbs and the body, associated, to different the animal (see Schmid and Partner Engineering AG, 2005; degrees, with tonic ﬂexion or extension. They were in all Trastoy-Rios et al., 2006) and connected to an EEG recorder.
cases followed by full recovery.
Animals in groups 2, 3, 5, and 6 were then irradiated for 2 hrwith either 900-MHz pulse-modulated GSM (0.26 W) or 900- Processing of Brain Tissue and Immunohistochemistry MHz nonmodulated radiation (0.26 W): the power level was One hour after the rats were irradiated, they were given chosen to be somewhat lower than the average power radiated an overdose of pentobarbital and preﬁxed by transcardial per- by a GSM mobile phone. During irradiation, the animals fusion with physiological saline followed by formaldehyde in were videotaped to record clinical signs of seizure, and their phosphate buffer (pH 7.4). The animals were slaughtered, and EEGs were recorded. Animals were exposed to radiation for 2 the brains were immediately removed from the skull, placed hr; this period was chosen because it has been widely used in in fresh ﬁxative solution for 4 hr at 48C, and then transferred other studies (Fritze et al., 1997), thus facilitating comparison.
to phosphate-buffered saline (PBS) for 12 hr at the same tem- The animals were then removed from the cage and sedated perature, after which transverse 50-lm sections were cut with with pentobarbital for transfer to the histochemistry labora- a vibrotome, collected freely ﬂoating in PBS, and processed as tory. Using this exposure system, we estimated (for each tis- follows. The sections were pretreated for 1 hr with normal sue) the 1-g-averaged peak SAR, i.e., the peak speciﬁc rabbit serum and Triton X-100 [10% and 0.25%, respectively, absorbed radiation considering a volume of tissue weighing 1 in 0.02% potassium phosphate-buffered saline (KPBS)] and g, and mean SAR, i.e., the average speciﬁc absorption rate.
then exposed overnight at room temperature to polyclonal These indices were estimated with the aid of SEMCAD soft- sheep anti-Fos antibody (from Cambridge Research Biochem- ware (Schmid and Partner Engineering AG, Zurich, Switzer- icals; 1:1,000 in KPBS), rinsed with KPBS, incubated for 1 hr land), by numerical simulation of absorption by the brain of a with biotinylated rabbit anti-sheep antibody (from Vector, 198-g ‘‘phantom'' rat and then extrapolation to the rats used Burlingame, CA; 1:500 in KPBS), rinsed three times with in the experiments (Schmid and Partner Engineering AG, KPBS, incubated for 30 min with an avidin-biotin-peroxidase 2005; Trastoy-Rios et al., 2006).
complex prepared following the instructions of the manufac- The estimated SAR (SARE) for the experimental ani- turer (Dako, Glostrup, Denmark), and ﬁnally labelled with mals, obtained by adjusting the simulated values to the actual weights of the individual rats and the actual absorbed power, H2O2 solution (Dako).
can be written as Regions of Interest Fos immunoreactivity was observed in cell nuclei in several cortical (Willoughby et al., 1995) and subcortical (Pax-inos and Watson, 1986; Willoughby et al., 1995) functional simulated SAR, PA,E power absorbed by regions related to convulsive seizures. Speciﬁc regions within power absorbed by the simulated rat, WE each major structure were selected for detailed analysis. For weight of the rat, and W 5 weight of the simulated rat.
the cerebrum, we considered 1) somatosensory areas, frontal A separate group of six rats not treated with picrotoxin motor cortex, and parietal somatosensorial cortex, grouped as (not one of the groups described above) was used to validate ‘‘neocortex,'' and 2) olfactory areas and piriform, primary sen- the results obtained by simulation. These rats (average weight sory, and integrative entorhinal cortices, grouped as ‘‘paleo- 195.5 g) absorbed an average power of 13.56 mW. The simu- cortex.'' We also considered cerebral subcortical structures: lated 200-g rat absorbed a power of 13.67 mW. Comparison hippocampal (dentate gyrus, CA1, and CA3) and thalamic of the two results gives a mean error (n 5 6 rats) of 0.8% in [the central medial (CM) and central lateral (CL) nuclei of the absorbed power and 2.3% in weight. This indicates very low intralaminar nuclear group] structures (Fig. 1A–C). These error for estimation of the SAR for each rat with equation 1.
cortical and subcortical structures included some areas that Because the software tool used for the simulation have previously been described as showing picrotoxin-induced (SEMCAD; Schmid and Partner Engineering AG) does not neuronal activation (Willoughby et al., 1995).
consider transmission of antenna signals other than pure sinu-soids, it was necessary to adapt the numerical results by simply Quantiﬁcation and Statistical Analysis dividing the average power that impinges on the transmittingantenna by the crest factor corresponding to GSM modula- The numbers of c-Fos-positive cells were counted by tion, namely, 8.3 (Huber et al., 2003).
investigators who were blind to exposure conditions. For eachrat, three or four sections were taken from each of the follow-ing areas for examination of c-Fos expression in localized Behavioral and Clinical Signs regions: 1) cortical areas (frontal motor, parietal motor, piri- All animals were continuously observed and videotaped form olfactory, and entorhinal olfactory cortices); 2) hippo- for 2 hr after intraperitoneal injection of picrotoxin. Behav- campal structures (dentate gyrus, CA1, and CA3); and 3) tha- ioral modiﬁcations, myoclonic jerks, and occurrence and la- lamic structures (centromedial and centrolateral nuclei; see tency of convulsive seizures were recorded. Myoclonic jerks Fig. 1). In each area, c-Fos-positive cells were counted in a Journal of Neuroscience Research Pulse-Modulated GSM Radiation Increases c-Fos Fig. 1. Schematic drawings of transverse sections through the rat brain, showing the anatomical regionsin which nuclear c-Fos expression was measured, at the 9.2-mm level (interaval coordinates) in corticalareas (frontal cortex, FR1; parietal cortex, PAR1; piriform cortex, PIR; A), the 5.7-mm level in hippo-campal areas (dentate gyrus, DG; CA1; CA3; and the central medial, CM, and central lateral, CL, nucleiof the thalamic intralaminar nuclear group; B), and the 4.2-mm level in entorhinal cortex (ENT; C).
Journal of Neuroscience Research Lo´pez-Martı´n et al.
0.32-30.24-mm ﬁeld magniﬁed 320 in a Nikon Eclipse ior was reﬂected in the EEG by the presence of short- E200 microscope connected to a computer running morpho- duration polyspikes or continuous spike-and-wave dis- metric software (from Kappa, Monrovia, CA). Counts per charges during the seizures (see EEG traces in Fig. 2A).
ﬁeld in each of the areas were expressed as means for individ- Nonpicrotoxin-treated rats exposed to unmodu- ual animals or experiments 6 SEM per group. The signiﬁ- lated radiation (no-PT/UNMOD) showed periods of cance of between-group differences in c-Fos-positive cell immobility alternating with activities such as sucking and counts was assessed by ANOVA: 1) considering all brain small head movements, with no abnormalities in the regions together, with factors treatment (picrotoxin or no pic- EEG recordings. Picrotoxin-treated rats exposed to rotoxin) and radiation (no radiation, modulated radiation, or unmodulated radiation (PT/UNMOD) showed aggres- unmodulated radiation); 2) considering each brain region sepa- siveness and locomotor activity starting a few minutes af- rately, with factors treatment, radiation, and area (i.e., areas ter administration of the picrotoxin and lasting about within that region); and 3) considering each brain area sepa- 10 min. Two rats showed occasional signs of myoclonic rately, with factors treatment and radiation. Natural logarithm head jerks and movements of forepaws after 20 min, but transformations were applied to data to obtain normality and only one rat showed generalized seizures. The EEGs of homoscedasticity. Differences between groups and areas were these rats showed short-duration polyspikes or continu- considered signiﬁcant at P < 0.05 after Bonferroni correction.
ous spike-and-wave discharges.
The mean power absorbed by rats in the four irra- With regard to the proportion of c-Fos-immu- diated groups, estimated with equation 1, are shown in Table II, along with the average weight, mean SAR in throughout the whole brain, two-way analysis of var- brain and body, and peak SAR averaged for 1 g of body iance (factors radiation and picrotoxin) indicated a signif- and brain. It can be seen that the SAR values are low, icant effect of the factor picrotoxin, with c-Fos expres- all of them below thermal values.
sion signiﬁcantly higher in picrotoxin-treated than innonpicrotoxin-treated animals (P < 0.001). Irradiation Clinical Behavior and Electroencephalography did not have the same effect in the different groups (between treated and nontreated considered together): c- Nonpicrotoxin-treated nonirradiated rats (no-PT/ Fos expression did not differ in the GSM groups (PT 1 no-RAD) showed initial stress attributable to immobili- GSM and no-PT 1 GSM) and the unmodulated radia- zation but did not exhibit any abnormal activity or signs tion (PT 1 UNMOD and no-PT 1 UNMOD) groups of seizure, and none of the EEGs recorded from these (P 5 0.181) but did differ signiﬁcantly between these groups showed abnormalities. Picrotoxin-treated nonirra- groups and the nonirradiated groups (P < 0.001; PT 1 diated rats (PT/no-RAD) showed bursts of locomotor no-RAD and no-PT 1 no-RAD; see Table III).
activity lasting between 5 and 10 min, after which the With regard to the picrotoxin-treated groups sepa- rats remained immobile but alert. EEGs showed isolated rately, c-Fos expression differed signiﬁcantly among the spikes or very short bursts of spikes but no more than PT/GSM, PT/UNMOD, and PT/no-RAD groups minimal signs of seizure.
(P < 0.001 in all comparisons). For the nonpicrotoxin- Nonpicrotoxin-treated GSM-irradiated rats (no- treated groups separately, c-Fos expression differed sig- PT/GSM) showed initial stress but did not exhibit any niﬁcantly between no-PT/GSM and no-PT/UNMOD abnormal activity or signs of myoclonic jerks. Picro- groups (P < 0.001) but not between the no-PT/GSM toxin-treated GSM-irradiated rats (PT/GSM) began to and no-PT/no-RAD groups (P 5 0.454; see Table IV).
exhibit myoclonic jerks of the head and the body within The effect of GSM radiation increased the magni- 10 min of administration of picrotoxin. The myoclonic tude of the difference in c-Fos expression between PT jerks persisted for long periods, but seizures were and non-PT rats (P < 0.001); expression was very high observed in only two animals, which showed intermit- in GSM/PT rats and low in GSM/no-PT rats. In con- tent generalized convulsions for 20–30 min. This behav- trast, there were no signiﬁcant differences between TABLE II. Mean Absorbed Power and SAR Values for the Four Experimental Groups* *The effects of the stainless-steel screws are not taken into account here. For acronyms, see Table I.
Journal of Neuroscience Research Pulse-Modulated GSM Radiation Increases c-Fos Fig. 2. EEG traces and c-Fos-positive cell counts in the different ex- shows a seizure (epileptic recruiting rhythm). The trace at lower perimental groups. A: EEG traces are representative traces for each rightshows symmetric paroxysmal fast activity. B: Histograms show- group. The column on the left shows traces for rats that did not ing c-Fos-positive cell counts in nonirradiated (no-RAD), GSM-irra- receive picrotoxin; none of these rats showed signiﬁcant EEG abnor- diated (GSM), and unmodulated-radiation-exposed (UNMOD) rats malities. The column on the right shows traces for rats that received previously treated with i.p. subconvulsive doses of picrotoxin. Values picrotoxin. The traces at upper right and lower right show general- shown are means for eight rats per group; whiskers indicate SEMs.
ized paroxysmal theta waves and fast activity. The trace in the middle UNMOD/PT and UNMOD/no-PT rats (P 5 0.322) the pontomesencephalic nuclei. In all areas, the mean c- or between no-RAD/PT and no-RAD/no-PT rats Fos expression in irradiated with GSM picrotoxin-treated (P 5 0.549; Fig. 2B).
(PT 1 GSM) rats was higher than in nonirradiated pic- c-Fos expression in different brain regions.
rotoxin-treated rats (PT 1 No-RAD), showing twofold All brain areas showed c-Fos expression after picrotoxin increases in some areas, with even greater differences in treatment, with or without posterior irradiation. In rats the hippocampus. In the neocortical areas, c-Fos expres- treated with picrotoxin, c-Fos-positive neurons were sion was similar for picrotoxin-treated rats exposed to observed systematically in the thalamus and cortical areas, GSM and unmodulated radiation (UNMOD), and in the and in some animals more or less scattered expression remaining areas c-Fos expression was higher in rats was also observed in the hippocampus, the amygdala, the exposed to GSM than in rats exposed to unmodulated putamen-caudate complex, and/or the hypothalamus and radiation in frontal areas. In rats not treated with picro- Journal of Neuroscience Research Lo´pez-Martı´n et al.
or parietal; P < 0.001) and by the interaction area 3 between the GSM and no-RAD groups but was mark- radiation (P < 0.001). Analyses of variance considering edly higher in the UNMOD group. Below we discuss the frontal and parietal areas separately in both cases the effects of the different treatments on c-Fos expression indicated that both picrotoxin and radiation, and the in the speciﬁc brain regions.
interaction between them, had signiﬁcant effects on c- c-Fos expression in the neocortex (frontal and parietal cor- Fos expression (P < 0.001 in all cases; see Table III).
Initial analysis indicated that c-Fos expression in In considering only picrotoxin-treated rats, radia- the neocortex was signiﬁcantly affected by area (frontal tion signiﬁcantly affected c-Fos expression in the frontalarea of picrotoxin-treated rats (GSM compared with no-RAD, P < 0.001; UNMOD compared with no-RAD, TABLE III. Results of Two-Way ANOVA (Treatment 3 P 5 0.005), but GSM modulation had no signiﬁcant Radiation) or Three-Way ANOVA (Treatment 3 Radiation 3 effect (GSM compared with UNMOD, P 5 0.192; Fig.
Area) for the Whole Brain and for Different Brain Regions* 3A–D). In the parietal area of picrotoxin-treated rats, there were signiﬁcant differences in c-Fos expressionbetween GSM and no-RAD and between UNMOD and no-RAD, but not between UNMOD and GSM (P 5 0.120; Fig. 4A–D, Table IV). In considering only nonpicrotoxin-treated rats, in frontal and parietal areas c- Fos expression in the UNMOD group differed signiﬁ- cantly from that in the GSM and no-RAD groups (P < 0.001), but there was no signiﬁcant difference between the GSM and the no-RAD groups (P 5 0.620; Fig.
The statistical analyses of the data for this region did not show signiﬁcant effects on c-Fos expression of area (P 5 0.242) or area 3 picrotoxin 3 Hippocampal areas radiation (P 5 0.086). Separate analyses of the different olfactory areas in the paleocortex indicated that, in both the piriform and the entorhinal areas, the factors picro- toxin and radiation and the interaction picrotoxin 3 radiation had signiﬁcant effects on c-Fos expression (P < 0.001 in all cases; see Table III).
GSM modulation had a marked effect in animals *F, Fisher-Snedecor F statistic; DF, degrees of freedom; P value: level of treated with picrotoxin or not treated with picrotoxin both in the piriform and in the entorhinal areas, with TABLE IV. Mean Values 6 SEM of the Cell Counts in Whole Brain and in Brain Areas and for the Different Treatments(With and Without Radiation and With and Without Picrotoxin)y Hippocampal areasGD Thalamic nucleiCM yTwo-way ANOVA (treatment 3 radiation) applied to data corresponding to whole brain and individual brain areas.
*Signiﬁcant differences in picrotoxin-treated vs. nontreated animals for each type of radiation between groups (at P < 0.05) following Bonferroni cor-rection.
1–3Signiﬁcant differences according to the radiation factor (1, NR; 2, GSM; 3, UNMOD) in PT or non-PT animals.
Journal of Neuroscience Research Fig. 3. A: c-Fos-positive cell counts in frontal cortex (FR1) in picrotoxin- and nonpicrotoxin-treated animals that subsequently were not irradiated (no-RAD), exposed to GSM radiation(GSM), or exposed to unmodulated radiation (UNMOD). Photomicrographs of DAB-stainedtransverse sections of the brains of picrotoxin-treated no-RAD (B), GSM (C), and UNMOD (D)rats in frontal cortex. The boxed area in each panel is enlarged in the inset. Scale bar 5 500 lm.
Fig. 4. A: c-Fos-positive cell counts in parietal cortex (PAR1) in picrotoxin- and nonpicrotoxin-treated animals that were subsequently not irradiated (no-RAD), exposed to GSM radiation(GSM), or exposed to unmodulated radiation (UNMOD). Photomicrographs of DAB-stainedtransverse sections of the brains of picrotoxin-treated no-RAD (B), GSM (C), and UNMOD (D)rats in parietal cortex. The boxed area in each panel is enlarged in the inset. Scale bar 5 500 lm.
Lo´pez-Martı´n et al.
Fig. 5. A: c-Fos-positive cell counts in piriform cortex (PIR) in picrotoxin- and nonpicrotoxin-treated animals that were subsequently not irradiated (no-RAD), exposed to GSM radiation (GSM),or exposed to unmodulated radiation (UNMOD). Photomicrographs of DAB-stained transversesections of the brains of picrotoxin-treated no-RAD (B), GSM (C), and UNMOD (D) rats in piri-form cortex. The boxed area in each panel is enlarged in the inset. Scale bar 5 500 lm.
signiﬁcant differences between GSM and UNMOD and signiﬁcantly between the GSM and the UNMOD between GSM and no-RAD in both areas (P < 0.001 groups (see Fig 7A–C,E,F). In nonpicrotoxin-treated in both cases), but there was a signiﬁcant difference only animals, c-Fos expression was highest in the UNMOD between GSM and no-RAD in the piriform area (P < group, in which counts differed signiﬁcantly from those 0.001) and not in the entorhinal area (P 5 0.473) in in the other two groups (P < 0.001); in contrast, the nonpicrotoxin-treated animals (Figs. 5, 6, Table IV).
counts in the GSM group differed only from those in Hippocampus: dentate gyrus, CA1, and CA3.
the no-RAD group in CA1 (P 5 0.043; see Table IV).
statistical analyses of the data for the hippocampus Thalamic nuclei.
Studies of the thalamus were per- revealed signiﬁcant effects of area, area and treatment, formed in the central medial (CM) and central lateral area and radiation, and area 3 radiation and area 3 (CL) nuclei of the intralaminar nuclei group. The inter- treatment 3 radiation interactions (P < 0.001 in all actions area 3 picrotoxin, area 3 radiation, and area 3 cases; see Table III). In considering each of the areas of picrotoxin 3 radiation all had signiﬁcant effects (P 5 the hippocampus separately and bearing in mind the 0.021, P < 0.0001, and P < 0.001, respectively) on c- observed picrotoxin 3 radiation interaction, (except for Fos expression. In considering each of the areas of the DG area with respect to treatment; P 5 0,394), multiple thalamus separately, there were signiﬁcant differences in comparisons indicated signiﬁcant differences between all both areas in relation to radiation and picrotoxin (P < irradiated groups compared (GSM compared with no- 0.05 in both cases), but signiﬁcant differences in relation RAD, UNMOD compared with no-RAD, and GSM to the interaction between these factors were only compared with UNMOD) both in picrotoxin- and in observed in the CL nucleus (see Table III).
nonpicrotoxin-treated animals (P < 0.001 in all cases).
Finally, the statistical analyses considering each nu- The effect of GSM modulation was marked in the den- cleus separately indicated that, in the CM nucleus, the tate gyrus in picrotoxin-treated animals, with very high effect of exposure to radiation (GSM compared with c-Fos expression in the GSM group, signiﬁcantly differ- no-RAD, UNMOD compared with no-RAD) was sig- ent from the counts in the UNMOD and no-RAD niﬁcant only in nonpicrotoxin-treated rats (P 5 0.049, groups (P < 0.001 in both cases; see Fig. 7A–D). In the P 5 0.001, respectively). In the CL nucleus, modulated CA1 and CA3, however, c-Fos expression did not differ radiation had a signiﬁcant effect on picrotoxin-treated Journal of Neuroscience Research Pulse-Modulated GSM Radiation Increases c-Fos Fig. 6. A: c-Fos-positive cell counts in entorhinal cortex (ENT) in picrotoxin- and nonpicro-toxin-treated animals that were subsequently not irradiated (no-RAD), exposed to GSM radiation(GSM), or exposed to unmodulated radiation (UNMOD). Photomicrographs of DAB-stainedtransverse sections of entorhinal cortex from picrotoxin-treated no-RAD (B), GSM (C), andUNMOD (D) rats. The boxed area in each panel is enlarged in the inset. Scale bar 5 500 lm.
rats (P 5 0.013), with signiﬁcantly higher counts in the dependent protein kinase C (PKC; Paulraj and Behari, GSM group than in the UNMOD group. (Fig. 7A–E, 2004), and cAMP-dependent protein kinase A (PKA; Byus et al., 1984). The suggestion that these effectsoccur speciﬁcally with ﬁelds that are amplitude modu-lated at extremely low frequencies is intriguing but difﬁ- cult to interpret (Stewart, 2000). Likewise, PKA protectsagainst acute picrotoxin-induced seizures (Va´zquez- Effects of GSM Compared With Unmodulated Lo´pez et al., 2005), and voltage-dependent calcium Radiation on Cerebral Activity channels are essential for picrotoxin-induced epileptic The present results indicate that for UNMOD radia- activity (Straub et al., 1994).
tion there was no signiﬁcant difference in c-Fos expres- The SAR for a given tissue and microwave radia- sion between nonpicrotoxin-treated rats tion exposure depends on physical parameters, including UNMOD) and picrotoxin-treated rats (PT 1 UNMOD); whether the microwave signal is pulsed or nonpulsed occasional discharges occurred in EEG recording, and sei- (Michaelson et al., 2007). The biological effects observed zure development was infrequent in treated animals.
in the present study in rats exposed to GSM are unlikely However, GSM radiation induced seizures and increased to have been of thermal origin, because the estimated c-Fos-positive neuron counts in animals made seizure- SAR levels (Table II) were well below the threshold for prone by picrotoxin treatment (see Fig. 2).
induction of hyperthermia (4 or 5 W/kg; Mickeley These ﬁndings (scarce clinical and EEG signs with- et al., 1994). GSM radiation is capable of triggering seiz- out GSM and seizures with GSM) suggest that the con- ures in seizure-prone rats through nonthermal mecha- vulsant drug picrotoxin and GSM radiation had parallel nisms (Lo´pez-Martı´n et al., 2006). In the absence of effects, or effects via a common mechanism, as has been pulse modulation (the UNMOD group), the estimated reported for other drugs (Michaelson et al., 2007). Am- SAR was higher (see Table II) but still not high enough plitude-modulation GSM-type radiation (at the fre- to trigger seizures by a thermal or other mechanism (see quency used, 271 kHz) has been found to cause changes Guy and Chou, 1982).
In the present study we also found that, in nonpi- (Blackman et al., 1980; Kittel et al., 1996), calcium- crotoxin-treated (nonseizure-prone) rats, c-Fos expres- Journal of Neuroscience Research Lo´pez-Martı´n et al.
Fig. 7. Photomicrographs of DAB-stained transverse sections of hippocampal areas of brains of pic-rotoxin-treated no-RAD (A), GSM (B), and UNMOD (C) rats: dentate gyrus (DG), CA1, andCA3. The boxed DG area in each panel is enlarged in the inset. c-Fos-positive cell counts in hip-pocampal areas of brains of picrotoxin- and nonpicrotoxin-treated no-RAD (D), GSM (E), andUNMOD (F) rats. Scale bar 5 500 lm.
sion was signiﬁcantly higher in the brains of those rats ﬁndings suggest the existence of a mechanism that is not exposed to nonmodulated radiation than in nonirradiated mediated by temperature variations (Curcio et al., 2005).
or GSM-exposed rats. In other studies the increased c- The present study may provide relevant information Fos expression has been attributed to immobilization about the effects of mobile-phone-type radiation on stress (Cullinan et al., 1995), because there is no differ- brain activity when the excitability of GABAergic neu- ence in the responses of GSM-irradiated and nonirradi- rons is increased (Hossmann and Herman, 2003; Ferri ated animals (Fritze et al., 1997; Finne, 2005). GSM et al., 2006).
radiation has previously been reported to affect geneexpression in brain cells (Belyaev et al., 2006), butincreased cerebral c-Fos expression has been reported in Comparison of the Effects of GSM normal (nonpicrotoxin-treated) rats only when the radia- and Unmodulated Radiation on Neuronal tion is applied at intensities high enough to cause hyper- Activation in Different Anatomical Regions thermia (Mickley et al., 1994; Morrissey et al., 1999).
Cortical areas: neocortex and paleocortex.
Pulse modulation appears to be essential for trigger- The results of the present study indicate that GSM radia- ing seizures and increasing the level of c-Fos, and these Journal of Neuroscience Research Pulse-Modulated GSM Radiation Increases c-Fos Fig. 8. Photomicrographs of DAB-stained transverse sections from the thalamic intralaminar nucleiof picrotoxin-treated no-RAD (A), GSM (B), and UNMOD (C) rats: central medial (CM) nu-cleus and central lateral (CL) nucleus. The boxed areas in each panel are enlarged in the inset. c-Fos-positive cell counts in hippocampal areas of brains of picrotoxin- and nonpicrotoxin-treatedno-RAD (D), GSM (E), and UNMOD (F) rats. Scale bar 5 500 lm.
region-speciﬁc effects on c-Fos expression in the neo- reduces GABAergic inhibition by interaction with the cortex and paleocortex. In the neocortex (speciﬁcally, chloride ionophore of the GABAA receptor (Olsen, the frontoparietal cortex), radiation caused an increase in 2006); however, the neocortex (or frontoparietal cortex) c-Fos expression, but GSM radiation caused no greater may play a constant role in the appearance of the thresh- increase than equivalent unmodulated radiation. By con- old of picrotoxin-induced convulsive seizures (Vergnes trast, in the paleocortex in the limbic system (speciﬁcally et al., 2000). In addition, at subconvulsive doses, this olfactory areas), GSM radiation had a greater effect on drug also increases the excitability of the sensorimotor c-Fos expression than unmodulated radiation. Neocorti- cortex in previous states of cortical stimulation (Koryn- cal GABaergic interneurons form complex networks tova et al., 2002). In humans, the activation of the c-Fos within the cerebral cortex and play an important role in gene in the neocortex after an epileptic crisis suggests inhibiting the activity of local circuit neurons. Small that this gene is directly modulated by the degree of epi- decreases in the efﬁcacy of the intracortical GABAergic leptic activity and may be a clinically relevant biological inhibition system can lead to propagation of synchron- marker (Sanjay et al., 2005).
ized discharges, which may play role in susceptibility to One possible explanation for the similar effects of epileptogenesis (Luhmann and Price, 1990). Picrotoxin GSM and unmodulated radiation in the motor cortex is Journal of Neuroscience Research Lo´pez-Martı´n et al.
the predominant intervention of other systems, such as some authors suggest that low doses of picrotoxin do the neuroendocrine system (Smith et al., 1997). It is also not facilitate the transmission of seizure activity within possible that the stimulation caused by the pulses directly the limbic system (Koryntova et al., 2002), in the present or indirectly in subcortical areas sends inputs to the neo- study we found higher c-Fos expression in limbic areas cortex, increasing the inhibition of the microcircuits of (especially in the dentate gyrus) that were more sensitive the frontal cortex by the hippocampal GABAergic sys- to the combined effect of pulse modulation and picro- tem (Mantovani et al., 2006; Takita et al., 2007).
toxin than the areas of the motor system (see Results).
In contrast, c-Fos-positive cell counts in the ento- There is some controversy regarding the reported rhinal and piriform cortices (in the limbic system) were effects that amplitude-modulated RF ﬁelds have on the markedly affected by GSM radiation, and c-Fos expression excitability of neurons in the hippocampus of rats in in these areas was associated with c-Fos expression in vitro (Tattersall et al., 2001), and it is possible that the other regions of the limbic system, with which it showed effects are due to an electrode-related artefact. In vivo a clear temporal relationship (as further discussed below).
RF studies showed effects in the hippocampus (Paulraj It is well known that stress may trigger or exacerbate seiz- et al., 2004) as well as on the metabolism of Ach (Kun- ures (Frucht et al., 2000). The entorhinal and piriform jilwar and Behari et al., 1993; Barteri et al., 2005) and cortices contain glucocorticoid receptors (Chao et al., altered cognitive functions in humans related to this ana- 1989); it is possible that these receptors are stimulated by tomical area (Koivisto et al., 2000; Eldesteyn and Older- shaw, 2002; Mainer et al., 2004).
pituitary-adrenal axis during seizures (Mraovitch and The expression of c-Fos showed marked activity in Calando, 1999) and also that picrotoxin may stimulate the limbic system; the effects on the dentate gyrus in cholinergic and GABAergic modulation in the hypothala- hippocampus and extrahippocampal regions (piriform mus (Singh et al., 1997). However, intraperitoneal sub- and entorhinal cortex) caused by GSM in seizure-prone convulsive doses of picrotoxin alone, and in the absence rats in the present study suggest, as do other studies with of radiation or some other trigger, probably do not GABAA noncompetitive antagonists (Shehab et al., induce seizures (Nutt et al., 1982) but may be used to 1992; Eells et al., 2004) or Ach agonists (Barone et al., inﬂuence GABAergic inhibition (Koryntova et al., 2002).
1993; Mraovitch and Calando, 1999), the following Our hypothesis is that the combined effect on the cortex excitatory circuitry in determining the anatomical struc- of the GABAergic antagonist and nonthermal GSM radia- tures recruited by propagating seizure activity: entorhinal tion is the origin of the observed electrical discharges, cortex ? dentate gyrus ? CA3 ? CA1 and via the although other factors may intervene in the propagation perforant pathway (via mossy ﬁbers and Schaffer's collat- of seizures to limbic or motor cortex circuits.
eral ﬁbers, respectively,) and the CA1 pathway to the Another ﬁnding of this study was that, in nonpi- connections of the entorhinal cortex, forming a hippo- crotoxin-treated animals, unmodulated radiation caused campal-parahippocampal loop. This excitatory circuitry an increase in c-Fos-positive neuron counts in most of may be one of the anatomical substrates of seizures the regions of the cortex studied, CM and CL being (Mraovitch and Calando, 1999).
exceptions. GSM radiation may have relatively subtle In the present study, GSM radiation had little effect effects (possibly inhibitory effects) on neuronal activa- on either the central medial or the central lateral nuclei; tion; however, in combination with GABAergic antago- the TIN does not appear to show great effects in limbic nists, there may be synergistic effects.
or motor convulsions, possibly because of the intervention GSM-type pulse modulation of electromagnetic of hippocampal structures (Nanobashvali et al., 2003).
ﬁelds affects neuronal activation in the cortex of picro- TIN neurons may participate with thalomocortical or cor- toxin-pretreated rats, leading to increased c-Fos expres- ticothalamic inputs and cause inhibition or activation, sion, modiﬁed behavior, and altered EEG traces. This depending on other neurotransmitters, such as acetylcho- acute and reversible increase in neuronal excitability in line or noradrenaline, which may be modiﬁed by the the cortex has been reported by other authors (see Eulitz combined action of picrotoxin and GSM. The indirect et al., 1998; Borbe´ly et al., 1999; Huber et al., 2002, interaction with other systems, such as the striatum and 2003, 2005; Ferri et al., 2006), suggesting an imbalance hypothalamus or locus coeruleus (with which CM nuclei between the neurotransmitters GABA and glutamate have the most connections), may trigger secondary regula- (Mausset-Bonafont et al., 2004) and acetylcholine (Lai tion of these systems (Mraovitch and Calando, 1999).
et al., 1991). In our opinion, these results indicate that The present data on the expression of c-Fos indicate an the paleocortex (notably the entorhinal cortex) is espe- important degree of participation of hippocampal anatom- cially sensitive to pulsed radiation, such as GSM, and ical structures in the neuronal activity triggered during may play an important role in the progression of neuro- seizure propagation. Thalamic structures play a more vari- nal activity and seizures.
able role in the synchronization of this neuronal circuit.
Subcortical areas: hippocampus and thalamic In hippocampal areas, the effect of GSM was apparent in CA1 and CA3 and very marked in the den- tate gyrus, where c-Fos expression in picrotoxin-treated Nine-hundred-mega-Hertz GSM radiation trig- animals was already very high (see Figs. 7, 8). Although gered a marked increase in neuronal excitability in sei- Journal of Neuroscience Research Pulse-Modulated GSM Radiation Increases c-Fos zure-prone rats, as manifested by behavioral indicators, Dubreuil D, Jay T, Edeline JM. 2003. Head-only exposure to GSM 900- EEG indicators, and neuronal c-Fos expression, with MHz electromagnetic ﬁelds does not alter rat's memory in spatial and respect to rats exposed to unmodulated radiation. Signal non-spatial tasks. Behav Brain Res 145:51–61.
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