Article
Maternal mobile phone exposure alters intrinsic electrophysiological properties of CA1 pyramidal neurons in rat offspring
Toxicology and Industrial Health 1–12 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714525497 tih.sagepub.com
Moazamehosadat Razavinasab1, Kasra Moazzami2 and Mohammad Shabani1 Abstract Some studies have shown that exposure to electromagnetic field (EMF) may result in structural damage to neurons. In this study, we have elucidated the alteration in the hippocampal function of offspring Wistar rats (n ¼ 8 rats in each group) that were chronically exposed to mobile phones during their gestational period by applying behavioral, histological, and electrophysiological tests. Rats in the EMF group were exposed to 900 MHz pulsed-EMF irradiation for 6 h/day. Whole cell recordings in hippocampal pyramidal cells in the mobile phone groups did show a decrease in neuronal excitability. Mobile phone exposure was mostly associated with a decrease in the number of action potentials fired in spontaneous activity and in response to current injection in both male and female groups. There was an increase in the amplitude of the afterhyperpolarization (AHP) in mobile phone rats compared with the control. The results of the passive avoidance and Morris water maze assessment of learning and memory performance showed that phone exposure significantly altered learning acquisition and memory retention in male and female rats compared with the control rats. Light microscopy study of brain sections of the control and mobile phone-exposed rats showed normal morphology. Our results suggest that exposure to mobile phones adversely affects the cognitive performance of both female and male offspring rats using behavioral and electrophysiological techniques. Keywords Mobile phone exposure, pregnancy, neural excitability, offspring, pyramidal neurons, rat
Introduction Mobile phones release ultrahigh-frequency electromagnetic fields (EMF) around them when in use (Carballo-Quinta´s et al., 2011; Scho¨nborn et al., 1998). The close proximity of mobile phones to the user’s head leads to the absorption of part of the EMF into the head and brain (Carballo-Quinta´s et al., 2011; Scho¨nborn et al., 1998). The rapid increase in the use of mobile phones over the past few years has been accompanied by increasing some possible health concerns for the effects from their use. In particular, the possible adverse effects of EMF on the brain and cognitive functioning have been the subject of investigation by many groups (Cosquer et al., 2005; Dubreuil et al., 2003; Foroozandeh et al., 2012; Lai et al., 2005; Narayanan et al., 2009; Ragbetli et al., 2010; Sonmez et al., 2010; Smythe and Costall, 2003).
Results regarding the effects of EMF on cognition are highly variable and contradictory partly due to the different experimental conditions and the intrinsic phenomenological complexity. In some studies, EMF exposure induced adverse cognitive effects that were recorded using histological and behavioral measures.
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Kerman Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Islamic Republic of Iran 2 Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Corresponding author: Mohammad Shabani, Kerman Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman 76198, Islamic Republic of Iran. Email: [email protected]
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These changes included diminished numbers of Purkinje cells and a tendency for granule cells to increase in cerebellum (Ragbetli et al., 2010; Sonmez et al., 2010), as well as disruption of spatial learning in a radial arm maze task and decreased acquisition of learned responses in rats (Lai et al., 2005; Narayanan et al., 2009). These observations, however, have not been supported by other groups, suggesting that EMF emitted from mobile phones does not decrease cognitive functioning (Cosquer et al., 2005; Dubreuil et al., 2003) or may even improve cognitive performance (Smythe and Costall, 2003). Previous reports suggest impairments in the development of the dentate gyrus by prenatal exposure to high-frequency EMF (Odaci et al., 2008). Recently, Haghani et al. (2013) also reported that maternal mobile phone exposure adversely affects the electrophysiological properties of Purkinje neurons in rat offspring. Also, 2.14 GHz signals applied for 20 h/day during the gestation period did not cause any adverse effects on pregnancy or the development of pups (Takahashi et al., 2010). However, detailed electrophysiological changes in rat offspring following EMF exposure have not been addressed adequately. Most studies assessing the effects of EMF on cognition have been focused on adult animals, while studies investigating the effects of occupational maternal exposure to EMF and the risk of cognitive impairment in offspring are lacking. Despite the fact that previous studies have shown lack of embryotoxicity and teratogenicity in rats receiving EMF (Ogawa et al., 2009; Takahashi et al., 2010), the effects on cognition has not been evaluated thoroughly. Therefore, the present study was designed to address the potential influences of gestational exposure to EMF on the cognitive performance in rats. Furthermore, since most cognitive tasks are widely accepted to have a substantial hippocampal involvement, the present study aimed to investigate the potential electrophysiological changes of CA1 pyramidal neurons of the hippocampus following prenatal EMF exposure.
Materials and methods Primiparous Wistar female rats, weighing 200–250 g, were housed for 2 weeks before mating at constant room temperature (22–25 C) with 12-h light and 12–h dark (7:00 a.m.–7:00 p.m.) and free access to food and water. Pairs of females were then placed with single male rats in the late afternoon. Vaginal smears or plugs were examined in the following
morning at 9:00 a.m. The day in which sperm was found has been considered as the gestation day (GD 0). Afterward, pregnant rats were randomly assigned to either a control group or to a mobile phone exposure group. The care of laboratory animals followed the guiding principles for care and use of laboratory animals of the Neuroscience Research Center of Kerman Medical University, Islamic Republic of Iran, and the study protocol was approved by the animal ethics committee of this institution [Code: EC/KNRC/88-34].
900 MHz Pulsed-EMF exposure conditions Pregnant rats were placed inside experimental cages on the first day of pregnancy. A total of 16 pregnant rats have been used, 8 rats in the control group and 8 rats in EMF-irradiated group. Control group was exposed to the same conditions as in the EMF group but without cellular phone. Pregnant rats were irradiated from 8 a.m. until 2.00 p.m. (each day in pregnancy period) for a total of 6 h/day, since the day of sperm detection (pregnancy start) until offspring birth. During radiofrequency exposure, rats were restrained in 5-mm thick Plexiglas with 25 cm length. Both the cellular phone and the Plexiglas cage were placed inside an aluminum Faraday cage. The field intensity was measured with a radiofrequency spectrum analyzer (Anritsu MS2711, Japan). The mobile phone was operated in test mode and was powered through a stabilized power supply, thus antenna power supply was constant. The measured specific absorption rate (SAR) value in our data was between 0.3 and 0.9 W/kg, a value without significant thermal effects. Temperature was measured before, during, and after the exposure by a rectal probe only in a series of pups that did not enter at behavioral and electrophysiological analysis. Before and after each experiment, a series of electromagnetic measurements (i.e. input power and reflected power) were performed to evaluate the correct operation of the exposure system. The average power density and SAR measurements were performed at an electronic mobile company. After each birth, mother and its offspring (both males and females) were removed from the Faraday cage and placed inside a separate Plexiglas cage. Pups were weaned at day 23 of age.
Slice preparation Male and female offspring (28–30-day-old) of either control (n ¼ 8) or maternal EMF-exposed
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group (n ¼ 8) was used. Animals were anesthetized with ether followed by decapitation. Brains were removed rapidly and placed in ice-cold artificial spinal fluid (ACSF) containing 206 mM sucrose, 2.8 mM potassium, chloride (KCl), 1 mM calcium chloride (CaCl2), 1 mM magnesium chloride (MgCl2), 2 mM magnesium sulfate (MgSO4), 1.25 mM monosodium phosphate (NaH2PO4), 26 mM sodium bicarbonate (NaHCO3), and 10 mM D-glucose and equilibrated to a pH of 7.4 (with 95% oxygen and 5% carbon dioxide); the osmolarity was adjusted to 295 mOsm. The hippocampi were dissected out of the brain, and 300 mm thick transverse slices were obtained using a vibrating microtome (752 M, Campden Instruments Ltd, UK). Following this procedure, slices were incubated in ACSF containing 124 mM sodium chloride, 2.8 mM KCl, 2 mM CaCl2, 2 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 10 mM D-glucose with pH 7.4 295 mOsm osmolarity for at least 60 min at 35 C and thereafter maintained at room temperature (22–25 C) before being transferred to a recording chamber. From each animal just one pyramidal neuron from one brain slice was recorded.
Whole-cell patch clamp recording Whole-cell patch clamp recordings in current clamp mode from pyramidal neurons were made, as described previously by Haghani et al. (2011), under direct visual control using differential interference contrast optics (BX 51WI; Olympus, Tokyo, Japan). CA1 pyramidal neurons were visualized with a 60 water immersion objective lens using Nomarski-type differential interference contrast imaging with infrared illumination. Images were captured with a charge-coupled device camera (ORCA, Hamamatsu, Japan). The slices were continuously superfused with normal ACSF (1–2 ml/min) at room temperature (22– 25 C). Whole-cell current clamp recordings were made from CA1 pyramidal neurons using Multiclamp 700B amplifiers (Axon Instruments, Foster City, California, USA) equipped with Digidata 1440 A/D converter (Axon Instruments). Electrophysiological responses were filtered at 5 kHz and sampled at 10 kHz and stored on a personal computer for offline analysis. Patch pipettes were pulled with an electrode puller (PC10; Narishige, Tokyo, Japan) and had a resistance of 3–6M when filled with internal solution containing 135 mM potassium methylsulfate, 10 mM KCl, 10 mM Hepes, 1 mM MgCl2, 2 mM adenosine triphosphate disodium salt, and 0.4 mM guanosine triphosphate sodium salt. The pH of the internal
solution was set to 7.3 by potassium hydroxide, and osmolarity was adjusted to 290 mOsm. To investigate the excitability of neurons, action potentials (APs) were induced in pyramidal cells from a holding potential of V ¼ 70 mV in 100 ms duration current steps ranging from 500 to þ500 pA in 100 pA increments. Before positive current steps, a negative prepulse protocol current with 300 pA was identified. To test the effect of EMF irradiation on excitability, the number of APs and APs rebound generated by these current injections and the amplitude of the corresponding after hyperpolarizations (AHP) were analyzed. AHP amplitude was taken as the absolute value of the difference between the voltage at holding and the voltage measured 10 ms after the peak of the AP using the 520 ms duration current injections. The AP amplitude and half-width were measured as peak distance from the resting membrane potential and the width at half amplitude, respectively. The AP threshold was defined as a measure of the voltage at the onset of the AP. The first-spike latency was defined as the time between the offset of the negative current steps and the peak of the first spike.
Behavioral tests Hippocampus-related behavioral dysfunctions in EMFexposed rats were analyzed using hippocampus functional tasks. Two behavioral tasks, namely, shuttle box and Morris water maze tasks were chosen.
Passive avoidance learning and memory test On postnatal day 30, the rats underwent passive avoidance training. The passive avoidance apparatus consisted of two equal-sized, connected chambers that were separated by a guillotine door. The rats were individually allowed to be habituated to the apparatus prior to testing. The rats from each experimental group were placed individually in the lighted chamber facing away from the entrance to the dark side; 10 s later, the guillotine door was raised, and the latency to enter the dark chamber was recorded. If the rats did not enter the dark chamber within 60 s, they were eliminated from the test and replaced with a new rat. The habituation trial was repeated after 5 min for the same interval. For the learning trial, after 2 h, the third adaption trial was administered in which an electrical stimulation (0.5 mA, 50 Hz, 2 s once) was delivered to the feet through the stainless steel floor after entering the dark chamber. After 20 s, the rats were removed from the dark compartment and returned to their own cage. After 5 min, the same test
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was conducted again, and if the rats did not enter the dark chamber by 300 s, the successful acquisition of passive avoidance response was recorded. In the retention trials that were performed after 1 day and 7 days of the learning trial (acquisition test) the rats were again individually placed in the illuminated chamber but no foot shock was administered if they entered the dark chamber. The rats were allowed to step into the dark compartment and then the latency to reenter the dark chamber was recorded. In both the learning and retention trials, the time to enter the dark section was recorded as the step-through latency. The maximum cutoff time for the step-through latency was 300 s when the rat did not enter the dark chamber in the retention trials (Van Der Stelt et al., 2006). Morris water maze task. The testing procedure was performed as previously described by Shabani et al. (2012b). The experimental apparatus consisted of a circular water tank (140 cm wide and 45 cm high). A platform, either visible or submerged, which is 15 cm wide and 35 cm high, was placed 1.5 cm above or below the surface of the water. The maze was located in a large and quiet test room, surrounded by many visual cues external to the maze, which were visible from within the pool and could be used by the rats for spatial location. The water temperature was 22 + 2 C. Data collection was automated by a video image motion analyzer (Ethovision, Noldus Information Technology, Netherlands). To assess for gross physical, sensory, motor, or motivational impairments, five rats from the control group and five rats from EMF-exposed group were first trained in a task with a visible escape platform. The rats were trained in the maze over 13 sessions on 4 consecutive days, 4 sessions on each day except on the last day, when only 1 session was given. The rats were towel dried on exiting the pool and then placed in pre-warmed cages with paper towel, where they were given a recuperation period of at least 30 min between trials. All the experimental groups were tested during the lights on period between 8:00 and 12:00 h. On each trial, rats were randomly released into the water from one of the four quadrants with their face toward the wall of the maze. During acquisition, the location of the platform remained constant, and the rats were allowed to swim for duration of 60 s to find the hidden platform. After the animal found the platform, it was allowed to remain there for 20–30 s and was then moved to an animal cage to wait for 20–30 s before the start of the next trial. The time and distance
needed to find the hidden platform were collected and analyzed later. A single probe trial was given 1 day after the last training trial to test the spatial memory in the water maze. In this trial, the platform was removed, and the rat was allowed to swim for 60 s. The time and distance spent in the target quadrant (quadrant 4) were analyzed as a measure of spatial memory retention (Shabani et al., 2012a, 2012b).
Histological evaluation Under deep anesthesia, the animals were killed and their brains were removed and immersed in 10% buffered formaldehyde at 4 C overnight. The brains were processed by standard method for light microscopy study. Coronal sections 1.6–2.8 mm posterior to bregma were cut at a thickness of 8 mm using a microtome. Neuronal injury in the hippocampus was assessed by staining sections with cresyl fast violet (Nissl staining).
Statistical analysis Initially, male and female rats in the control group were compared using unpaired sample t test, the results of which did not show any difference. Following maternal mobile phone exposure, sex differences were compared using two-way analysis of variance (ANOVA) analyses for all groups. To investigate the effect of the sex and learning trial in Morris water maze test, two-way ANOVAs for repeated measures were applied to measure the time and distance required to locate the hidden platform in both sexes. Two-way ANOVAs were used to calculate the effect of sex and 900 MHz pulsed-EMF exposure on the passive avoidance learning test, to probe data in shuttle box and Morris water maze tests, and finally to address the electrophysiological changes in intracellular recording. Individual comparisons were measured using Tukey’s comparisons test where appropriate. Data are expressed as means + SEM. Statistical significance was defined at p < 0.05. All computations were made using the Statistical Package for Social Sciences software package (Version 16.0; SPSS Inc., Chicago, Illinois, USA).
Results Behavioral tests Passive avoidance learning and memory test. The results of the passive avoidance assessment of learning and memory performance showed that the shock number
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Figure 1. Mobile phone exposure affected the acquisition of learned responses and retention latencies in the passive avoidance learning and memory test. (a) The phone-exposed animals exhibited higher shock number than the control animals in learning performance. (b,c) Mobile phone exposure significantly decreased 1 day and 7 days testing retention latencies. *p < 0.05, compared with the control groups. Saline and mobile M/F: Saline and mobile-exposed male/female.
was increased significantly in rats exposed to EMF in comparison with the control group (p < 0.05; Figure 1(a)). When the testing was performed 1 day after the shock experience (30 days post-EMF exposure), the step-through latency was significantly decreased in EMF-exposed rats (male: 144.9 + 35.2 s, female: 85.05 + 25.3 s) compared with the control rats (male: 234 + 30.4 s, female: 212.8 + 42.09 s; p < 0.05; Figure 1(b)). When the testing was performed 7 days following the shock experience (on postnatal day 37), the step-through latency was also significantly decreased in EMF-exposed rats (male: 62.07 + 7.09 s, female: 49.8 + 18.6 s) compared with the control rats (male: 130.8 + 12.02 s, female: 111.7 + 19.2 s; p < 0.05; Figure 1(c)). Prenatal exposure to mobile phone did not alter the time in dark compartment (in seconds) or crossing number in either days 1 or 7 following the shock experience (data not shown).
Morris water maze. The results of the training trial are depicted in Figure 2. The mobile phone-irradiated rats and the controls took progressively less time to locate the hidden platform over the course of the 12 trials during the training period. Data for the block effect in all rats showed a reduction in escape latencies and the swimming distance to locate the platform across blocks of trials, indicating spatial acquisition (p < 0.05). The path length and time latency at the second and third blocks for the male and female mobile phoneirradiated rats were significantly higher than for the control rats (at least p < 0.01; Figure 2(a) and (b)). There were no significant differences in the swimming speeds (Figure 2(c)) among the groups during all periods, indicating that the swimming speed did not have influence on the latencies. In the probe test, the percentage of path length traveled (male and female, p < 0.05; Figure 2(d)) together with the mean percentage of time spent (male
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Figure 2. (a) Morris water maze distance; (b) escape latencies; and (c) swimming speed. All groups of animals improved their water maze acquisition performance, that is, decreased their latency to find the platform from the first to the last trial. However, there was a notable rise in the distance and latency times of the rats exposed to mobile phones compared with the control rats during the training trails. (d) Effect of mobile phone exposure on percentage traveled; (e) time spent; and (f) crossing number in the target quadrant. Mobile phone animals spent less time in the target quadrant and needed more time to reach the target quadrant. Results are expressed as mean + SEM for four groups of animals according to trial group. *p < 0.05; **p < 0.01, ***p < 0.001 compared with the control groups.
and female, p < 0.5, Figure 2(e)) in the correct quadrant by EMF-exposed rats was significantly shorter when compared with the control groups. There was no significant difference between the phone-irradiated and control groups for the crossing number (Figure 2(f)) to the correct quadrant.
Electrophysiology studies The mean resting membrane potentials (male: 68.01 + 4.8 mV, female: 66.9 + 3.7 mV, n ¼ 8) in mobile phone-exposed rats were not significantly different compared with the control group (male: 63.31
+ 4.9 mV, female: 65.06 + 5.1, n ¼ 8). There was no significant difference between the groups regarding the mean membrane input resistance (control, male: 160.1 + 11.3 M, female: 156.0 + 10.4 M, n ¼ 8; EMF, male: 181.03 + 17.3 M, female: 172.0 + 22.4 M, n ¼ 8). Since there were no differences between male and female groups, data were presented as control and mobile phone groups. Comparison of the AP characteristics between pyramidal neurons recorded from the control and mobile phoneexposed rats revealed significant differences (Figures 3 and 4). Following 20–22 days of mobile phone irradiation, EMF-exposed rats showed a significant
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Figure 3. Whole-cell patch clamp recordings revealed that chronic EMF exposure with mobile phone during gestational period decreases the firing rate and increase AHP amplitude and AP half width in hippocampal pyramidal neurons. EMF: electromagnetic field; AHP: after hyperpolarization; AP: action potential.
increase in the amplitude of AHP in spontaneous activity (control: 2.08 + 0.13 mV; EMF:4.6 + 1.1 mV; p < 0.01, Figure 3), after positive injection currents (control: 4.1 + 0.2 mV; EMF:7.2 + 1.2 mV; p < 0.01, Figure 4) and AP duration at half-amplitude (control: 1.4 + 0.05 ms; EMF-exposed group: 2.2 + 0.06 ms; p < 0.05, Figure 3). Similarly, using a prepulse current injection protocol (Figure 4(b)), EMF exposure was associated with a reduction in number of AP elicited by all currents injection and this reduction was significant (n ¼ 8, p < 0.05). However, the amplitude and time to peak of the AP was not significantly altered in mobile phoneexposed rats compared with the control rats. Moreover, in the positive current protocol (Figure 4(a)), EMF exposure was associated with an approximately 25% reduction in AP number, but this change was not significant (p > 0.05).
Analysis of the repetitive firing properties of CA1 pyramidal neurons of EMF-exposed rats also revealed a significant decrease in the AP firing rate recorded from pyramidal neuronal in response to five consecutive depolarizing pulses with fixed amplitude (200 pA) and duration (200 ms). Figure 5 demonstrates the representative traces showing a decrease in the active properties of hippocampal pyramidal neurons in EMF-exposed rats. In the 520 ms negative current protocol (Figure 6), mobile phone exposure was associated with a significant reduction (p < 0.01) of 50% decrease in the number of AP elicited by a 500-pA current injection. Moreover, first-spike latency was increased in EMFexposed group rats in comparison with the control rats (p < 0.01). No difference was observed among male and female rats in either control or EMF groups.
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Figure 4. Mobile phone during gestational period decreases neuronal excitability: (a) representative records of action potentials evoked by current injection (100–500 pA, 520 ms); (b) in current clamp, the voltage responses to negative current prepulse (300 pA, followed by 100–500 pA positive currents, 520 ms) were recorded from pyramidal neurons. Action potential number decreases with increasing current intensity under all conditions.
Histological studies Light microscopy study of brain sections of the control and mobile phone-exposed rats showed normal morphology (Figure 7). The dentate gyrus, CA1, CA2, and CA3 of the hippocampus from male and female control and mobile phone-exposed rats were intact.
Discussion With a rapid increase in the number of users of mobile phones in many countries, especially in the developed world, there is a growing interest in today’s society for the potential deleterious effects of radio frequency EMFs to the health of the general public. Moreover, since a great proportion of these EMFs are absorbed into the head and brain, there is an increasing concern for the effects of electromagnetic radiation on the development and function of brain. The aim of the present study was to elucidate the potential alterations in cognitive function of offspring rats exposed to chronic mobile phone exposure during their gestational period. We found that EMF exposure adversely affects the cognitive performance of rats using behavioral and electrophysiological techniques.
The effect of EMF on the cognitive function of adult animals has resulted in conflicting results (Cosquer et al., 2005; Dubreuil et al., 2003; Lai et al., 2005; Narayanan et al., 2009; Sonmez et al., 2010; Smythe and Costall, 2003), which may be partly attributed to the heterogeneous setting of these experiments. Nevertheless, studies investigating the effects of mobile-induced EMF on the cognitive performance of offspring rats exposed during the embryonic period are scarce. In two studies investigating the effects of EMF exposure to rat fetus, the authors did not observe any signs of embryotoxicity or teratogenicity in offspring (Ogawa et al., 2009; Takahashi et al., 2010). Arber and Lin previously showed that continuous exposure of neurons to microwaves for 60 min inhibited spontaneous activity (Arber and Lin, 1985). In a study carried out by Haghani et al. (2013), it was shown that maternal mobile phone exposure was capable to decrease the spontaneous activity in rat offspring. In contrast to our results, Wachtel et al. (1975) reported that firing rate increases following exposure to 1.5 GHz microwaves. However, it should be noted that the authors did not focus on the cognitive performance of
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Figure 5. Representative traces showing the differences in the action potential firing rate recorded from pyramidal neuronal somata in response to five consecutive depolarizing pulses with fixed amplitude (200 pA) and duration (200 ms). Action potential number decreases in all consecutive depolarizing pulses in mobile phone animals compared with the control groups.
animals. Our results are in line with these studies showing no significant alterations between the control and mobile phone-exposed rats in the light microscopic evaluations of brain sections of either. However, our study revealed altered electrophysiological characteristics among EMF-exposed fetus, which was also translated to behavioral changes in vivo. The effect of EMF on cell excitability is important in particular for the nerve cells (Tombini et al., 2012). The present study showed a decrease in neuronal excitability in the offspring of pregnant rats exposed to mobile phones. Our results indicate that mobile phone exposure was mostly associated with a decrease in the number of APs fired in spontaneous activity and in response to current injection in both
male and female groups and an increase in the amplitude of the AHP in mobile phone rats, thus significantly dampening neuronal excitability. These changes in excitability indicate that EMF is associated with impairment in neuronal function when hippocampus slices are used as in vitro measurements (Tattersall et al., 2001). Changes in neuronal excitability that are often related with learning (Sanchez-Vives et al., 2000; Seroussi et al., 2002) could be determined by modulating the amplitude and duration of AHP that follow the APs in nerve cells. In hippocampus pyramidal neurons, calcium ion-activated potassium (Kþ) channels are responsible for a fast AHP occurring immediately after APs (Shao et al., 2004). Lockridge and Yuan (2011) reported A-type Kþ channels that have been implicated in learning and memory
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Figure 6. Recordings of the latency to the first spike in a representative cell in control and mobile phone-exposed conditions. Hippocampal CA1 pyramidal neurons in mobile phone-exposed rats exhibited a long delay to the first spike compared with either control groups. Rebound action potential number decreases with increasing current intensity in 500 pA condition.
Figure 7. Light microscopy study of brain sections of the control and mobile phone-exposed rats showed normal morphology.
could also influence the amplitude of AHP in rat hippocampal CA1 pyramidal neurons. In another study, Xu et al. (2010) reported chronic exposure (15 min/
day for 8 days) to cell phone signal (1800 MHz, 2.4 W/kg) in rat-cultured hippocampal neurons decreased both amplitude of a-amino-3-hydroxy-5-methyl-4isoxazole propionic acid miniature excitatory postsynaptic currents and the expression of postsynaptic density. Neural excitability is determined based on the interplay between synaptic inputs and intrinsic membrane characteristics (Antri et al., 2009). The electrophysiological findings of the present study demonstrated that maternal exposure to mobile phone irradiation led to profound alterations in the intrinsic membrane properties of rat CA1 pyramidal neurons. Changes in either synaptic activities or membrane intrinsic properties affected the output of the neuron (Schulz et al., 2006). These data indicate that chronic exposure to mobile phone significantly decreases excitatory activity in rat CA1 neurons. Maternal exposure with mobile phone irradiation caused a significant increase in the first-spike latency and a significant reduction in the AP rebound in prepulse current injections. Pyramidal neuronal excitability decreased in response to five consecutive depolarizing pulses. Mobile phone exposure also induced a significant increase in the latency to the first spike that was accompanied by a significant decrease in the rebound spike firing at the offset of hyperpolarization. The activation of A-type Kþ channels has been shown to regulate neuronal firing by affecting the AP onset time and threshold (Storm, 1988). This finding suggests a possible enhancement of transient Kþ outward channel currents in pyramidal cells from offsprings prenatally exposed to EMF. Recent studies have shown that blockade of IA channels with 4-APs in neurons affects not only the first-spike latency but also AHP and the frequency of spikes (Goudarzi et al., 2010; Haghani et al., 2012). Transient Kþ outward channels also play an important role in shaping the AP waveform and in controlling repetitive firing in nerve cells (Yuan et al., 2005). In our study, under current clamp conditions, the AP firing rate recorded from pyramidal neuronal in response to five consecutive depolarizing pulses was significantly decreased in the maternal mobile-exposed rats compared with control. In conclusion, EMF is able to induce a series of disruptions in learning and memory. Both male and female fetuses were sensitive to many behavioral effects following maternal exposure to mobile phone irradiation. While we did not provide sufficient evidence to support an exact mechanism of EMF interaction, the findings of the present study appear to be
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sufficiently robust to enable further studies to investigate this question in better detail. Acknowledgments The present article is the product of a research project that was approved by the Kerman University of Medical Science as a grant for the MsC thesis. The author would like to appreciate the heads of Kimia Mobin Company (Mobin Aghpour) for device design.
Conflict of interest The authors declared no conflict of interest. The author alone is responsible for the content and writing of the paper.
Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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altered intrinsic repetitive firing properties induced by Ab neurotoxicity. Neuroscience Letters 507: 33–37. Haghani M, Shabani M and Moazzami K (2013) Maternal mobile phone exposure adversely affects the electrophysiological properties of Purkinje neurons in rat offspring. Neuroscience 250: 588–598. Haghani M, Shabani M, Javan M, et al. (2012) CB1 cannabinoid receptor activation rescues amyloid b-induced alterations in behaviour and intrinsic electrophysiological properties of rat hippocampal CA1 pyramidal neurones. Cellular Physiology and Biochemistry 29: 391–406. Lai H, Horita A and Guy AW (2005) Microwave irradiation affects radial-arm maze performance in the rat. Bioelectromagnetics 15: 95–104. Lockridge A and Yuan LL (2011) Spatial learning deficits in mice lacking A-type Kþ channel subunits. Hippocampus 21: 1152–1156. Narayanan SN, Kumar RS, Potu BK, et al. (2009) Spatial memory perfomance of wistar rats exposed to mobile phone. Clinics 64: 231–234. Odaci E, Bas O and Kaplan S (2008) Effects of prenatal exposure to a 900 MHz electromagnetic field on the dentate gyrus of rats: a stereological and histopathological study. Brain Research 1238: 224–229. Ogawa K, Nabae K, Wang J, et al. (2009) Effects of gestational exposure to 1.95-GHz W-CDMA signals for IMT2000 cellular phones: lack of embryotoxicity and teratogenicity in rats. Bioelectromagnetics 30: 205–212. Ragbetli MC, Aydinlioglu A, Koyun N, et al. (2010) The effect of mobile phone on the number of Purkinje cells: a stereological study. International Journal of Radiation Biology 86: 548–554. Sanchez-Vives MV, Nowak LG and Mccormick DA (2000) Cellular mechanisms of long-lasting adaptation in visual cortical neurons in vitro. The Journal of Neuroscience 20: 4286–4299. Scho¨nborn F, Burkhardt M and Kuster N (1998) Differences in energy absorption between heads of adults and children in the near field of sources. Health Physics 74: 160–168. Schulz DJ, Baines RA, Hempel CM, et al. (2006) Cellular excitability and the regulation of functional neuronal identity: from gene expression to neuromodulation. The Journal of Neuroscience 26: 10362–10367. Seroussi Y, Brosh I and Barkai E (2002) Short communication learning-induced reduction in post-burst afterhyperpolarization (AHP) is mediated by activation of PKC. European Journal of Neuroscience 16: 965–969. Shabani M, Larizadeh MH, Parsania S, et al. (2012a) Profound destructive effects of adolescent exposure to vincristine accompanied with some sex differences in
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Maternal mobile phone exposure alters intrinsic electrophysiological properties of CA1 pyramidal neurons in rat offspring
Toxicology and Industrial Health 1–12 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233714525497 tih.sagepub.com
Moazamehosadat Razavinasab1, Kasra Moazzami2 and Mohammad Shabani1 Abstract Some studies have shown that exposure to electromagnetic field (EMF) may result in structural damage to neurons. In this study, we have elucidated the alteration in the hippocampal function of offspring Wistar rats (n ¼ 8 rats in each group) that were chronically exposed to mobile phones during their gestational period by applying behavioral, histological, and electrophysiological tests. Rats in the EMF group were exposed to 900 MHz pulsed-EMF irradiation for 6 h/day. Whole cell recordings in hippocampal pyramidal cells in the mobile phone groups did show a decrease in neuronal excitability. Mobile phone exposure was mostly associated with a decrease in the number of action potentials fired in spontaneous activity and in response to current injection in both male and female groups. There was an increase in the amplitude of the afterhyperpolarization (AHP) in mobile phone rats compared with the control. The results of the passive avoidance and Morris water maze assessment of learning and memory performance showed that phone exposure significantly altered learning acquisition and memory retention in male and female rats compared with the control rats. Light microscopy study of brain sections of the control and mobile phone-exposed rats showed normal morphology. Our results suggest that exposure to mobile phones adversely affects the cognitive performance of both female and male offspring rats using behavioral and electrophysiological techniques. Keywords Mobile phone exposure, pregnancy, neural excitability, offspring, pyramidal neurons, rat
Introduction Mobile phones release ultrahigh-frequency electromagnetic fields (EMF) around them when in use (Carballo-Quinta´s et al., 2011; Scho¨nborn et al., 1998). The close proximity of mobile phones to the user’s head leads to the absorption of part of the EMF into the head and brain (Carballo-Quinta´s et al., 2011; Scho¨nborn et al., 1998). The rapid increase in the use of mobile phones over the past few years has been accompanied by increasing some possible health concerns for the effects from their use. In particular, the possible adverse effects of EMF on the brain and cognitive functioning have been the subject of investigation by many groups (Cosquer et al., 2005; Dubreuil et al., 2003; Foroozandeh et al., 2012; Lai et al., 2005; Narayanan et al., 2009; Ragbetli et al., 2010; Sonmez et al., 2010; Smythe and Costall, 2003).
Results regarding the effects of EMF on cognition are highly variable and contradictory partly due to the different experimental conditions and the intrinsic phenomenological complexity. In some studies, EMF exposure induced adverse cognitive effects that were recorded using histological and behavioral measures.
1
Kerman Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Islamic Republic of Iran 2 Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Corresponding author: Mohammad Shabani, Kerman Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman 76198, Islamic Republic of Iran. Email: [email protected]
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These changes included diminished numbers of Purkinje cells and a tendency for granule cells to increase in cerebellum (Ragbetli et al., 2010; Sonmez et al., 2010), as well as disruption of spatial learning in a radial arm maze task and decreased acquisition of learned responses in rats (Lai et al., 2005; Narayanan et al., 2009). These observations, however, have not been supported by other groups, suggesting that EMF emitted from mobile phones does not decrease cognitive functioning (Cosquer et al., 2005; Dubreuil et al., 2003) or may even improve cognitive performance (Smythe and Costall, 2003). Previous reports suggest impairments in the development of the dentate gyrus by prenatal exposure to high-frequency EMF (Odaci et al., 2008). Recently, Haghani et al. (2013) also reported that maternal mobile phone exposure adversely affects the electrophysiological properties of Purkinje neurons in rat offspring. Also, 2.14 GHz signals applied for 20 h/day during the gestation period did not cause any adverse effects on pregnancy or the development of pups (Takahashi et al., 2010). However, detailed electrophysiological changes in rat offspring following EMF exposure have not been addressed adequately. Most studies assessing the effects of EMF on cognition have been focused on adult animals, while studies investigating the effects of occupational maternal exposure to EMF and the risk of cognitive impairment in offspring are lacking. Despite the fact that previous studies have shown lack of embryotoxicity and teratogenicity in rats receiving EMF (Ogawa et al., 2009; Takahashi et al., 2010), the effects on cognition has not been evaluated thoroughly. Therefore, the present study was designed to address the potential influences of gestational exposure to EMF on the cognitive performance in rats. Furthermore, since most cognitive tasks are widely accepted to have a substantial hippocampal involvement, the present study aimed to investigate the potential electrophysiological changes of CA1 pyramidal neurons of the hippocampus following prenatal EMF exposure.
Materials and methods Primiparous Wistar female rats, weighing 200–250 g, were housed for 2 weeks before mating at constant room temperature (22–25 C) with 12-h light and 12–h dark (7:00 a.m.–7:00 p.m.) and free access to food and water. Pairs of females were then placed with single male rats in the late afternoon. Vaginal smears or plugs were examined in the following
morning at 9:00 a.m. The day in which sperm was found has been considered as the gestation day (GD 0). Afterward, pregnant rats were randomly assigned to either a control group or to a mobile phone exposure group. The care of laboratory animals followed the guiding principles for care and use of laboratory animals of the Neuroscience Research Center of Kerman Medical University, Islamic Republic of Iran, and the study protocol was approved by the animal ethics committee of this institution [Code: EC/KNRC/88-34].
900 MHz Pulsed-EMF exposure conditions Pregnant rats were placed inside experimental cages on the first day of pregnancy. A total of 16 pregnant rats have been used, 8 rats in the control group and 8 rats in EMF-irradiated group. Control group was exposed to the same conditions as in the EMF group but without cellular phone. Pregnant rats were irradiated from 8 a.m. until 2.00 p.m. (each day in pregnancy period) for a total of 6 h/day, since the day of sperm detection (pregnancy start) until offspring birth. During radiofrequency exposure, rats were restrained in 5-mm thick Plexiglas with 25 cm length. Both the cellular phone and the Plexiglas cage were placed inside an aluminum Faraday cage. The field intensity was measured with a radiofrequency spectrum analyzer (Anritsu MS2711, Japan). The mobile phone was operated in test mode and was powered through a stabilized power supply, thus antenna power supply was constant. The measured specific absorption rate (SAR) value in our data was between 0.3 and 0.9 W/kg, a value without significant thermal effects. Temperature was measured before, during, and after the exposure by a rectal probe only in a series of pups that did not enter at behavioral and electrophysiological analysis. Before and after each experiment, a series of electromagnetic measurements (i.e. input power and reflected power) were performed to evaluate the correct operation of the exposure system. The average power density and SAR measurements were performed at an electronic mobile company. After each birth, mother and its offspring (both males and females) were removed from the Faraday cage and placed inside a separate Plexiglas cage. Pups were weaned at day 23 of age.
Slice preparation Male and female offspring (28–30-day-old) of either control (n ¼ 8) or maternal EMF-exposed
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group (n ¼ 8) was used. Animals were anesthetized with ether followed by decapitation. Brains were removed rapidly and placed in ice-cold artificial spinal fluid (ACSF) containing 206 mM sucrose, 2.8 mM potassium, chloride (KCl), 1 mM calcium chloride (CaCl2), 1 mM magnesium chloride (MgCl2), 2 mM magnesium sulfate (MgSO4), 1.25 mM monosodium phosphate (NaH2PO4), 26 mM sodium bicarbonate (NaHCO3), and 10 mM D-glucose and equilibrated to a pH of 7.4 (with 95% oxygen and 5% carbon dioxide); the osmolarity was adjusted to 295 mOsm. The hippocampi were dissected out of the brain, and 300 mm thick transverse slices were obtained using a vibrating microtome (752 M, Campden Instruments Ltd, UK). Following this procedure, slices were incubated in ACSF containing 124 mM sodium chloride, 2.8 mM KCl, 2 mM CaCl2, 2 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 10 mM D-glucose with pH 7.4 295 mOsm osmolarity for at least 60 min at 35 C and thereafter maintained at room temperature (22–25 C) before being transferred to a recording chamber. From each animal just one pyramidal neuron from one brain slice was recorded.
Whole-cell patch clamp recording Whole-cell patch clamp recordings in current clamp mode from pyramidal neurons were made, as described previously by Haghani et al. (2011), under direct visual control using differential interference contrast optics (BX 51WI; Olympus, Tokyo, Japan). CA1 pyramidal neurons were visualized with a 60 water immersion objective lens using Nomarski-type differential interference contrast imaging with infrared illumination. Images were captured with a charge-coupled device camera (ORCA, Hamamatsu, Japan). The slices were continuously superfused with normal ACSF (1–2 ml/min) at room temperature (22– 25 C). Whole-cell current clamp recordings were made from CA1 pyramidal neurons using Multiclamp 700B amplifiers (Axon Instruments, Foster City, California, USA) equipped with Digidata 1440 A/D converter (Axon Instruments). Electrophysiological responses were filtered at 5 kHz and sampled at 10 kHz and stored on a personal computer for offline analysis. Patch pipettes were pulled with an electrode puller (PC10; Narishige, Tokyo, Japan) and had a resistance of 3–6M when filled with internal solution containing 135 mM potassium methylsulfate, 10 mM KCl, 10 mM Hepes, 1 mM MgCl2, 2 mM adenosine triphosphate disodium salt, and 0.4 mM guanosine triphosphate sodium salt. The pH of the internal
solution was set to 7.3 by potassium hydroxide, and osmolarity was adjusted to 290 mOsm. To investigate the excitability of neurons, action potentials (APs) were induced in pyramidal cells from a holding potential of V ¼ 70 mV in 100 ms duration current steps ranging from 500 to þ500 pA in 100 pA increments. Before positive current steps, a negative prepulse protocol current with 300 pA was identified. To test the effect of EMF irradiation on excitability, the number of APs and APs rebound generated by these current injections and the amplitude of the corresponding after hyperpolarizations (AHP) were analyzed. AHP amplitude was taken as the absolute value of the difference between the voltage at holding and the voltage measured 10 ms after the peak of the AP using the 520 ms duration current injections. The AP amplitude and half-width were measured as peak distance from the resting membrane potential and the width at half amplitude, respectively. The AP threshold was defined as a measure of the voltage at the onset of the AP. The first-spike latency was defined as the time between the offset of the negative current steps and the peak of the first spike.
Behavioral tests Hippocampus-related behavioral dysfunctions in EMFexposed rats were analyzed using hippocampus functional tasks. Two behavioral tasks, namely, shuttle box and Morris water maze tasks were chosen.
Passive avoidance learning and memory test On postnatal day 30, the rats underwent passive avoidance training. The passive avoidance apparatus consisted of two equal-sized, connected chambers that were separated by a guillotine door. The rats were individually allowed to be habituated to the apparatus prior to testing. The rats from each experimental group were placed individually in the lighted chamber facing away from the entrance to the dark side; 10 s later, the guillotine door was raised, and the latency to enter the dark chamber was recorded. If the rats did not enter the dark chamber within 60 s, they were eliminated from the test and replaced with a new rat. The habituation trial was repeated after 5 min for the same interval. For the learning trial, after 2 h, the third adaption trial was administered in which an electrical stimulation (0.5 mA, 50 Hz, 2 s once) was delivered to the feet through the stainless steel floor after entering the dark chamber. After 20 s, the rats were removed from the dark compartment and returned to their own cage. After 5 min, the same test
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was conducted again, and if the rats did not enter the dark chamber by 300 s, the successful acquisition of passive avoidance response was recorded. In the retention trials that were performed after 1 day and 7 days of the learning trial (acquisition test) the rats were again individually placed in the illuminated chamber but no foot shock was administered if they entered the dark chamber. The rats were allowed to step into the dark compartment and then the latency to reenter the dark chamber was recorded. In both the learning and retention trials, the time to enter the dark section was recorded as the step-through latency. The maximum cutoff time for the step-through latency was 300 s when the rat did not enter the dark chamber in the retention trials (Van Der Stelt et al., 2006). Morris water maze task. The testing procedure was performed as previously described by Shabani et al. (2012b). The experimental apparatus consisted of a circular water tank (140 cm wide and 45 cm high). A platform, either visible or submerged, which is 15 cm wide and 35 cm high, was placed 1.5 cm above or below the surface of the water. The maze was located in a large and quiet test room, surrounded by many visual cues external to the maze, which were visible from within the pool and could be used by the rats for spatial location. The water temperature was 22 + 2 C. Data collection was automated by a video image motion analyzer (Ethovision, Noldus Information Technology, Netherlands). To assess for gross physical, sensory, motor, or motivational impairments, five rats from the control group and five rats from EMF-exposed group were first trained in a task with a visible escape platform. The rats were trained in the maze over 13 sessions on 4 consecutive days, 4 sessions on each day except on the last day, when only 1 session was given. The rats were towel dried on exiting the pool and then placed in pre-warmed cages with paper towel, where they were given a recuperation period of at least 30 min between trials. All the experimental groups were tested during the lights on period between 8:00 and 12:00 h. On each trial, rats were randomly released into the water from one of the four quadrants with their face toward the wall of the maze. During acquisition, the location of the platform remained constant, and the rats were allowed to swim for duration of 60 s to find the hidden platform. After the animal found the platform, it was allowed to remain there for 20–30 s and was then moved to an animal cage to wait for 20–30 s before the start of the next trial. The time and distance
needed to find the hidden platform were collected and analyzed later. A single probe trial was given 1 day after the last training trial to test the spatial memory in the water maze. In this trial, the platform was removed, and the rat was allowed to swim for 60 s. The time and distance spent in the target quadrant (quadrant 4) were analyzed as a measure of spatial memory retention (Shabani et al., 2012a, 2012b).
Histological evaluation Under deep anesthesia, the animals were killed and their brains were removed and immersed in 10% buffered formaldehyde at 4 C overnight. The brains were processed by standard method for light microscopy study. Coronal sections 1.6–2.8 mm posterior to bregma were cut at a thickness of 8 mm using a microtome. Neuronal injury in the hippocampus was assessed by staining sections with cresyl fast violet (Nissl staining).
Statistical analysis Initially, male and female rats in the control group were compared using unpaired sample t test, the results of which did not show any difference. Following maternal mobile phone exposure, sex differences were compared using two-way analysis of variance (ANOVA) analyses for all groups. To investigate the effect of the sex and learning trial in Morris water maze test, two-way ANOVAs for repeated measures were applied to measure the time and distance required to locate the hidden platform in both sexes. Two-way ANOVAs were used to calculate the effect of sex and 900 MHz pulsed-EMF exposure on the passive avoidance learning test, to probe data in shuttle box and Morris water maze tests, and finally to address the electrophysiological changes in intracellular recording. Individual comparisons were measured using Tukey’s comparisons test where appropriate. Data are expressed as means + SEM. Statistical significance was defined at p < 0.05. All computations were made using the Statistical Package for Social Sciences software package (Version 16.0; SPSS Inc., Chicago, Illinois, USA).
Results Behavioral tests Passive avoidance learning and memory test. The results of the passive avoidance assessment of learning and memory performance showed that the shock number
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Figure 1. Mobile phone exposure affected the acquisition of learned responses and retention latencies in the passive avoidance learning and memory test. (a) The phone-exposed animals exhibited higher shock number than the control animals in learning performance. (b,c) Mobile phone exposure significantly decreased 1 day and 7 days testing retention latencies. *p < 0.05, compared with the control groups. Saline and mobile M/F: Saline and mobile-exposed male/female.
was increased significantly in rats exposed to EMF in comparison with the control group (p < 0.05; Figure 1(a)). When the testing was performed 1 day after the shock experience (30 days post-EMF exposure), the step-through latency was significantly decreased in EMF-exposed rats (male: 144.9 + 35.2 s, female: 85.05 + 25.3 s) compared with the control rats (male: 234 + 30.4 s, female: 212.8 + 42.09 s; p < 0.05; Figure 1(b)). When the testing was performed 7 days following the shock experience (on postnatal day 37), the step-through latency was also significantly decreased in EMF-exposed rats (male: 62.07 + 7.09 s, female: 49.8 + 18.6 s) compared with the control rats (male: 130.8 + 12.02 s, female: 111.7 + 19.2 s; p < 0.05; Figure 1(c)). Prenatal exposure to mobile phone did not alter the time in dark compartment (in seconds) or crossing number in either days 1 or 7 following the shock experience (data not shown).
Morris water maze. The results of the training trial are depicted in Figure 2. The mobile phone-irradiated rats and the controls took progressively less time to locate the hidden platform over the course of the 12 trials during the training period. Data for the block effect in all rats showed a reduction in escape latencies and the swimming distance to locate the platform across blocks of trials, indicating spatial acquisition (p < 0.05). The path length and time latency at the second and third blocks for the male and female mobile phoneirradiated rats were significantly higher than for the control rats (at least p < 0.01; Figure 2(a) and (b)). There were no significant differences in the swimming speeds (Figure 2(c)) among the groups during all periods, indicating that the swimming speed did not have influence on the latencies. In the probe test, the percentage of path length traveled (male and female, p < 0.05; Figure 2(d)) together with the mean percentage of time spent (male
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Figure 2. (a) Morris water maze distance; (b) escape latencies; and (c) swimming speed. All groups of animals improved their water maze acquisition performance, that is, decreased their latency to find the platform from the first to the last trial. However, there was a notable rise in the distance and latency times of the rats exposed to mobile phones compared with the control rats during the training trails. (d) Effect of mobile phone exposure on percentage traveled; (e) time spent; and (f) crossing number in the target quadrant. Mobile phone animals spent less time in the target quadrant and needed more time to reach the target quadrant. Results are expressed as mean + SEM for four groups of animals according to trial group. *p < 0.05; **p < 0.01, ***p < 0.001 compared with the control groups.
and female, p < 0.5, Figure 2(e)) in the correct quadrant by EMF-exposed rats was significantly shorter when compared with the control groups. There was no significant difference between the phone-irradiated and control groups for the crossing number (Figure 2(f)) to the correct quadrant.
Electrophysiology studies The mean resting membrane potentials (male: 68.01 + 4.8 mV, female: 66.9 + 3.7 mV, n ¼ 8) in mobile phone-exposed rats were not significantly different compared with the control group (male: 63.31
+ 4.9 mV, female: 65.06 + 5.1, n ¼ 8). There was no significant difference between the groups regarding the mean membrane input resistance (control, male: 160.1 + 11.3 M, female: 156.0 + 10.4 M, n ¼ 8; EMF, male: 181.03 + 17.3 M, female: 172.0 + 22.4 M, n ¼ 8). Since there were no differences between male and female groups, data were presented as control and mobile phone groups. Comparison of the AP characteristics between pyramidal neurons recorded from the control and mobile phoneexposed rats revealed significant differences (Figures 3 and 4). Following 20–22 days of mobile phone irradiation, EMF-exposed rats showed a significant
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Figure 3. Whole-cell patch clamp recordings revealed that chronic EMF exposure with mobile phone during gestational period decreases the firing rate and increase AHP amplitude and AP half width in hippocampal pyramidal neurons. EMF: electromagnetic field; AHP: after hyperpolarization; AP: action potential.
increase in the amplitude of AHP in spontaneous activity (control: 2.08 + 0.13 mV; EMF:4.6 + 1.1 mV; p < 0.01, Figure 3), after positive injection currents (control: 4.1 + 0.2 mV; EMF:7.2 + 1.2 mV; p < 0.01, Figure 4) and AP duration at half-amplitude (control: 1.4 + 0.05 ms; EMF-exposed group: 2.2 + 0.06 ms; p < 0.05, Figure 3). Similarly, using a prepulse current injection protocol (Figure 4(b)), EMF exposure was associated with a reduction in number of AP elicited by all currents injection and this reduction was significant (n ¼ 8, p < 0.05). However, the amplitude and time to peak of the AP was not significantly altered in mobile phoneexposed rats compared with the control rats. Moreover, in the positive current protocol (Figure 4(a)), EMF exposure was associated with an approximately 25% reduction in AP number, but this change was not significant (p > 0.05).
Analysis of the repetitive firing properties of CA1 pyramidal neurons of EMF-exposed rats also revealed a significant decrease in the AP firing rate recorded from pyramidal neuronal in response to five consecutive depolarizing pulses with fixed amplitude (200 pA) and duration (200 ms). Figure 5 demonstrates the representative traces showing a decrease in the active properties of hippocampal pyramidal neurons in EMF-exposed rats. In the 520 ms negative current protocol (Figure 6), mobile phone exposure was associated with a significant reduction (p < 0.01) of 50% decrease in the number of AP elicited by a 500-pA current injection. Moreover, first-spike latency was increased in EMFexposed group rats in comparison with the control rats (p < 0.01). No difference was observed among male and female rats in either control or EMF groups.
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Figure 4. Mobile phone during gestational period decreases neuronal excitability: (a) representative records of action potentials evoked by current injection (100–500 pA, 520 ms); (b) in current clamp, the voltage responses to negative current prepulse (300 pA, followed by 100–500 pA positive currents, 520 ms) were recorded from pyramidal neurons. Action potential number decreases with increasing current intensity under all conditions.
Histological studies Light microscopy study of brain sections of the control and mobile phone-exposed rats showed normal morphology (Figure 7). The dentate gyrus, CA1, CA2, and CA3 of the hippocampus from male and female control and mobile phone-exposed rats were intact.
Discussion With a rapid increase in the number of users of mobile phones in many countries, especially in the developed world, there is a growing interest in today’s society for the potential deleterious effects of radio frequency EMFs to the health of the general public. Moreover, since a great proportion of these EMFs are absorbed into the head and brain, there is an increasing concern for the effects of electromagnetic radiation on the development and function of brain. The aim of the present study was to elucidate the potential alterations in cognitive function of offspring rats exposed to chronic mobile phone exposure during their gestational period. We found that EMF exposure adversely affects the cognitive performance of rats using behavioral and electrophysiological techniques.
The effect of EMF on the cognitive function of adult animals has resulted in conflicting results (Cosquer et al., 2005; Dubreuil et al., 2003; Lai et al., 2005; Narayanan et al., 2009; Sonmez et al., 2010; Smythe and Costall, 2003), which may be partly attributed to the heterogeneous setting of these experiments. Nevertheless, studies investigating the effects of mobile-induced EMF on the cognitive performance of offspring rats exposed during the embryonic period are scarce. In two studies investigating the effects of EMF exposure to rat fetus, the authors did not observe any signs of embryotoxicity or teratogenicity in offspring (Ogawa et al., 2009; Takahashi et al., 2010). Arber and Lin previously showed that continuous exposure of neurons to microwaves for 60 min inhibited spontaneous activity (Arber and Lin, 1985). In a study carried out by Haghani et al. (2013), it was shown that maternal mobile phone exposure was capable to decrease the spontaneous activity in rat offspring. In contrast to our results, Wachtel et al. (1975) reported that firing rate increases following exposure to 1.5 GHz microwaves. However, it should be noted that the authors did not focus on the cognitive performance of
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Figure 5. Representative traces showing the differences in the action potential firing rate recorded from pyramidal neuronal somata in response to five consecutive depolarizing pulses with fixed amplitude (200 pA) and duration (200 ms). Action potential number decreases in all consecutive depolarizing pulses in mobile phone animals compared with the control groups.
animals. Our results are in line with these studies showing no significant alterations between the control and mobile phone-exposed rats in the light microscopic evaluations of brain sections of either. However, our study revealed altered electrophysiological characteristics among EMF-exposed fetus, which was also translated to behavioral changes in vivo. The effect of EMF on cell excitability is important in particular for the nerve cells (Tombini et al., 2012). The present study showed a decrease in neuronal excitability in the offspring of pregnant rats exposed to mobile phones. Our results indicate that mobile phone exposure was mostly associated with a decrease in the number of APs fired in spontaneous activity and in response to current injection in both
male and female groups and an increase in the amplitude of the AHP in mobile phone rats, thus significantly dampening neuronal excitability. These changes in excitability indicate that EMF is associated with impairment in neuronal function when hippocampus slices are used as in vitro measurements (Tattersall et al., 2001). Changes in neuronal excitability that are often related with learning (Sanchez-Vives et al., 2000; Seroussi et al., 2002) could be determined by modulating the amplitude and duration of AHP that follow the APs in nerve cells. In hippocampus pyramidal neurons, calcium ion-activated potassium (Kþ) channels are responsible for a fast AHP occurring immediately after APs (Shao et al., 2004). Lockridge and Yuan (2011) reported A-type Kþ channels that have been implicated in learning and memory
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Figure 6. Recordings of the latency to the first spike in a representative cell in control and mobile phone-exposed conditions. Hippocampal CA1 pyramidal neurons in mobile phone-exposed rats exhibited a long delay to the first spike compared with either control groups. Rebound action potential number decreases with increasing current intensity in 500 pA condition.
Figure 7. Light microscopy study of brain sections of the control and mobile phone-exposed rats showed normal morphology.
could also influence the amplitude of AHP in rat hippocampal CA1 pyramidal neurons. In another study, Xu et al. (2010) reported chronic exposure (15 min/
day for 8 days) to cell phone signal (1800 MHz, 2.4 W/kg) in rat-cultured hippocampal neurons decreased both amplitude of a-amino-3-hydroxy-5-methyl-4isoxazole propionic acid miniature excitatory postsynaptic currents and the expression of postsynaptic density. Neural excitability is determined based on the interplay between synaptic inputs and intrinsic membrane characteristics (Antri et al., 2009). The electrophysiological findings of the present study demonstrated that maternal exposure to mobile phone irradiation led to profound alterations in the intrinsic membrane properties of rat CA1 pyramidal neurons. Changes in either synaptic activities or membrane intrinsic properties affected the output of the neuron (Schulz et al., 2006). These data indicate that chronic exposure to mobile phone significantly decreases excitatory activity in rat CA1 neurons. Maternal exposure with mobile phone irradiation caused a significant increase in the first-spike latency and a significant reduction in the AP rebound in prepulse current injections. Pyramidal neuronal excitability decreased in response to five consecutive depolarizing pulses. Mobile phone exposure also induced a significant increase in the latency to the first spike that was accompanied by a significant decrease in the rebound spike firing at the offset of hyperpolarization. The activation of A-type Kþ channels has been shown to regulate neuronal firing by affecting the AP onset time and threshold (Storm, 1988). This finding suggests a possible enhancement of transient Kþ outward channel currents in pyramidal cells from offsprings prenatally exposed to EMF. Recent studies have shown that blockade of IA channels with 4-APs in neurons affects not only the first-spike latency but also AHP and the frequency of spikes (Goudarzi et al., 2010; Haghani et al., 2012). Transient Kþ outward channels also play an important role in shaping the AP waveform and in controlling repetitive firing in nerve cells (Yuan et al., 2005). In our study, under current clamp conditions, the AP firing rate recorded from pyramidal neuronal in response to five consecutive depolarizing pulses was significantly decreased in the maternal mobile-exposed rats compared with control. In conclusion, EMF is able to induce a series of disruptions in learning and memory. Both male and female fetuses were sensitive to many behavioral effects following maternal exposure to mobile phone irradiation. While we did not provide sufficient evidence to support an exact mechanism of EMF interaction, the findings of the present study appear to be
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sufficiently robust to enable further studies to investigate this question in better detail. Acknowledgments The present article is the product of a research project that was approved by the Kerman University of Medical Science as a grant for the MsC thesis. The author would like to appreciate the heads of Kimia Mobin Company (Mobin Aghpour) for device design.
Conflict of interest The authors declared no conflict of interest. The author alone is responsible for the content and writing of the paper.
Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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