In order to determine if in-utero cell phone radiation exposure affects behavior we chose to conduct a battery of tests that identify impairments in memory, hyperactivity, anxiety and fear, which are often associated with ADHD. Thirty-three female mice were exposed throughout gestation (days 1–17) to radiation from muted and silenced 800–1900 Mhz cellular phones with a SAR of 1.6 W/kg. The phones were positioned above each cage over the feeding bottle area at a distance of 4.5–22.3 cm from each mouse, depending on the location of the animal within the cage and placed on an uninterrupted active call for the duration of the trial. A control group of forty-two female mice was kept concurrently under the same conditions, however using a deactivated phone. Parturition was not different between groups and occurred at 19 days ± 1 day. In order to evaluate memory in the exposed and unexposed mice, 161 progeny were given a standard object recognition memory test in three different cohorts at 8, 12 and 16 weeks of age (82 experimental and 79 control mice). The mice were allowed to explore two identical objects for 15 minutes per day for two days and on the third day one object was replaced with a novel object. On day 3 the mice were filmed for 5 minutes exploring the novel and familiar objects. Three observers, blinded to the treatment, viewed the footage and recorded the exploration time for the novel and familiar objects. The preference index was defined as the time spent exploring the new object divided by the time spent exploring both the new and old object, multiplied by one hundred. A decrease in preference index indicates diminished memory. The preference index of the experimental group at 8, 12 and 16 weeks was less than the control and the results were significant at each time point [Figure 1]. The mean preference index in the exposed group was 56.8, 69.4 and 63.5 compared to 66.5, 71.7 and 71.2 in the control group at 8, 12 and 16 weeks, respectively. The experimental group had a cumulative mean preference index of 63.0% and the control group 69.9% (p = 0.003, n = 161, t test). Compared to the control group, the exposed mice had a significantly lower mean preference index suggesting impairment in memory [Figure 1]. In order to ensure that our findings are in fact due to memory deficits and not distractibility or hyperactivity we calculated the percent time spent idle - not exploring either of the objects. The mean idle time in the exposed group was 90.06, 90.53 and 96.48 compared to 92.12, 91.89 and 97.07 in the control group at 8, 12 and 16 weeks, respectively. The control group had a cumulative mean idle time of 90.8% while the experimental group had a cumulative mean idle time of 90.4% and the difference between the two groups was not statistically significant (p = .58).

Figure 1 Behavioral testing in exposed and control mice. The left column displays the data determined in mice at several ages after exposure. The right column demonstrates the cumulative average. To test memory the Standard object recognition memory test was used and a Preference Index (percent of total exploration time spent exploring the new object) shown at 8, 12 and 16 weeks of age. The cumulative mean preference index of the experimental group was 63.0% and the control group 69.9% (*p = 0.003, n = 161). To test hyperactivity we used the Light/Dark box test and display transitions at 12, 15 and 18 weeks of age. The cumulative mean number of transitions in the experimental group was 24.4 and the control group 16.4 (*p = <0.001, n = 141). To test anxiety we measured time spent in the dark at 12, 15 and 18 weeks of age. The cumulative average time spent in the dark in the experimental group was 207 seconds and in the control was 234 seconds (*p < 0.001, n = 141). To measure fear we used the Step down assay and display the time spent on the platform at 12 weeks and adulthood. The cumulative mean time spent on the platform in the experimental group was 16.7 seconds and in the control was 18.5 seconds (p = 0.59, n = 98). Full size image

To explore fearful behavior we performed the light/dark box test measuring hyperactivity/anxiety and the step down assay assessing fear of exploring the environment. The light/dark box test measures anxiety using a rodent's natural aversion to bright light14. The box contained two compartments: one white compartment that was illuminated and one black compartment that remained dark. The number of transitions between the two compartments was used to determine locomotion and in turn hyperactivity15. Anxious behavior is measured by recording the time spent in each compartment15. A total of 141 progeny were given the light/dark box test in three different cohorts at 12, 15 and 18 weeks of age (71 experimental and 70 control mice). Each mouse was placed in the light/dark box for 5 minutes and filmed. Three observers, blinded to the treatment regimen, viewed the footage and recorded the time spent in the dark compartment along with the number of transitions between each compartment. The average number of transitions in the experimental group at 12, 15 and 18 weeks was fewer than in respective controls and the results were significant at each time point [Figure 1]. The average number of transitions in exposed mice was 29.9, 32.5 and 14.8 compared to 23.9, 13.8 and 6.5 in the control group at 12, 15 and 18 weeks, respectively. The experimental group showed a cumulative mean of 24.4 transitions and the control group showed a mean of 16.4 transitions (p <0.001). Compared to the control group, the greater number of transitions between the two compartments in the experimental group suggested hyperactive behavior [Figure 1].

To identify whether anxiety might be a factor contributing to the behavioral phenotype reported in the light/dark box experiment, we first compared the duration of time in the dark versus the time spent in the light. An increased time in the dark indicates anxious behavior15. At 12, 15 and 18 weeks the experimental group spent less time in the dark and the results were significant at each time point [Figure 1]. The duration of time in darkness of the exposed group was 210.8, 187.0 and 235.8 seconds compared to 225.6, 215.5 and 270.6 seconds in the control group at 12, 15 and 18 weeks, respectively. The mice exposed in utero spent a cumulative average of 207 seconds in the dark while the control mice spent an average of 234 seconds in the dark indicating decreased anxiety in the cellular phone exposed mice (p < 0.001) [Figure 1].

The Step Down Assay was performed on 98 mice at 12 weeks and in adulthood to determine fear of exploring the environment (51 control and 47 experimental mice). The test is performed by recording the time spent on a standard platform. A greater period of time on the platform indicates increased fearfulness. Exposed mice showed no significant difference in time spent on the platform when compared to the controls [Figure 1]. The control mice spent an average of 18.5 seconds while the experimental group spent an average of 16.7 seconds (p = 0.59) [Figure 1].

Overall, the mice exposed in-utero to radiation were hyperactive, had decreased memory and decreased anxiety.

To understand the mechanisms underlying the changes in the memory and hyperactivity in animals exposed to radiation in-utero, we examined whether changes in the neuronal circuitry occurred in brain areas responsible for these compromised behaviors. Specifically, we asked whether changes in the synaptic transmission in CNS neurons are responsible for impaired memory and hyperactivity in radiation-exposed animals. The prefrontal cortex (PFC) is responsible for executive functions by screening distractions and maintaining attention in goal-oriented behaviors. Impairment of the PFC leads to dysregulated behavior/emotion such as ADHD16. The pyramidal neurons, the primary cell type in this structure, regulate attention and behavior through a complex and interconnected network. Whole cell patch clamp recordings of miniature excitatory postsynaptic currents (mEPSCs) were performed in pyramidal neurons of the PFC in control and cell phone-exposed mice. mEPSCs were generated by random vesicle release of glutamate from presynaptic neurons in the absence of stimulation. The measurement of mEPSCs is used to analyze the efficacy of synaptic transmission. Changes in mEPSC frequency are thought to result from modification of the presynaptic component of synaptic transmission, while amplitude changes indicate alterations in the postsynaptic component17,18. Coronal prefrontal cortex slices (300 μm) were prepared from 3–4 week old mice. mEPSCs were recorded in layer V pyramidal neurons in the prefrontal cortex in mice exposed to in-utero radiation for 9, 15 and 24 hours/day throughout gestation; the detection and analysis of mEPSC frequency and amplitude were performed as we described previously18. In animals exposed to in-utero radiation for 24 hours/day, a decrease in the frequency of mEPSCs was seen (control: 1.00 ± 0.12 Hz, n = 40; 24 hours/day: 0.72 ± 0.06 Hz, n = 43, p<0.05, t test, Figure 2A and B). The cumulative probability curves for the amplitude of mEPSC events recorded from the in utero cell phone-exposed mice (24 hours/day) shifted significantly to the left relative to those recorded from the controls (P<0.01, Kolmogorov-Smirnov test; control: 2765 events, cell phone exposure: 2224 events), indicating that the amplitude of mEPSCs was decreased [Figure 2C]. In a subset of experiments, we examined whether the reduction of mEPSC frequency depended on dosages of exposure in mice prenatally exposed 0, 9, 15 and 24 hours per day [Figure 3]. The trend of the dose-dependent decrease in the frequency of mEPSCs (0 hour/day: 1.37±0.41, n = 9; 9 hours/day: 1.27 ± 0.21 Hz, n = 9; 15 hours/day: 1.04 ± 0.20 Hz, n = 10; 24 hours/day: 0.72± 0.13, n = 11) was statistically significant (linear correlation: Correlation Coef = −0.97, Unadjusted r2 = 0.94, P<0.05).

Figure 2 Synaptic efficacy of glutamatergic synapses is decreased in brain neurons of mice after prenatal exposure to cell phone radiation. A–C, mEPSCs were recorded in layer V pyramidal neurons of the prefrontal cortex. Representative traces of mEPSCs from control and cell phone exposure groups are shown in A. mEPSC frequency and cumulative probability of mEPSC amplitude from both groups are shown in B (*, P<0.05, t test) and C (**, P<0.01, Kolmogorov-Smirnov test; controls, 2225 events; Exposed, 2766 events). D–F, representative traces, frequency and amplitude of mEPSCs recorded in neurons in the VMH are shown. *, P<0.05, Kolmogorov-Smirnov test; Control: 2161 events, Cell phone group: 2261 events. Full size image

Figure 3 A dose-dependent attenuation in the frequency of mEPSCs in layer V pyramidal neurons in mice. The frequency of mEPSCs recorded in mice prenatally exposed to cell phone radiation at of dose of 0, 9, 15 and 24 hrs per day are shown. Error bars are SEM. The dose responsive relationship is determined using regression analysis (Correlation coefficient, −0.97; r2, 0.94; P<0.05). Full size image

Altogether, these results indicate that synaptic efficacy of glutamatergic transmission decreases at both pre- and postsynaptic sites in layer V pyramidal neurons. Thus, we demonstrate impairment in glutamatergic transmission (release from nerve terminals and glutamate receptor response) onto pyramidal neurons in the PFC after in-utero exposure to radiation from cellular telephones.

In a parallel experiment we examined whether in-utero radiation exposure led to changes in synaptic transmission in another brain area. mEPSCs were recorded in neurons in the ventral medial hypothalamus (VMH), a brain area implicated in the regulation of energy homeostasis19,20. Our results indicated that in mice exposed to radiation for 24 hours/day, the frequency of mEPSCs (control: 8.13±1.20 Hz, n = 14; cell phone radiation: 8.32±1.17 Hz, n = 14) was not significantly different from that in control mice (P>0.05, t test, Figure 2D and E). However, the cumulative probability of mEPSC amplitude recorded in radiation-exposed mice significantly shifted to the left (P<0.05, Kolmogorov-Smirnov test; control: 2161 events, cell phone group: 2261 events; Figure 2F), suggesting that the amplitude of mEPSCs is smaller in the cell-phone exposed group than in controls. This result implies that an impairment of glutamatergic transmission occurs at the postsynaptic site. In summary, our results suggest that the effects of prenatal exposure to the cell phone radiation were not limited to the cortex.

Maternal stress can alter fetal development by increasing offspring exposure to corticosterone, causing cognitive deficits, hyperactivity and alterations of the hypothalamo-pituitary-adrenal axis21. In order to exclude the possibility that impaired memory and behavior in exposed mice was caused by stress resulting from experimental manipulation, we measured serum corticosterone levels on day twelve of gestation using an ELISA assay. The mean corticosterone level in the exposed female mice (69.91 ng/ml, n = 6) was not significantly different from that in the control females (69.94 ng/ml, n = 6) [Figure 4], eliminating stress as a source of the observed behavioral and electrophysiologic differences.