Decreased dopamine in striatum and difficult locomotor recovery from MPTP insult after exposure to radiofrequency electromagnetic fields

Abstract

Concern is growing about possible neuronal effects of human exposure to radiofrequency electromagnetic fields because of the increasing usage of cell phones and the close proximity of these devices to the brain when in use. We found that exposure to a radiofrequency electromagnetic field (RF-EMF) of 835 MHz (4.0 W/kg specific absorption rate [SAR] for 5 h/day for 12 weeks) affects striatal neurons in C57BL/6 mice. The number of synaptic vesicles (SVs) in striatal presynaptic boutons was significantly decreased after RF-EMF exposure. The expression levels of synapsin I and II were also significantly decreased in the striatum of the RF-EMF-exposed group. RF-EMF exposure led to a reduction in dopamine concentration in the striatum and also to a decrease in the expression of tyrosine hydroxylase in striatal neurons. Furthermore, in behavioral tests, exposure to RF-EMF impeded the recovery of locomotor activities after repeated treatments with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). These results suggest that the observed decrease in dopamine concentration in the striatum was caused by both a reduction in the number of dopaminergic neurons and a decline in the number of SVs. The decreased dopamine neuron numbers and concentration seen after RF-EMF exposure would have caused the difficult recovery after MPTP treatment. In summary, our results strongly suggest that exposing the brain to RF-EMF can decrease the number of SVs and dopaminergic neurons in the striatum. These primary changes impair the recovery of locomotor activities following MPTP damage to the striatum.

Figure 1

Expression levels of synapsin genes in the mouse striatum change after RF-EMF exposure. (A–C) Striatal total RNA was extracted from sham-exposed and RF-EMF-exposed mice and analyzed for synapsin I/II/III expression levels. The relative mRNA levels of synapsin I/II/III were normalized to the expression level of GAPDH by the 2^−ΔΔCt method. (D) Shown are the expression levels of synapsin genes by sqRT-PCR followed by electrophoresis on 1.5% agarose gel. Cropped, representative gel images are shown. The expression values in the striatum of RF-EMF-exposed mice were normalized to those of the sham-exposed mice. Each bar represents the mean ± SEM of three independent experiments. Statistical significance was evaluated using a two-tailed, unpaired Student’s t-test (*P < 0.05; **P < 0.01).

Figure 2

Expression levels of synapsin I/II proteins in the striatum of RF-EMF-exposed mice. (A) Total proteins extracted from the striatum of mice were subjected to SDS-PAGE electrophoresis and immunoblotted with antibodies against synapsin I and synapsin II. The expression levels of synapsin I/II are significantly reduced by RF-EMF exposure. Cropped, representative gel images are shown. (B) The protein levels of synapsin I/II were normalized relative to α-tubulin. Each bar shows the mean ± SEM of three independent experiments. (C) Representative immunofluorescence images of synapsin I/II in striatum. Scale bar = 100 μm. (D) Relative optical density (ROD). The ratio of the ROD was calibrated as %, with the sham designated as 100%. Each bar represents the mean ± SEM. Statistical significance was evaluated using two-tailed unpaired Student’s t-test (**P < 0.01, ****P < 0.0001).

Figure 3

Changes in the number and size of SVs in mouse striatum after RF-EMF exposure. (A) Representative TEM micrographs of the synaptic region in the striatum were acquired from sham- (a, b and c) and RF-EMF-exposed (RF) mice (d, e and f), respectively. M, mitochondria; Pre-SN, pre synaptic neuron; Post-SN, post synaptic neuron; SVs, synaptic vesicles; scale bars, 500 nm. (B,C) Comparison of SV density and size between sham- and RF-EMF exposed mice. 30–31 synapses were randomly chosen in each condition. (B) Shown are numbers of SVs per square micron (C) Shown are the cross-sectional areas (nm²) of the SVs. Each bar represents the mean ± SEM. Statistical significance was evaluated using two-tailed unpaired Student’s t-test (****P < 0.0001).

Figure 4

Striatal dopamine concentration decreases after RF-EMF exposure. (A) Shown are the measured striatal dopamine levels. (B) Internal calibration for dopamine analysis. Each bar represents the mean ± SEM. Statistical significance was evaluated using two-tailed, unpaired Student’s t-test (*P < 0.05).

Figure 5

Striatal TH is down-regulated after RF-EMF exposure. (A) Total proteins were subjected to SDS-PAGE electrophoresis and immunoblotted with anti-TH antibody. Cropped, representative gel images are shown. (B) Shown is quantified band intensity. The TH protein level was normalized to α-tubulin. Each bar shows the mean ± SEM of three independent experiments. Statistical significance was evaluated using two-tailed unpaired Student’s t-test (**P < 0.01).

Figure 6

Striatal dopaminergic terminals are reduced after RF-EMF exposure. TH immunohistochemical analysis in substantia nigra pars compacta (SNpc) (A,B) and striatum (C,D) in sham-exposed (A,C) and RF-EMF exposed (B,D) mice. TH immunoreactivities are markedly decreased in the SNpc and striatum in the RF-EMF-exposed group. Scale bar = 100 μm. (E) Relative optical density (ROD) as % for TH-immunoreactive structures in the striatum in the sham- and RF-EMF groups. Bars indicate mean ± SEM. Statistical significance was evaluated using two-tailed, unpaired Student’s t-test (**P < 0.01; vs. sham).

Figure 7

General locomotor activities and basic motor activities before and after MPTP administration. Sham-exposed and RF-EMF exposed mice were treated with 20 mg/kg MPTP for 3 days at regular intervals and examined over five days for deficits of locomotor activity and basic motor activity before/after MPTP administration. General locomotor activity measured total moving distance (A), mean moving distance (B), total moving duration (C), total moving duration (D), mean moving duration (E), and rearing frequency (F) in the open field behavioral test and basic motor activity measured latency to fall (G) by rotarod test. Each bar shows the mean ± SEM of 8 mice. Statistical significance was evaluated using the Student’s t-test (*P < 0.05; **P < 0.01; both sham vs. RF-EMF-exposed group).