Exposure to RF-EMF Alters Postsynaptic Structure and Hinders Neurite Outgrowth in Developing Hippocampal Neurons of Early Postnatal Mice

Abstract

Exposure to radiofrequency electromagnetic fields (RF-EMFs) has increased rapidly in children, but information on the effects of RF-EMF exposure to the central nervous system in children is limited. In this study, pups and dams were exposed to whole-body RF-EMF at 4.0 W/kg specific absorption rate (SAR) for 5 h per day for 4 weeks (from postnatal day (P) 1 to P28). The effects of RF-EMF exposure on neurons were evaluated by using both pups' hippocampus and primary cultured hippocampal neurons. The total number of dendritic spines showed statistically significant decreases in the dentate gyrus (DG) but was not altered in the cornu ammonis (CA1) in hippocampal neurons. In particular, the number of mushroom-type dendritic spines showed statistically significant decreases in the CA1 and DG. The expression of glutamate receptors was decreased in mushroom-type dendritic spines in the CA1 and DG of hippocampal neurons following RF-EMF exposure. The expression of brain-derived neurotrophic factor (BDNF) in the CA1 and DG was significantly lower statistically in RF-EMF-exposed mice. The number of post-synaptic density protein 95 (PSD95) puncta gradually increased over time but was significantly decreased statistically at days in vitro (DIV) 5, 7, and 9 following RF-EMF exposure. Decreased BDNF expression was restricted to the soma and was not observed in neurites of hippocampal neurons following RF-EMF exposure. The length of neurite outgrowth and number of branches showed statistically significant decreases, but no changes in the soma size of hippocampal neurons were observed. Further, the memory index showed statistically significant decreases in RF-EMF-exposed mice, suggesting that decreased synaptic density following RF-EMF exposure at early developmental stages may affect memory function. Collectively, these data suggest that hindered neuronal outgrowth following RF-EMF exposure may decrease overall synaptic density during early neurite development of hippocampal neurons.

Keywords: RF-EMF; dendritic spine; glutamate receptor; hippocampus; neurite outgrowth; neuron.

Figure 1

Changes in dendritic spines in hippocampal CA1 and DG of RF-EMF-exposed early postnatal mice. (A). Representative TEM images of dendritic spines of hippocampal CA1 (a,c) and DG (b,d) in control (a,b) and RF-EMF-exposed mice (c,d). The number of thin (b), stubby (c), and mushroom-type (d) dendritic spines and the total number of dendritic spines (a) were analyzed in hippocampal CA1 (B) and DG (C). Data are expressed as means ± SD. Statistical significance was evaluated using the Student’s t-test. * p < 0.05, ** p < 0.01 vs. control (control n = 4 mice, RF-EMF n = 4 mice). M, mushroom; T, thin; S, Stubby; ● Control; ■ RF-EMF. Scale bars = 500 nm.

Figure 2

Expression levels of AMPAR and NMDAR in the hippocampus of RF-EMF-exposed early postnatal mice. (A). Protein expression levels of AMPAR (control n = 7 mice, RF-EMF n = 7 mice) and NMDAR (control n = 6 mice, RF-EMF n = 6 mice) in mouse hippocampus. Total protein was immunoblotted with antibodies against AMPAR (a) and NMDAR (b). The intensities of bands were quantified by densitometry. Protein levels were normalized relative to α-tubulin. (B,C). Representative immunogold staining for glutamate receptors, AMPAR (B) and NMDAR (C), in dendritic spines of hippocampal CA1 (a,c) and DG (b,d) in control (a,b) and RF-EMF-exposed mice (c,d). Gold particles labeling AMPAR and NMDAR were observed by transmission electron microscopy. The total number of gold particles labeling AMPAR (Bg) and NMDAR (Cg) in hippocampal CA1 (e) and DG (f) were quantified in mushroom-type dendritic spines. Data are expressed as means ± SD. Statistical significance was evaluated using the Student’s t-test. ** p < 0.01, *** p < 0.001 vs. control (control n = 4 mice, RF-EMF n = 4 mice). M, mushroom; ● Control; ■ RF-EMF. Scale bars = 200 nm.

Figure 3

Expression levels of BDNF in the hippocampus of RF-EMF-exposed early postnatal mice. (A). Protein expression levels of BDNF in mouse hippocampus (control n = 5 mice, RF-EMF n = 5 mice). Total protein was immunoblotted with an antibody against BDNF (a). The intensities of bands were quantified by densitometry (b). Protein levels were normalized relative to α-tubulin. (B). Representative immunogold staining for BDNF in hippocampal CA1 (a,c) and DG (b,d) in control (a,b) and RF-EMF-exposed mice (c,d). Gold particles labeling BDNF were observed by transmission electron microscopy. (C). The total number of gold particles labeling BDNF (c) in hippocampal CA1 (a) and DG (b) were quantified in hippocampal neurons. Data are expressed as means ± SD. Statistical significance was evaluated using the Student’s t-test. * p < 0.05, ** p < 0.01 vs. control (control n = 4 mice, RF-EMF n = 4 mice). M, mushroom; ● Control; ■ RF-EMF. Scale bars = 200 nm.

Figure 4

Exposure to RF-EMF decreased synaptic densities in mouse hippocampal neurons. (A). Confocal images show hippocampal neurons expressing PSD95 (green) and MAP2 (red). Images of PSD95 and MAP2 were analyzed using ImageJ. (B). Time-dependent changes in PSD95-positive punctae in dendrites of mouse hippocampal neurons. (C). Summary of the changes in PSD95 punctae as a percentage of control. Data are expressed as means ± SD. Statistical significance was evaluated using either ANOVA (B) or Student’s t-test (C). * p < 0.05, ** p < 0.01 vs. control (control n = 20 mice, RF-EMF n = 16 mice). Scale bars = 15 µm.

Figure 5

Exposure to RF-EMF decreased the expression of NMDAR and AMPAR. (A). Confocal images display hippocampal neurons expressing AMPAR (GluR1, green) or NMDAR (NR1, green) with MAP2 (red). Images of GluR1 and NR1 with MAP2 were analyzed using ImageJ. (B,C). Summary of changes in the number of GluR1 and NR1 punctae. Data are expressed as means ± SD. Statistical significance was evaluated using the Student’s t-test. * p < 0.05, **** p < 0.0001 vs. control (control n = 18 mice, RF-EMF n = 18 mice). Scale bars = 15 µm.

Figure 6

Exposure to RF-EMF decreased somatic BDNF expression in primary cultured hippocampal neurons. (A). Confocal images display hippocampal neurons expressing BDNF (green), MAP2 (red), and nuclei (DAPI, blue). Expression of BDNF in hippocampal neurons was analyzed on DIV 9. (B,C). Summary of changes in BDNF expression in the soma and neurites of cultured hippocampal neurons. Data are expressed as means ± SD. Statistical significance was evaluated using the Student’s t-test. ** p < 0.01 vs. control (control n = 18 mice, RF-EMF n = 18 mice). Scale bar = 15 µm.

Figure 7

Exposure to RF-EMF decreased neurite outgrowth in developing hippocampal neurons. (A). Confocal images display neurite outgrowth (MAP2, red) in developing neurons. (B). Time-dependent changes in neurite length C. Number of branches D. Soma size. Data are expressed as means ± SD. Statistical significance was evaluated using ANOVA (B) or Student’s t-test (C,D). * p < 0.05, ** p < 0.01 vs. control (control n = 20 mice, RF-EMF n = 16 mice). Scale bar = 15 µm.

Figure 8

Novel object recognition test in RF-EMF-exposed early postnatal mice. Mice were exposed to 1850 MHz RF-EMF at a SAR of 4.0 W/kg for 5 h per day for 4 weeks (from P1 to P28). The memory index was decreased in RF-EMF-exposed mice (control n = 17 mice, RF-EMF n = 16 mice). The memory index was calculated by measuring interaction time with the novel object (A). The discrimination ratio was calculated by measuring the exploration time for the novel object compared to the old object as a proportion of the total exploration time (B). Data are expressed as means ± SD. Statistical significance was evaluated using the Student’s t-test. * p < 0.05.