Fig 1.
Ultrastructure of synaptic vesicles in the cerebral cortex of mice after 835 MHz radiofrequency (RF) exposure.
(A) Representative TEM micrographs of the synapse region in the cerebral cortex were acquired from 12 week sham exposed (a and b) and RF-EMF exposed mice (c and d). M, mitochondria; Pre-SN, pre synaptic neuron; Post-SN, post synaptic neuron; SVs, synaptic vesicles; Size bars: 500 nm. Comparisons of synaptic vesicle number (Ba, SVs per unit area (mm2)) and size (Bb, the cross-section area (nm2)) at the presynaptic terminals in the cerebral cortex between sham-control and RF-EMF exposed mice for 4- and 12-week periods. Each bar represents the mean ± SEM. Statistical significance was evaluated using Student's t-test, *p <0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 2.
Electron tomography of presynaptic terminals in mice cerebral cortex following 12-weeks of RF-EMF exposure.
Electron tomography process from two dimensional (2D) tilt series imaging to three dimensional (3D) modeling in the cerebral cortex of sham-exposed mouse (A) and RF-EMF exposed mouse (B). The tilt series of synapse image containing 121 images were recorded over a tilt range of -60° to +60°, with an interval of 1° using Bio-HVEM (Aa and Ba); the 0° reference image with Bio-HVEM (Ab and Bb); the virtual digital slice extracted from the 3D tomogram which was generated by alignment of tilt series images using Composer (Ac and Bc); boundaries of the region of interest that were visible in each tomographic slice were traced as contours overlaid on the image by AMIRA (FEI) (Ad and Bd); tomographic slices of surface of objects, including SVs, stacked to generate 3D models using AMIRA (Ae and Be). The SV membranes in pre-synaptic terminals are represented in violet color. Size bars: 1 μm.
Fig 3.
3D electron tomographic analysis of synaptic vesicle number at the presynaptic terminal in cerebral cortex after sham and 12-week RF-EMF exposure.
The five 190 nm3 cubes were randomly extracted from the pre-synapse 3D model of sham-exposed (A Control) and RF-EMF (A RF-EMF) exposed condition, respectively. Rendering of tomographic slice (Aa and c), 3D model with cube (Ab and d), Cubes (Control 1–5 and RF-EMF 1–5). Size bars: 1 μm (a—d), 200 nm (cube 1–5). The SVs in each cube were counted and statistically analyzed. Each bar represents the mean ± SEM. Statistical significance was evaluated using Student's t-TEST: *p <0.05.
Fig 4.
Expression levels of synapsins in the cerebral cortex of mice following RF-EMF exposure for 4 or 12 weeks.
(A) Cortical total RNA extracted from sham and RF-EMF-exposed mice were analyzed for the expression level of synapsins I/II/III using quantitative real-time PCR (qRT-PCR). The relative mRNA levels of synapsins I/II/III (a-c) as calculated by normalizing to expression of GAPDH with the 2-ΔΔCt method (n = 10). Each bar represents the mean ± SEM. Statistical significance was evaluated using two-tailed unpaired Student t-test (*p<0.05). (B) Levels of synapsin I/II proteins in the cerebral cortex of mice after RF-EMF exposure for 4 weeks (4W) or 12 weeks (12W). Representative immunoblots of synapsin I and synapsin II (a). The intensity of the bands was quantified (b and c). The protein level of synapsin I/II was normalized to α-tubulin. Each bar shows mean with SEM. Statistical significance was evaluated using two-tailed unpaired Student's t-test: *p<0.05, **p<0.01.