Repeated Head Exposures to a 5G-3.5 GHz Signal Do Not Alter Behavior but Modify Intracortical Gene Expression in Adult Male Mice.

The introduction and expansion of 5G telecommunications systems requires a reassessment of the technology's biological impact on the brain. Rodent models can be used to assess spontaneous behaviors and emotional states or cognitive abilities, such as learning and memory. These models can also analyze the cellular and molecular mechanisms responsible for behavioral changes triggered by environmental influences. Regarding the effects of radiofrequency electromagnetic fields (RF-EMF) on learning and spatial memory in rodents, published papers show inconsistent results. Some report no effect, while others report significant changes in learning or memory abilities. This diversity most likely reflects the influence of a combination of experimental parameters that vary from study to study. While 2G, 3G, and 4G systems are still in use, the use of 5G mobile communications is growing rapidly and is expected to reach 2.3 billion subscriptions by the end of 2024. This study investigated whether chronic exposure to a 5G 3.5 GHz signal for one month (1 hour per day; 5 days per week) can alter the behavior, memory, and intracerebral gene expression in mice.

The experiments were performed with seven-week-old male C57BL/6 J mice (n = 32). Exposure was performed on awake, head-tied animals to ensure homogeneous daily head exposure. Each day, a dipole antenna was positioned 5 mm from the animals' heads in a fixed, standardized position near the right temporal cortex for one hour. The exposure system included a radiofrequency generator that produced a "real" 5G-modulated 3.5 GHz signal compliant with the 5G NR standard. Each exposed mouse (n = 12) was compared to a pseudo-exposed (PSD) mouse (n = 12) under identical conditions. Specific absorption rates (SAR) were determined both numerically using a simulated mouse model and experimentally using a homogeneous mouse model with a Luxtron probe. The difference between the two SAR values was less than 30%. After 20 days of 5G exposure (1 hour per day), the mice's locomotor activity was compared to that of the pseudo-exposed mice in an open field (OF) test. Learning and memory abilities were also tested using object recognition and object localization tasks. A camera connected to a video tracking system was placed above the OF to record the mice's activity. Once behavioral testing was complete, the brains were rapidly removed from the skulls of the anesthetized mice. mRNA sequencing was then performed using tissue collected from 5G-exposed (n = 7) and pseudo-exposed (n = 8) animals that underwent 27 hours of exposure or PSD exposure over 6 weeks. Whole-genome mRNA profiling was performed in symmetrical areas of the right and left cerebral cortex, which differed based on local energy deposition. Differences in transcript levels were analyzed using the Benjamini-Hochberg false discovery rate (FDR) control method.

Intracerebral SAR values were highest in the ventral cortical areas ipsilateral to the antenna, reaching 0.43 ± 0.12 W/kg. SAR values in the contralateral (left) cortex dropped to 0.14 ± 0.05 W/kg. The averaged SAR of the whole brain was 0.19 ± 0.12 W/kg. All SAR values are below the International Commission on Non-Ionizing Radiation Protection (ICNIRP) limits and are in the non-thermal range.

Mice exposed to 5G showed no hyperactivity or abnormal anxiety, and their level of open field activity and object exploration was similar to that of pseudo-exposed mice. In addition, long-term object recognition memory and long-term memory for the spatial position of objects were not impaired by 27 hours of chronic 5G exposure. As expected, the transcriptome profiles in the right and left cortex of pseudo-exposed animals were almost identical. Significant differences in transcript levels (FC > 1.2, FDR-adjusted p < 0.05) between the right and left cortex in pseudo-exposed mice were limited to five genes out of 12,423 detectable genes in these brain regions. Comparing 5G-exposed and PSD-exposed animals revealed significant gene modulation triggered by the 5G 3.5 GHz signal. In the right cortex, the changes in expression level (FC > 1.2, FDR-adjusted p < 0.05) were limited to 77 genes, i.e. less than 0.7% of the expressed genes in this region. These differentially expressed genes (DEGs) included 40 upregulated and 37 downregulated genes in response to chronic 5G exposure. In the left cortex, the total number of DEGs was slightly higher (84 genes), despite the lower SAR value compared to the right cortex. These 84 DEGs were divided into 30 upregulated genes and 54 downregulated genes in response to 5G exposure. The identities of the DEGs differed significantly between the right and left cortices, which had only eight DEGs in common; seven of these were downregulated in both regions. In the right cortex, the significant enrichments of gene ontology (GO) terms were mainly associated with the mitochondrial oxidative phosphorylation system (OXPHOS). The mitochondrial genome contains 13 genes that encode protein subunits of the ATP-producing OXPHOS. Ten of these genes were upregulated upon exposure to the 5G signal. These genes encode central subunits of the enzyme complexes I, III, IV, and V of OXPHOS, which are embedded in or anchored to the inner mitochondrial membrane. In addition, 9 and 11 DEGs encoded components of glutamatergic synapses in the right and left cortices, respectively.

The results of this study do not show a significant effect of the 5G signal on locomotion, anxiety levels, or memory. However, they reveal biological responses in the form of modulations in gene expression in the cerebral cortex. Due to the asymmetric head exposure, the authors compared gene expression in the right and left cortices exposed to different levels of the 5G 3.5 GHz signal. They observed that a threefold decrease in the mean SAR value was associated with significant changes in DEG identity without a decrease in the number of responding genes. This result suggests that the magnitude of the gene response does not increase proportionally with the amount of energy absorbed by the tissue. Despite the significant differences in gene identities, DEG profiles in the right and left cortices showed substantial enrichment of genes associated with glutamatergic synapses, which facilitate excitatory neurotransmission between the hippocampus and cortical regions. Numerous studies have investigated the ability of RF-EMF to promote reactive oxygen species (ROS) generation and oxidative stress in tissues. Consistent with the hypothesis that a 5G-modulated 3.5 GHz signal could promote ROS production, this study shows that 5G exposure increases the expression of mitochondrial genes that encode subunits of four of the five OXPHOS protein complexes, including ROS-producing complexes I and III. Overall, the results show that 5G exposure over a six-week period significantly alters the expression of a limited number of genes, which could affect glutamatergic synapses and mitochondrial function.

Editor's note:

Like the other French study discussed here (Dahon et al., 2025), this study stands out for its optimal execution and application of the latest methods. The study also used a "real" 5G signal. However, as the authors state, further research is needed to clarify whether and how quickly the observed changes in the transcriptome translate to the proteome and at what point observable changes in glutamatergic neurotransmission and mitochondrial function occur after 5G exposure. (AT)