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The ever-increasing demand for mobile telephony communication has led to the continuous emergence of wireless technologies (G), which may have different impacts on biological systems.To test this, we exposed rats to a single-head exposure to a 4G long-term evolution (LTE)-1800 MHz electromagnetic field (EMF) for 2 hours.We then assessed the effect of lipopolysaccharide-induced acute neuroinflammation on microglia spatial coverage and electrophysiological neuronal activity in the primary auditory cortex (ACx).The average SAR in ACx is 0.5 W/kg.Multi-unit recordings show that LTE-EMF triggers a reduction in the intensity of the response to pure tones and natural vocalizations, while an increase in the acoustic threshold for low and mid-range frequencies.Iba1 immunohistochemistry showed no changes in the area covered by microglial bodies and processes.In healthy rats, the same LTE exposure did not induce changes in response intensity and acoustic thresholds.Our data demonstrate that acute neuroinflammation sensitizes neurons to LTE-EMF, resulting in altered processing of acoustic stimuli in ACx.
The electromagnetic environment of mankind has changed dramatically over the past three decades due to the continuous expansion of wireless communications.Currently, more than two-thirds of the population are considered mobile phone (MP) users.The large-scale spread of this technology has sparked concerns and debate about the potentially dangerous effects of pulsed electromagnetic fields (EMFs) in the radio frequency (RF) range, which are emitted by MPs or base stations and encode communications.This public health issue has inspired a number of experimental studies devoted to investigating the effects of radiofrequency absorption in biological tissues1.Some of these studies have looked for changes in neuronal network activity and cognitive processes, given the proximity of the brain to RF sources under the pervasive use of MP.Many reported studies address the effects of pulse modulated signals used in second generation (2G) global system for mobile communications (GSM) or wideband code division multiple access (WCDMA)/third generation universal mobile telecommunications systems (WCDMA/3G UMTS)2 ,3,4,5.Little is known about the effects of radio frequency signals used in fourth generation (4G) mobile services, which rely on an all-digital Internet Protocol technology called Long Term Evolution (LTE) technology.Launched in 2011, LTE handset service is expected to reach 6.6 billion global LTE subscribers in January 2022 (GSMA: //gsacom.com).Compared to GSM (2G) and WCDMA (3G) systems based on single-carrier modulation schemes, LTE uses Orthogonal Frequency Division Multiplexing (OFDM) as the basic signal format6.Worldwide, LTE mobile services use a range of different frequency bands between 450 and 3700 MHz, including the 900 and 1800 MHz bands also used in GSM.
The ability of RF exposure to affect biological processes is largely determined by the specific absorption rate (SAR) expressed in W/kg, which measures the energy absorbed in biological tissue.The effects of acute 30-minute head exposure to 2.573 GHz LTE signals on global neuronal network activity were recently explored in healthy human volunteers.Using resting state fMRI, it was observed that LTE exposure can induce spontaneous slow frequency fluctuations and alterations in intra- or inter-regional connectivity, while spatial peak SAR levels averaged over 10 g of tissue were estimated to vary between 0.42 and 1.52 W/ kg, according to topics 7, 8, 9.EEG analysis under similar exposure conditions (30 min duration, estimated peak SAR level of 1.34 W/kg using a representative human head model) demonstrated reduced spectral power and hemispheric coherence in the alpha and beta bands.However, two other studies based on EEG analysis found that 20 or 30 minutes of LTE head exposure, with maximum local SAR levels set at around 2 W/kg, either had no detectable effect11 or resulted in spectral power in the alpha band decreased, while cognition did not change in function assessed with the Stroop test 12 .Significant differences were also found in the results of EEG or cognitive studies specifically looking at the effects of GSM or UMTS EMF exposure.They are thought to arise from variations in method design and experimental parameters, including signal type and modulation, exposure intensity and duration, or from heterogeneity in human subjects with respect to age, anatomy, or gender.
So far, few animal studies have been used to determine how exposure to LTE signaling affects brain function.It has recently been reported that systemic exposure of developing mice from late embryonic stage to weaning (30 min/day, 5 days/week, with a mean whole-body SAR of 0.5 or 1 W/kg) resulted in altered motor and appetite behaviors in adulthood 14. Repeated systemic exposure (2 ha per day for 6 weeks) in adult rats was found to induce oxidative stress and reduce the amplitude of visual evoked potentials obtained from the optic nerve, with a maximum SAR estimated to be as low as 10 mW/kg15.
In addition to analysis at multiple scales, including the cellular and molecular levels, rodent models can be used to study the effects of RF exposure during disease, as previously focused on GSM or WCDMA/3G UMTS EMF in the context of acute neuroinflammation. Studies have shown the effects of seizures, neurodegenerative diseases or gliomas 16,17,18,19,20.
Lipopolysaccharide (LPS)-injected rodents are a classic preclinical model of acute neuroinflammatory responses associated with benign infectious diseases caused by viruses or bacteria that affect the majority of the population each year.This inflammatory state leads to a reversible disease and depressive behavioral syndrome characterized by fever, loss of appetite, and reduced social interaction.Resident CNS phagocytes such as microglia are key effector cells of this neuroinflammatory response.Treatment of rodents with LPS triggers activation of microglia characterized by remodeling of their shape and cellular processes and profound changes in the transcriptome profile, including upregulation of genes encoding pro-inflammatory cytokines or enzymes, which affect neuronal networks Activities 22, 23, 24.
Studying the effects of a single 2-hour head exposure to GSM-1800 MHz EMF in LPS-treated rats, we found that GSM signaling triggers cellular responses in the cerebral cortex, affecting gene expression, glutamate receptor phosphorylation, neuronal Meta-evoked firing and morphology of microglia in the cerebral cortex.These effects were not detected in healthy rats that received the same GSM exposure, suggesting that the LPS-triggered neuroinflammatory state sensitizes CNS cells to GSM signaling.Focusing on the auditory cortex (ACx) of LPS-treated rats, where the local SAR averaged 1.55 W/kg, we observed that GSM exposure resulted in an increase in the length or branching of microglial processes and a decrease in neuronal responses evoked by pure tones and .Natural Stimulation 28.
In the current study, we aimed to examine whether head-only exposure to LTE-1800 MHz signals could also alter microglial morphology and neuronal activity in ACx, reducing the power of exposure by two-thirds.We show here that LTE signaling had no effect on microglial processes but still triggered a significant reduction in sound-evoked cortical activity in the ACx of LPS-treated rats with a SAR value of 0.5 W/kg.
Given previous evidence that exposure to GSM-1800 MHz altered microglial morphology under pro-inflammatory conditions, we investigated this effect after exposure to LTE signaling.
Adult rats were injected with LPS 24 hours before head-only sham exposure or exposure to LTE-1800 MHz.Upon exposure, LPS-triggered neuroinflammatory responses were established in the cerebral cortex, as shown by upregulation of proinflammatory genes and changes in cortical microglia morphology (Figure 1).The power exposed by the LTE head was set to obtain an average SAR level of 0.5 W/kg in ACx (Figure 2).To determine whether LPS-activated microglia were responsive to LTE EMF, we analyzed cortical sections stained with anti-Iba1 that selectively labeled these cells.As shown in Figure 3a, in ACx sections fixed 3 to 4 hours after sham or LTE exposure, microglia looked remarkably similar, showing a "dense-like" cell morphology elicited by LPS pro-inflammatory treatment (Figure 1).Consistent with the absence of morphological responses, quantitative image analysis revealed no significant differences in total area (unpaired t-test, p = 0.308) or area (p = 0.196) and density (p = 0.061) of Iba1 immunoreactivity when comparing exposure to Iba 1-stained cell bodies in LTE rats versus sham-exposed animals (Fig. 3b-d).
Effects of LPS ip injection on cortical microglia morphology.Representative view of microglia in a coronal section of the cerebral cortex (dorsomedial region) 24 hours after intraperitoneal injection of LPS or vehicle (control).Cells were stained with anti-Iba1 antibody as previously described.LPS pro-inflammatory treatment resulted in changes in microglia morphology, including proximal thickening and increased short secondary branches of cellular processes, resulting in a "dense-like" appearance.Scale bar: 20 µm.
Dosimetric analysis of specific absorption rate (SAR) in rat brain during exposure to 1800 MHz LTE.A previously described heterogeneous model of phantom rat and loop antenna62 was used to assess local SAR in the brain, with a 0.5 mm3 cubic grid.(a) Global view of a rat model in an exposure setting with a loop antenna above the head and a metallic thermal pad (yellow) below the body.(b) Distribution of SAR values ​​in the adult brain at 0.5 mm3 spatial resolution.The area delimited by the black outline in the sagittal section corresponds to the primary auditory cortex where microglial and neuronal activity is analyzed.The color-coded scale of SAR values ​​applies to all numerical simulations shown in the figure.
LPS-injected microglia in rat auditory cortex following LTE or Sham exposure.(a) Representative stacked view of microglia stained with anti-Iba1 antibody in coronal sections of LPS-perfused rat auditory cortex 3 to 4 hours after Sham or LTE exposure (exposure).Scale bar: 20 µm.(bd) Morphometric assessment of microglia 3 to 4 hours after sham (open dots) or LTE exposure (exposed, black dots).(b, c) Spatial coverage (b) of the microglia marker Iba1 and areas of Iba1-positive cell bodies (c).Data represent anti-Iba1 staining area normalized to the mean from Sham-exposed animals.(d) Count of anti-Iba1-stained microglial cell bodies.Differences between Sham (n = 5) and LTE (n = 6) animals were not significant (p > 0.05, unpaired t-test).The top and bottom of the box, the upper and lower lines represent the 25th-75th percentile and the 5-95th percentile, respectively.The mean value is marked in red in the box.
Table 1 summarizes the animal numbers and multi-unit recordings obtained in the primary auditory cortex of four groups of rats (Sham, Exposed, Sham-LPS, Exposed-LPS).In the results below, we include all recordings that exhibit a significant spectral temporal receptive field (STRF), ie, tone-evoked responses at least 6 standard deviations higher than spontaneous firing rates (see Table 1).Applying this criterion, we selected 266 records for the Sham group, 273 records for the Exposed group, 299 records for the Sham-LPS group, and 295 records for the Exposed-LPS group.
In the following paragraphs, we will first describe the parameters extracted from the spectral-temporal receptive field (that is, the response to pure tones) and the response to xenogeneic specific vocalizations.We will then describe the quantification of the frequency response area obtained for each group.Considering the presence of "nested data"30 in our experimental design, all statistical analyses were performed based on the number of positions in the electrode array (last row in Table 1), but all effects described below were also based on the number of positions in each group. Total number of multiunit recordings collected (third row in Table 1).
Figure 4a shows the optimal frequency distribution (BF, eliciting maximal response at 75 dB SPL) of cortical neurons obtained in LPS-treated Sham and exposed animals.The frequency range of BF in both groups was extended from 1 kHz to 36 kHz.Statistical analysis showed that these distributions were similar (chi-square, p = 0.278), suggesting that comparisons between the two groups could be made without sampling bias.
Effects of LTE exposure on quantified parameters of cortical responses in LPS-treated animals.(a) BF distribution in cortical neurons of LPS-treated animals exposed to LTE (black) and sham-exposed to LTE (white).There is no difference between the two distributions.(bf) The effect of LTE exposure on parameters quantifying the spectral temporal receptive field (STRF).Response strength was significantly reduced (*p < 0.05, unpaired t-test) across both STRF (total response strength) and optimal frequencies (b,c).Response duration, response bandwidth, and bandwidth constant (df).Both the strength and temporal reliability of responses to vocalizations were reduced (g, h).Spontaneous activity was not significantly reduced (i).(*p < 0.05, unpaired t-test).(j,k) Effects of LTE exposure on cortical thresholds.Mean thresholds were significantly higher in LTE-exposed rats compared to sham-exposed rats.This effect is more pronounced in the low and mid frequencies.
Figures 4b-f show the distribution of parameters derived from the STRF for these animals (means indicated by red lines).The effects of LTE exposure on LPS-treated animals appeared to indicate decreased neuronal excitability.First, overall response intensity and responses were significantly lower in BF compared with Sham-LPS animals (Fig. 4b,c unpaired t-test, p = 0.0017; and p = 0.0445).Likewise, responses to communication sounds decreased in both response strength and inter-trial reliability (Fig. 4g,h; unpaired t-test, p = 0.043).Spontaneous activity was reduced, but this effect was not significant (Fig. 4i; p = 0.0745).Response duration, tuning bandwidth, and response latency were not affected by LTE exposure in LPS-treated animals (Fig. 4d–f), indicating that frequency selectivity and precision of onset responses were not affected by LTE exposure in LPS-treated animals.
We next assessed whether pure tone cortical thresholds were altered by LTE exposure.From the frequency response area (FRA) obtained from each recording, we determined auditory thresholds for each frequency and averaged these thresholds for both groups of animals.Figure 4j shows the mean (± sem) thresholds from 1.1 to 36 kHz in LPS-treated rats.Comparing the auditory thresholds of the Sham and Exposed groups showed a substantial increase in thresholds in exposed animals compared with Sham animals (Fig. 4j), an effect that was more pronounced in low and mid frequencies.More precisely, at low frequencies (< 2.25 kHz), the proportion of A1 neurons with high threshold increased, while the proportion of low and medium threshold neurons decreased (chi-square = 43.85; p < 0.0001; Fig. 4k, left Figure) . The same effect was seen at mid-frequency (2.25 < Freq(kHz) < 11): a higher proportion of cortical recordings with intermediate thresholds and a smaller proportion of neurons with low thresholds compared to the unexposed group (Chi - Square = 71.17; p < 0.001; Figure 4k, middle panel).There was also a significant difference in threshold for high-frequency neurons (≥ 11 kHz, p = 0.0059); the proportion of low-threshold neurons decreased and the proportion of mid-high threshold increased (chi-square = 10.853; p = 0.04 Figure 4k, right panel).
Figure 5a shows the optimal frequency distribution (BF, eliciting maximum response at 75 dB SPL) of cortical neurons obtained in healthy animals for the Sham and Exposed groups.Statistical analysis showed that the two distributions were similar (chi-square, p = 0.157), suggesting that comparisons between the two groups could be made without sampling bias.
Effects of LTE exposure on quantified parameters of cortical responses in healthy animals.(a) BF distribution in cortical neurons of healthy animals exposed to LTE (dark blue) and sham-exposed to LTE (light blue).There is no difference between the two distributions.(bf) The effect of LTE exposure on parameters quantifying the spectral temporal receptive field (STRF).There was no significant change in the response intensity across the STRF and optimal frequencies (b,c).There is a slight increase in response duration (d), but no change in response bandwidth and bandwidth (e, f).Neither the strength nor the temporal reliability of the responses to vocalizations changed (g, h).There was no significant change in spontaneous activity (i).(*p < 0.05 unpaired t-test).(j,k) Effects of LTE exposure on cortical thresholds.On average, thresholds were not significantly changed in LTE-exposed rats compared to Sham-exposed rats, but higher frequency thresholds were slightly lower in exposed animals.
Figures 5b-f show boxplots representing the distribution and mean (red line) of parameters derived from the two sets of STRFs.In healthy animals, LTE exposure itself had little effect on the mean value of STRF parameters.Compared with the Sham group (light vs dark blue boxes for the exposed group), LTE exposure did not alter either the total response intensity nor the response of BF (Fig. 5b,c; unpaired t-test, p = 0.2176, and p = 0.8696 respectively).There was also no effect on spectral bandwidth and latency (p = 0.6764 and p = 0.7129, respectively), but there was a significant increase in response duration (p = 0.047).There was also no effect on the strength of vocalization responses (Fig. 5g, p = 0.4375), the inter-trial reliability of these responses (Fig. 5h, p = 0.3412), and spontaneous activity (Fig. 5).5i; p = 0.3256).
Figure 5j shows the mean (± sem) thresholds from 1.1 to 36 kHz in healthy rats.It did not show a significant difference between sham and exposed rats, except for a slightly lower threshold in exposed animals at high frequencies (11–36 kHz) (unpaired t-test, p = 0.0083).This effect reflects the fact that in exposed animals, in this frequency range (chi-square = 18.312, p = 0.001; Fig. 5k), there were slightly more neurons with low and medium thresholds (while high thresholds) fewer neurons).
In conclusion, when healthy animals were exposed to LTE, there was no effect on the response strength to pure tones and complex sounds such as vocalizations.Furthermore, in healthy animals, cortical auditory thresholds were similar between exposed and sham animals, whereas in LPS-treated animals, LTE exposure resulted in a substantial increase in cortical thresholds, especially in the low and mid-frequency range.
Our study showed that in adult male rats experiencing acute neuroinflammation, exposure to LTE-1800 MHz with a local SARACx of 0.5 W/kg (see Methods) resulted in a significant reduction in the intensity of sound-evoked responses in primary recordings of communication.These changes in neuronal activity occurred without any apparent change in the extent of the spatial domain covered by microglial processes.This effect of LTE on the intensity of cortical evoked responses was not observed in healthy rats.Considering the similarity in optimal frequency distribution between recording units in LTE-exposed and sham-exposed animals, the differences in neuronal reactivity can be attributed to biological effects of LTE signals rather than sampling bias (Fig. 4a).Furthermore, the absence of changes in response latency and spectral tuning bandwidth in LTE-exposed rats suggests that, most likely, these recordings were sampled from the same cortical layers, which are located in the primary ACx rather than secondary regions.
To our knowledge, the effect of LTE signaling on neuronal responses has not been previously reported.However, previous studies have documented the ability of GSM-1800 MHz or 1800 MHz continuous wave (CW) to alter neuronal excitability, albeit with significant differences depending on the experimental approach.Shortly after exposure to 1800 MHz CW at a SAR level of 8.2 W/Kg, recordings from snail ganglia showed decreased thresholds for triggering action potentials and neuronal modulation.On the other hand, spiking and bursting activity in primary neuronal cultures derived from rat brain was reduced by exposure to GSM-1800 MHz or 1800 MHz CW for 15 minutes at a SAR of 4.6 W/kg.This inhibition was only partially reversible within 30 minutes of exposure.Complete silencing of neurons was achieved at a SAR of 9.2 W/kg.Dose-response analysis showed that GSM-1800 MHz was more effective than 1800 MHz CW in suppressing burst activity, suggesting that neuronal responses depend on RF signal modulation.
In our setting, cortical evoked responses were collected in vivo 3 to 6 hours after the 2-hour head-only exposure ended.In a previous study, we investigated the effect of GSM-1800 MHz at SARACx of 1.55 W/kg and found no significant effect on sound-evoked cortical responses in healthy rats.Here, the only significant effect evoked in healthy rats by exposure to LTE-1800 at 0.5 W/kg SARACx was a slight increase in the duration of the response upon presentation of pure tones.This effect is difficult to explain because it is not accompanied by an increase in response intensity, suggesting that this longer response duration occurs with the same total number of action potentials fired by cortical neurons.One explanation might be that LTE exposure may reduce the activity of some inhibitory interneurons, as it has been documented that in primary ACx feedforward inhibition controls the duration of pyramidal cell responses triggered by excitatory thalamic input33,34, 35, 36, 37.
In contrast, in rats subjected to LPS-triggered neuroinflammation, LTE exposure had no effect on the duration of sound-evoked neuronal firing, but significant effects were detected on the strength of the evoked responses.In fact, compared to neuronal responses recorded in LPS-sham-exposed rats, neurons in LPS-treated rats exposed to LTE exhibited a reduction in the intensity of their responses, an effect observed both when presenting pure tones and natural vocalizations .The reduction in the intensity of the response to pure tones occurred without a narrowing of the spectral tuning bandwidth of 75 dB, and since it occurred at all sound intensities, it resulted in an increase in the acoustic thresholds of cortical neurons at low and mid frequencies.
The reduction in evoked response strength indicated that the effect of LTE signaling at SARACx of 0.5 W/kg in LPS-treated animals was similar to that of GSM-1800 MHz applied at three times higher SARACx (1.55 W/kg) 28 .As for GSM signaling, head exposure to LTE-1800 MHz may reduce neuronal excitability in rat ACx neurons subjected to LPS-triggered neuroinflammation.In line with this hypothesis, we also observed a trend toward decreased trial reliability of neuronal responses to vocalization (Fig. 4h) and decreased spontaneous activity (Fig. 4i).However, it has been difficult to determine in vivo whether LTE signaling reduces neuronal intrinsic excitability or reduces synaptic input, thereby controlling neuronal responses in ACx.
First, these weaker responses may be due to the intrinsically reduced excitability of cortical cells after exposure to LTE 1800 MHz.Supporting this idea, GSM-1800 MHz and 1800 MHz-CW reduced burst activity when applied directly to primary cultures of cortical rat neurons with SAR levels of 3.2 W/kg and 4.6 W/kg, respectively , but a threshold SAR level was required to significantly reduce burst activity.Advocating for reduced intrinsic excitability, we also observed lower rates of spontaneous firing in exposed animals than in sham-exposed animals.
Second, LTE exposure may also affect synaptic transmission from thalamo-cortical or cortical-cortical synapses.Numerous records now show that, in the auditory cortex, the breadth of spectral tuning is not solely determined by afferent thalamic projections, but that intracortical connections confer additional spectral input to cortical sites39,40.In our experiments, the fact that cortical STRF showed similar bandwidths in exposed and sham-exposed animals indirectly suggested that the effects of LTE exposure were not effects on cortical-cortical connectivity.This also suggests that higher connectivity in other cortical regions exposed at SAR than measured in ACx (Fig. 2) may not be responsible for the altered responses reported here.
Here, a greater proportion of LPS-exposed cortical recordings showed high thresholds compared to LPS-sham-exposed animals.Given that it has been proposed that the cortical acoustic threshold is primarily controlled by the strength of the thalamo-cortical synapse39,40, it can be suspected that thalamo-cortical transmission is partially reduced by exposure, either presynaptic (reduced glutamate release) or postsynaptic level (reduced receptor number or affinity).
Similar to the effects of GSM-1800 MHz, LTE-induced altered neuronal responses occurred in the context of LPS-triggered neuroinflammation, characterized by microglial responses.Current evidence suggests that microglia strongly influence the activity of neuronal networks in normal and pathological brains41,42,43.Their ability to modulate neurotransmission depends not only on the production of compounds they produce that may or may limit neurotransmission, but also on the high motility of their cellular processes.In the cerebral cortex, both increased and decreased activity of neuronal networks trigger rapid expansion of the microglial spatial domain due to the growth of microglial processes44,45.In particular, microglial protrusions are recruited near activated thalamocortical synapses and can inhibit the activity of excitatory synapses through mechanisms involving microglia-mediated local adenosine production.
In LPS-treated rats submitted to GSM-1800 MHz with SARACx at 1.55 W/kg, decreased activity of ACx neurons occurred with the growth of microglial processes marked by significant Iba1-stained areas in ACx28 Increase.This observation suggests that microglial remodeling triggered by GSM exposure can actively contribute to the GSM-induced reduction in sound-evoked neuronal responses.Our current study argues against this hypothesis in the context of LTE head exposure with SARACx limited to 0.5 W/kg, as we found no increase in the spatial domain covered by microglial processes.However, this does not rule out any effect of LTE signaling on LPS-activated microglia, which may in turn affect neuronal activity.Further studies are needed to answer this question and to determine the mechanisms by which acute neuroinflammation alters neuronal responses to LTE signaling.
To our knowledge, the effect of LTE signals on auditory processing has not been studied before.Our previous studies 26,28 and the current study showed that in the setting of acute inflammation, exposure of the head alone to GSM-1800 MHz or LTE-1800 MHz resulted in functional alterations in neuronal responses in ACx, as shown by the increase in hearing threshold.For at least two main reasons, cochlear function should not be affected by our LTE exposure.First, as shown in the dosimetry study shown in Figure 2, the highest levels of SAR (close to 1 W/kg) are located in the dorsomedial cortex (below the antenna), and they decrease substantially as one moves more laterally and laterally.The ventral part of the head.It can be estimated to be about 0.1 W/kg at the level of the rat pinna (below the ear canal).Second, when guinea pig ears were exposed for 2 months at GSM 900 MHz (5 days/week, 1 hour/day, SAR between 1 and 4 W/kg), there were no detectable changes in the magnitude of the distortion product otoacoustic Thresholds for Emission and Auditory Brainstem Responses 47.Furthermore, repeated head exposure to GSM 900 or 1800 MHz at a local SAR of 2 W/kg did not affect cochlear outer hair cell function in healthy rats48,49.These results echo data obtained in humans, where investigations have shown that 10- to 30-minute exposure to EMF from GSM cell phones has no consistent effect on auditory processing as assessed at the cochlear50,51,52​​or brainstem level53,54 .
In our study, LTE-triggered neuronal firing changes were observed in vivo 3 to 6 hours after exposure ended.In a previous study on the dorsomedial part of the cortex, several effects induced by GSM-1800 MHz observed at 24 hours after exposure were no longer detectable at 72 hours after exposure.This is the case with expansion of microglial processes, downregulation of the IL-1ß gene and post-translational modification of AMPA receptors.Considering that the auditory cortex has a lower SAR value (0.5W/kg) than the dorsomedial region (2.94W/kg26), the changes in neuronal activity reported here appear to be transient.
Our data should take into account the qualifying SAR limits and estimates of the actual SAR values ​​achieved in the cerebral cortex of mobile phone users.Current standards used to protect the public set the SAR limit to 2 W/kg for localized head or torso exposure to radio frequencies in the 100 kHz and 6 GHz RF range.
Dose simulations have been performed using different human head models to determine RF power absorption in different tissues of the head during general head or mobile phone communication.In addition to the diversity of human head models, these simulations highlight significant differences or uncertainties in estimating energy absorbed by the brain based on anatomical or histological parameters such as the external or internal shape of the skull, thickness, or water content.Different head tissues vary widely according to age, sex, or individual 56,57,58.Furthermore, cell phone characteristics, such as the internal location of the antenna and the position of the cell phone relative to the user's head, strongly influence the level and distribution of SAR values ​​in the cerebral cortex59,60.However, considering the reported SAR distributions in the human cerebral cortex, which were established from cell phone models emitting radio frequencies in the 1800 MHz range58, 59, 60, it appears that the SAR levels achieved in the human auditory cortex are still under-applied half of the human cerebral cortex.Our study (SARACx 0.5 W/kg).Therefore, our data do not challenge the current limits of SAR values ​​applicable to the public.
In conclusion, our study shows that a single head-only exposure to LTE-1800 MHz interferes with the neuronal responses of cortical neurons to sensory stimuli.Consistent with previous characterizations of the effects of GSM signaling, our results suggest that the effects of LTE signaling on neuronal activity vary by health status.Acute neuroinflammation sensitizes neurons to LTE-1800 MHz, resulting in altered cortical processing of auditory stimuli.
Data were collected at 55 days of age from the cerebral cortex of 31 adult male Wistar rats obtained in the Janvier laboratory.Rats were housed in a humidity (50-55%) and temperature (22-24 °C) controlled facility with a light/dark cycle of 12 h/12 ​​h (lights on at 7:30am) with free access to food and water.All experiments were performed in accordance with the guidelines established by the Council of the European Communities Directive (2010/63/EU Council Directive), which are similar to those described in the Society for Neuroscience Guidelines for the Use of Animals in Neuroscience Research.This protocol was approved by the Ethics Committee Paris-Sud and Center (CEEA N°59, Project 2014-25, National Protocol 03729.02) using procedures validated by this committee 32-2011 and 34-2012.
Animals were habituated to colony chambers for at least 1 week prior to LPS treatment and exposure (or sham exposure) to LTE-EMF.
Twenty-two rats were injected intraperitoneally (ip) with E. coli LPS (250 µg/kg, serotype 0127:B8, SIGMA) diluted with sterile endotoxin-free isotonic saline 24 hours before LTE or sham exposure (n per group). = 11).In 2-month-old Wistar male rats, this LPS treatment produces a neuroinflammatory response that is marked in the cerebral cortex by several pro-inflammatory genes (tumor necrosis factor-alpha, interleukin 1ß, CCL2, NOX2 , NOS2) were up-regulated 24 hours after LPS injection, including a 4- and 12-fold increase in the levels of transcripts encoding the NOX2 enzyme and interleukin 1ß, respectively.At this 24-h time point, cortical microglia displayed the typical "dense" cell morphology expected by LPS-triggered pro-inflammatory activation of cells (Figure 1), which is in contrast to LPS-triggered activation by others. Cellular pro-inflammatory activation corresponds to 24, 61.
Head-only exposure to LTE EMF was performed using the experimental setup previously used to evaluate the effect of GSM EMF26.LTE exposure was performed 24 hours after LPS injection (11 animals) or no LPS treatment (5 animals).Animals were lightly anesthetized with ketamine/xylazine (ketamine 80 mg/kg, ip; xylazine 10 mg/kg, ip) prior to exposure to prevent movement and to ensure the animal's head was in the loop antenna emitting the LTE signal Reproducible location below.Half of the rats from the same cage served as controls (11 sham-exposed animals, out of 22 rats pretreated with LPS): they were placed under the loop antenna and the energy of the LTE signal was set to zero.Weights of exposed and sham exposed animals were similar (p = 0.558, unpaired t-test, ns).All anesthetized animals were placed on a metal-free heating pad to maintain their body temperature around 37°C throughout the experiment.As in the previous experiments, the exposure time was set to 2 hours.After exposure, place the animal on another heating pad in the operating room.The same exposure procedure was applied to 10 healthy rats (untreated with LPS), half of which were sham-exposed from the same cage (p = 0.694).
The exposure system was similar to the systems 25, 62 described in previous studies, with the radio frequency generator replaced to generate LTE instead of GSM electromagnetic fields.Briefly, an RF generator (SMBV100A, 3.2 GHz, Rohde & Schwarz, Germany) emitting an LTE - 1800 MHz electromagnetic field was connected to a power amplifier (ZHL-4W-422+, Mini-Circuits, USA), a circulator ( D3 1719-N, Sodhy, France), a two-way coupler (CD D 1824-2, − 30 dB, Sodhy, France) and a four-way power divider (DC D 0922-4N, Sodhy, France), allowing simultaneous Expose four animals.A power meter (N1921A, Agilent, USA) connected to a bidirectional coupler allowed continuous measurement and monitoring of incident and reflected power within the device.Each output was connected to a loop antenna (Sama-Sistemi srl; Roma), enabling partial exposure of the animal's head.The loop antenna consists of a printed circuit with two metal lines (dielectric constant εr = 4.6) engraved on an insulating epoxy substrate.At one end, the device consists of a 1 mm wide wire forming a ring placed close to the animal's head.As in previous studies26,62, the specific absorption rate (SAR) was determined numerically using a numerical rat model and a finite difference time domain (FDTD) method63,64,65.They were also determined experimentally in a homogeneous rat model using Luxtron probes to measure temperature rise.In this case, SAR in W/kg is calculated using the formula: SAR = C ΔT/Δt, where C is the heat capacity in J/(kg K), ΔT, in °K and Δt Temperature change, time in seconds.The numerically determined SAR values ​​were compared with experimental SAR values ​​obtained using a homogeneous model, especially in equivalent rat brain regions.The difference between the numerical SAR measurements and the experimentally detected SAR values ​​is less than 30%.
Figure 2a shows the SAR distribution in the rat brain in the rat model, which matches the distribution in terms of body weight and size of the rats used in our study.Brain mean SAR was 0.37 ± 0.23 W/kg (mean ± SD).SAR values ​​are highest in the cortical area just below the loop antenna.The local SAR in ACx (SARACx) was 0.50 ± 0.08 W/kg (mean ± SD) (Fig. 2b).Since the body weights of exposed rats are homogeneous and differences in head tissue thickness are negligible, the actual SAR of ACx or other cortical areas is expected to be very similar between one exposed animal and another.
At the end of exposure, animals were supplemented with additional doses of ketamine (20 mg/kg, ip) and xylazine (4 mg/kg, ip) until no reflex movements were observed after pinching the hind paw.A local anesthetic (Xylocain 2%) was injected subcutaneously into the skin and temporalis muscle above the skull, and the animals were placed on a metal-free heating system.After placing the animal in the stereotaxic frame, a craniotomy was performed over the left temporal cortex.As in our previous study66, starting from the junction of the parietal and temporal bones, the opening was 9 mm wide and 5 mm high.The dura above the ACx was carefully removed under binocular control without damaging the blood vessels.At the end of the procedure, a base was constructed in dental acrylic cement for atraumatic fixation of the animal's head during recording.Place the stereotaxic frame supporting the animal in an acoustic attenuation chamber (IAC, model AC1).
Data were obtained from multi-unit recordings in the primary auditory cortex of 20 rats, including 10 animals pretreated with LPS.Extracellular recordings were obtained from an array of 16 tungsten electrodes (TDT, ø: 33 µm, < 1 MΩ) consisting of two rows of 8 electrodes spaced 1000 µm apart (350 µm between electrodes in the same row).A silver wire (ø: 300 µm) for grounding was inserted between the temporal bone and the contralateral dura.The estimated location of the primary ACx is 4-7 mm posterior to the bregma and 3 mm ventral to the supratemporal suture.The raw signal was amplified 10,000 times (TDT Medusa) and then processed by a multi-channel data acquisition system (RX5, TDT).Signals collected from each electrode were filtered (610–10,000 Hz) to extract multi-unit activity (MUA).Trigger levels were carefully set for each electrode (by coauthors blinded to exposed or sham-exposed states) to select the largest action potential from the signal.On-line and off-line inspection of the waveforms showed that the MUA collected here consisted of action potentials generated by 3 to 6 neurons near the electrodes.At the beginning of each experiment, we set the position of the electrode array so that two rows of eight electrodes could sample neurons, from low to high frequency responses when performed in the rostral orientation.
Acoustic stimuli were generated in Matlab, transmitted to an RP2.1 based sound delivery system (TDT) and sent to a Fostex loudspeaker (FE87E).The loudspeaker was placed 2 cm from the rat's right ear, at which distance the loudspeaker produced a flat frequency spectrum (± 3 dB) between 140 Hz and 36 kHz.Loudspeaker calibration was performed using noise and pure tones recorded with a Bruel and Kjaer microphone 4133 coupled to a preamplifier B&K 2169 and digital recorder Marantz PMD671.The Spectral Time Receptive Field (STRF) was determined using 97 gamma-tone frequencies, covering 8 (0.14–36 kHz) octaves, presented in random order at 75 dB SPL at 4.15 Hz.The Frequency Response Area (FRA) is determined using the same set of tones and presented in random order at 2 Hz from 75 to 5 dB SPL.Each frequency is presented eight times at each intensity.
Responses to natural stimuli were also assessed.In previous studies, we observed that rat vocalizations rarely elicited strong responses in ACx, regardless of the neuronal optimal frequency (BF), whereas xenograft-specific (eg, songbird or guinea pig vocalizations) typically The entire tone map.Therefore, we tested cortical responses to vocalizations in guinea pigs (the whistle used in 36 was connected to 1 s of stimuli, presented 25 times).

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