The Neural Mechanisms Underlying The Effect Of Extremely Low-frequency Magnetic Field On Spatial Learning And Memory In Entorhinal Cortex

Recently, there has been increasing public concern on potential health risks from electromagnetic field, including power-frequency ?elds, also known as extremely low frequency electromagnetic ?elds(ELF), and radiofrequency/microwave radiation emissions(RF) from wireless communications. It is one of the prominent types of pollutions in modern industrial society. ELF and/or RF have/has been reported to be associated with many diseases, including childhood leukaemia, brain tumours, genotoxic effects, neurological effects and neurodegenerative diseases, immune system deregulation, allergic and in?ammatory responses, breast cancer, miscarriage and some cardiovascular effects. The central nervous system(CNS) appears to be a potential target organ system for adverse ELF exposure. In addition to reports of ELF-related health problems, such as amyotrophic lateral sclerosis, Alzheimer’s disease, insomnia, headaches, sexual dysfunction, chronic fatigue, and assorted other maladies, there is increasing evidence to suggest that memory problems may also result from ELF exposure. However, the mechanism that how the ELF influences the function of learning and memory is still not well-know.The entorhinal cortex(EC) is regarded as a bidirectional gateway between cortical areas and the hippocampus. The sensory inputs mainly converge onto neurons in the super?cial layers(layers II–III) of the EC, where the principal neurons in the super?cial layers of the EC convey them to the hippocampus by way of a ?ber tract well known as the perforant pathway. The information reaches all four sub?elds of the hippocampal formation(dentate gyrus, areas CA3 and CA1, and the subiculum). The perforant pathway innervations exhibit regional specifity: ?bers originating from layer II neurons almost exclusively target the dentate gyrus and CA3, whereas layer III neurons project selectively to CA1 and the subiculum. From CA1, the information also travels back to layers III, V and VI of the EC.Beside the entorhinal–hippocampus connections, the EC also connects with a wide array of cortical and subcortical areas. Brie?y, the cortical inputs form two groups: those that terminate in the super?cial layers II and III and those that are preferentially distributed to the deep layers(V–VI). These anatomical propertie of the EC makes it to be a key “hub” in the neocortex-hippocampus-neocortex memory circuitries, and is closely related to consolidation and retrieval of memory, especially for the spatial memory. The medial EC(MEC) transfer the spatial information, while the lateral EC(LEC) is known to convey the non-spatial information to hippocampus. Thus, MEC may be more important for the spatial learning and memory.In the present study, we firstly investigated the effects of chronic exposure to a 0.5 m T 50 Hz ELF on the dendritic spine density and shape in the superfici al layers of the MEC. Then we used whole-cell patch-clamp recordings to test the electrophysiological effects of ELF on the stellate neurons and the pyramidal neurons of brain slices in the EC. At last, we determined the effects of ELF on the motor function and spatial refenrece memory through open field and water maze tests. The results are as follows:1. The Changes of Dendritic Spine Density and Morphology in the Superficial Layers of the MEC Induced by Extremely Low-Frequency Magnetic Field Exposure.We performed Golgi staining to reveal the dendritic spines of the principal neurons in rats. The results showed that ELF exposure induced a decrease in the spine density in the dendrites of stellate neurons and the basal dendrites of pyramidal neurons at both 14 days(86.42 ± 2.77% of control, P < 0.001) and 28 days(87.34 ± 1.10% of control, P < 0.001), which was largely due to the loss of the thin and branched spines. The alteration in the density of mushroom and stubby spines post ELF exposure was cell-type specific. For the stellate neurons, ELF exposure slightly increased the density of stubby spines at 28 days(121.96 ± 1.07% of control, P < 0.05), while it did not affect the density of mushroom spines(106.54 ± 0.91% of control, P = 0.45) at the same time. In the basal dendrites of pyramidal neurons, we observed a significant decrease in the mushroo m spine density only at the later time point post ELF exposure(14 days: 115.59 ± 1.99% of control, P = 0.23; 28 days: 88.64 ± 0.70% of control, P =0.21), while the stubby spine density was reduced at 14 days(78.26 ± 1.86% of control, P < 0.05) and partially restored at 28 days(93.73 ± 0.68% of control, P = 0.53) post ELF exposure. ELF exposure-induced reduction in the spine density in the apical dendrites of pyramidal neurons was only observed at 28 days, reflecting the distinct vulnerability of spines in the apical and basal dendrites. Considering the changes in spine number and shape are involved in synaptic plasticity and the MEC is a part of neural network that is closely related to learning and memory, these findings may be helpful for explaining the ELF exposure-induced impairment in cognitive functions.2. The resting membrane potential and membrane resistance of stellate and pyramidal neurons in the superficial layers of EC are not affected by Extremely Low-Frequency Magnetic Field ExposureUnder the current-clamp model, we set the membrane current at 0 p A, then the resting membrane potential(RMP) was obtained. The results show that the RMP of stellate neurons(n = 6, P = 0.303) and pyramidal neurons(n = 5, P = 0.781) were not affected by ELF exposure. Then the cell was evoked by +20 p A current steps(0.5 s) from-150 p A holding current to +250 p A, and the I-V curve was obtained to calculate the membrane resistance. ELF exposure did not change the membrane resistance of stellate neuron(n = 6, P = 0.936) or pyramidal neuron(n = 5, P = 0.657), indicating that the main membrane conductance is not influenced by the ELF exposure.3. The whole-cell currents of stellate and pyramidal neurons in the superficial layers of EC are not affected by Extremely Low-Frequency Magnetic Field ExposureThen the stimulation of +10 m V voltage steps(0.5 s) from-60 m V holding voltage to +30 m V was applied. Whole-cell outward current mainly contain the transient outward current and sustained outward current. We found that ELF did not affect the inward current of stellate neuron(control n = 6, exposure n = 5, P = 0.428) or pyramidal neuron(n = 5, P = 0.310). At the same time, the results show that the transient outward current of stellate neuron(control n = 8, exposure n = 5, P = 0.199) and pyramidal neuron(control n = 8, exposure n = 5, P = 0.358) were not influenced by the ELF exposure. The sustained outward current of stellate neuron(control n = 7, exposure n = 5, P = 0.409) and pyramidal neuron(control n = 6, exposure n = 5, P = 0.256) have similar result. These results indicate that ELF exposure does not affect the activation of voltage-depended Na+ and K+ channels.4. The ELF exposure does not affect the excitability of principal neurons in the superficial layers of EC.The spikes of the neurons were evoked by simulation of increment of +20 p A, depolarizing steps at different current intensities. The results show that the firing frequency of action potential of stellate neurons(control n = 11, exposure n = 8, P = 0.614) and pyramidal neurons(control n = 6, exposure n = 5, P = 0.343) was not affected by ELF exposure. These data indicate that ELF does not influence the intrinsic excitability.5. The effect of ELF to the exploratory behavior and motor functionLastly, we tested the effects of ELF exposure on rat exploratory behavior and motor function. We found that after 14 days exposure, the displacement distance(n = 10, P = 0.226) and rearing time(n = 10, P = 0.596) were not influenced by the ELF exposure. Similar for the 28 days, there was no difference in the displacement distance(n = 9, P = 0.241) and rearing time(n = 9, P = 0.163). These results indicate that ELF exposure does not affect the exploratory behavior and motor function of animals.6. The effect of ELF to the spatial reference memoryWe wondered whether ELF would affect the rat spatial reference learning and memory in the water maze. Analysis of the escape latencies revealed no signi?cant difference between the ELF and sham exposure groups [14 days:F(1,18) = 0.27,P = 0.61; 28days:F(1,16) = 0.60,P = 0.45]. The signi?cant effect of trials [14 days:F(7,126) = 6.71, P < 0.001; 28days:F(7,112) = 12.24, P < 0.001] and no signi?cant interaction between trial and group [14 days:F(7,126) = 0.37, P = 0.95; 28 days:F(7,112) = 0.68, P = 0.69] indicate the equivalent improvement in spatial learning and memory for sham and ELF exposure rats. Neither 14 [F(1,18) = 0.17,P = 0.67] nor 28 [F(1,16) = 1.56,P = 0.23] day ELF exposure did affect the rat swimming speed, indicating the ELF does not in?uence the rat motor function.