Research On The Long Term Effects Of Microwave Radiation Induced Learning And Memory Deficits Based On The Role Of NMDARs Regulation On Synaptic Plasticity

Objective: With microwave application in communications, medical, transportation, industrial, and military and other fields, its biological effects are also increasingly attracted attention. Studies have shown that the brain is one of the most sensitive targets of the microwave radiation. Meanwhile, microwave radiation can cause changes in learning and memory function, but the mechanism is unknown. Synaptic plasticity is the neural basis of learning and memory. N-methyl-D-aspartic receptors(NMDARs) played an indispensable role on synaptic plasticity and long-term potentiation(LTP) induction and maintenance. Previous studies have mainly focused on the immediate or the recent effect and mechanism of microwave radiation, less attention paid on the long-term effect and mechanism, especially the role of NMDARs regulation on synaptic plasticity. Therefore, this study was firstly designed to establish a long-term animal injuried model of synaptic plasticity impairments and learning and memory disorders aiming at exploring the role of LTP and NMDARs on the learning and memory impairment and synaptic plasticity injuries induced by microwave radiation. Then we intended to copy the microwave radiation induced cell synaptic plasticity damage model and to explore the microwave induced change of NMDARs current by the patch clamp technique. Ultimately, we intended to focus on the relationship between the changes of NMDARs important subunits(NR1, NR2 A and NR2B) and synaptic plasticity injuries after microwave exposure, aiming at finding the sensitive targets for microwave radiation induced brain damages.Materials and methods:(1) The male Wistar rats were exposed to microwave at 0, 5, 10 and 50 m W/cm~2 for 6 min. We respectively detected the changes at the acute phase(6h, 1d, 7d and 14d) and the later period(1m, 3m, 6m, 9m, 12 m and 18m) after microwave exposure. Morris water maze was applied to detect the spatial learning and memory function. High performance liquid chromatography(HPLC) was used to detect the amino acids neurotransmitters in rat hippocampus. The basic structure and synapse ultrastructure of rats’ hippocampus were observed by light and electron microscope. Changes of LTP induction and its relationship with synaptic plasticity were detected by the rat hippocampal LTP recording at the acute phase(immediate-6h) after microwave exposure. The expression of NMDARs important subunits NR1, NR2 A and NR2 B and their roles in synaptic plasticity were detected by Western blot at the later period(1m-18m) after microwave exposure.(2) The rat primary hippocampal neurons were exposed to microwave at 0 and 50 m W/cm~2 for 5min. Neurites were observed by the confocal laser scanning microscope(CLSM) using Vybrant CM-Dil cell-labeling at 6h, 1d and 5d after microwave radiation. The neuronal structure was observed by the scanning electron microscopy(SEM) at 5 d after microwave exposure. Immediately after radiation, the whole-cell patch clamp recording was applied to detect the NMDARs currents. The Western blot and real time PCR were used to detect the protein and m RNA expression. The level of cytoplasmic Ca2+ was detected by Fluo 4-AM staining.Results:(1) The changes of learning and memory at acute phase after microwave exposure: At 6h, 1d and 3d, rats only in 10 and 50 m W/cm~2 groups showed significant longer escape latency and less number of platform crossings.(2) The changes of amino acid neurotransmitters at acute phase after microwave exposure: 5m W/cm~2 microwave mainly induced the elevated levels of gly and GABA neurotransmitters at 1d and 7d. The 10 and 50 m W/cm~2 microwave can firstly increase the four kinds of amino acid neurotransmitters increased at 7d and then decrease them at 14 d.(3) The changes of hippocampal structure at acute phase after microwave exposure: The 5m W/cm~2 microwave radiation cannot induce changes in the organizational structure and ultrastructure of the hippocampus. At 7d, in the 10 and 50 m W/cm~2 groups, however, hippocampus neurons were significantly irregularly arranged, karyopyknosis occurred in most neurons and the nuclei shrinked into blue pieces. At 7d, the 50 m W/cm~2 microwave can cause blurred synaptic cleft, reduced vesicles, increased dense postsynaptic, besides mitochondria cavitation and with cristae breakdown, endoplasmic reticulum, and the the perineural cell phenomenon were also observed.(4) LTP changes after microwave exposure: we found that rats in the 10 m W/cm~2 and 50 m W/cm~2 groups showed a significant decline in PS amplitude during 6 h after microwave exposure(P<0.05 or P<0.01). There were no significant changes in the 5 m W/cm~2 group. At 24 h after 10 m W/cm~2 radiation, PS amplitude of rats after high-frequency stimulation increased, but still did not recover to the normal level.(5) The changes of learning and memory at later period after microwave exposure: The 5 m W/cm~2 microwave cannot cause changes of learning and memory function. However, rats in the 10 and 50 m W/cm~2 groups exhibited varyingly prolonged degrees of escape latency at 1-18m(P <0.05 or P <0.01).There was a clear dose-effect relationship.(6) The changes of amino acid neurotransmitters at later period after microwave exposure: The 5, 10 and 50 m W/cm~2 microwave radiation can mainly decrease the amino acid neurotransmitter(P <0.05 or P <0.01), with a clear dose-effect relationship.(7) The changes of hippocampal structure at later period after microwave exposure: The hippocampal structure did not change significantly in the 5 m W/cm~2 group. The 10 m W/cm~2 group can cause karyopyknosis at 1m and 3m and then showed a tendency to recover. The karyopyknosis of hippocampal neurons and widened perivascular gap were observed in the 50 m W/cm~2 group. At 6m-18 m the above injuries showed a recovery trend, but a small amount of neuron injuries were also observed at 18 m.(8) The expression of NR1, NR2 A and NR2 B in the rat hippocampus: The NR1 expression increased at 1m and then decrease from 3m to 18 m. The NR2 A expression increased, while the NR2 B expression decreased from 3m to 18 m.(9) The primary rat hippocampal neurons structural changes after 50 m W/cm~2 microwave radiation: At 5d, the number and length of neuritis significantly reduced and shorted. The fracture of connections between neurons and the neuritis were observed by SEM.(10) The NMDARs currents after microwave exposure in primary hippocampal neurons: The NMDARs current density decreases in the rat primary hippocampal neurons immediately after microwave exposure.(11) The protein and m RNA expression of NR1, NR2 A and NR2 B of the primary hippocampal neurons: The protein expression of NR2 B increased at 6h. The NR1, NR2 A NR2B expression increased at 12 h. The m RNA expression of NR1 increased at 1h, 6h and 12 h. The m RNA expression of NR2 A increased at 12 h, while the NR2 B m RNA expression increased at 1h and 12h(P<0.01).(12) The level of Ca2+ in primary hippocampal neurons: The Ca2+ level increased in rat primary hippocampal neuronal cytoplasm after 50 m W/cm~2 microwave radiation(P <0.01).Conclusions:(1) The 5m W/cm~2 microwave radiation did not cause obviously abnormalities in learning and memory and synaptic plasticity. However, the 10 and 50 m W/cm~2 microwave radiation can cause learning and memory impairment and synaptic plasticity damage, including the spatial learning and memory impairment, the metabolism disorders of amino acid neurotransmitters and organizational structure and ultrastructure damage with a clear dose of radiation.(2) The impairment of LTP induction and NMDARs subunit changes may be reasons of microwave induced learning and memory deficits.(3) The number and length of neuritis significantly reduced and shorted in the 50 m W/cm~2 group. Meanwhile, the fracture of connections between primary hippocampal neurons and the neuritis were also observed in the 50 m W/cm~2 group. The current density of NMDARs decreased after microwave exposure.(4) The 50 m W/cm~2 microwave increased of the protein and m RNA expression of NR1, NR2 A and NR2 B. Meanwhile, the increase of Ca2+ level was also observed in primary hippocampal neurons after microwave exposure.