| ObjectiveMicrowaves exist widely in people's life and work environment,the health hazards of which are of concern.Brain is one of the target organs sensitive to microwaves.It has been demonstrated that microwave exposure can cause neuronal synaptic plasticity damage,but related mechanisms are unknown.As a lysosome-dependent degradation pathway,autophagy removes intracellular aging or damaged organelles and proteins,and its role in synaptic plasticity is gradually taken seriously.Additionally,there is strong consensus that AMPK/mTOR signaling plays import roles in autophagy regulation.However,the study of the changes in neuronal autophagy after microwave exposure is still blank,and related molecular mechanisms have yet to be revealed.This study was to identify the changes in autophagy in neurons,related mechanisms and its influence on synaptic plasticity after microwave exposure,in order to provide evidence for identifying potential,causal molecular mechanisms.Materials and methods1.Study on the effects of microwave exposure on autophagy and AMPK/mTOR pathway in rat hippocampal neurons.A total of 110 male Wistar rats were randomly divided into two groups:microwave-exposed(MW)group and sham-exposed(SH)group.Rats of MW group were exposed to microwave source(average power density,30 mW/cm~2)for 15 min every other day three times.Rats in SH group were processed in parallel with those in MW group,but with microwave source switched off.The body surface and rectal temperatures of rats were monitored before and immediately following exposure.At 6 h,7 d,14 d,1 m,2 m and 3 m after exposure,probe trials were conducted.To evaluate the changes in rat cognitive function and hippocampal neuronal synaptic plasticity after microwave radiation,Morris water maze,biopac system,in vivo long-term potentiation(LTP)recording system,high performance liquid chromatography(HPLC),H&E staining and transmission electron microscope(TEM)were used to detect the spatial learning and memory ability,electroencephalogram(EEG),hippocampal LTP of rats,neurotransmitters and structures in rat hippocampus,respectively.Autophagy activity in rat hippocampus was assessed by autophagy ultrastructure,molecular markers(LC3-I/II,Beclin1 and LAMP1)and related genes(Atg5,Atg7 and Atg9)detected by TEM,Western Blot and immunohistochemistry(IHC).Immunofluorescence(IF)staining was used to show cell nuclei(labeled with DAPI),synaptophysin I(labeled with FITC)and LC3(labeled with TRITC)in rat hippocampus.Additionally,the key signal molecules of the AMPK/mTOR pathway(p-AMPK?Thr172,AMPK?,p-mTOR Ser2448,mTOR)in rat hippocampus were detected by Western Blot.2.Study on the regulatory role of AMPK/mTOR on neuronal autophagy induced by microwave exposure.Primary rat hippocampal neurons and PC12-derived neuron-like cells(neuron-like cells)were randomly divided into MW group and SH group.Cells in MW group were exposed to microwave source(average power density,30 mW/cm2)for 15 min.Cells in SH group were treated as the MW group,but without microwave exposure.The cells were harvested 1 h,6 h,12 h and 24 h after exposure.Synaptic plasticity related indicators of neurons in vitro,including synaptic structures,excitatory postsynaptic current(EPSC)and neurotransmitters,were measured by scanning electron microscope(SEM),patch clamp and HPLC,respectively.In neuron-like cells,autophagy structures,LC3 flux,molecular markers and related genes(as listed before)were detected by TEM,IF and Western Blot.And the key molecules of the AMPK/mTOR pathway in neuron-like cells were also tested.Thereafter,dorsomorphin and rapamycin were added to the culture medium of neuron-like cells 1 h before microwave exposure to inhibit AMPK and mTOR,respectively,the phosphorylation of AMPK/mTOR and LC3 flux in neuron-like cells were detected 6 h after exposure.3.Study on the effects of autophagy on synaptic plasticity injury induced by microwave exposure.LY294002 and Rapamycin were added to the culture medium of neuron-like cells 1 h before exposure,and LC3 flux and synaptic plasticity related indicators(structures and neurotransmitters)of neuron-like cells were then detected 6 h after exposure.Results1.Changes in rat temperatures.There were no significant differences observed in the surface temperatures and the rectal temperatures of rats between MW group and SH group before and after exposure(P>0.05).2.Changes in rat cognitive function and synaptic plasticity in rat hippocampal neurons.(1)Spatial learning and memory ability.The average escape latency(AEL)of rats was prolonged at 7 d and 14 d(P<0.05 or P<0.01),the percentage of time in the target quadrant was decreased at 7 d,14 d and 1 m(P<0.05 or P<0.01),and the number of crossings in 60 seconds was reduced at 14 d(P<0.01),after microwave exposure.(2)EEG.For the MW group,the gravity frequency of EEGs was decreased at 6 h and 7d(P<0.05 or P<0.01),the?wave power was decreased at 6 h(P<0.05),the?wave power was increased at 6 h(P<0.01),and the?wave power was increased at 6 h and 7 d after exposure(P<0.01).(3)LTP:Rats in the MW group showed a significant decline in PS amplitude at 6 h after exposure(P<0.01),indicating that the LTP in rat hippocampus was inhibited by microwave radiation.(4)Glu and GABA:Glu in rat hippocampus was down-regulated at 7 d and 14 d(P<0.05),GABA was up-regulated at 14 d(P<0.05),and the ratio of Glu to GABA was decreased at 7 d and 14 d(P<0.05 or P<0.01),after microwave exposure.(5)Structures:Rat hippocampal neurons of SH group showed normal structures.In the MW group,narrowed neurons,eosinophilic cytoplasm,attenuated triangular or spindle nuclei under light microscope,and decreased synaptic vesicles,blurred synaptic gaps and increased postsynaptic density under TEM,were observed at 7 d,14 d and 1 m.3.Changes of autophagy structures,molecular markers and related genes in rat hippocampal neurons.(1)Structures.The amount of autophagic vacuoles in rat hippocampal neurons was obviously increased at 7 d,14 d and 1 m following microwave exposure.In addition,large numbers of synaptic vesicles were encapsulated by autophagosomes,a phenomenon more obvious in the MW group.(2)Autophagy markers and related genes.Compared with that of SH group,the ratio of LC3-II to LC3-I in rat hippocampus of MW group was elevated at 14 d and 1 m(P<0.01),Beclin1 was increased at 7 d and 14 d(P<0.05 or P<0.01),LAMP1 was increased at 7 d,14 d and 1 m(P<0.01),Atg5 was increased at 7 d and 14 d(P<0.05 or P<0.01),and Atg9 was increased at 7 d(P<0.01).4.Changes in the colocation of autophagosomes and synaptic vesicles in rat hippocampus.After LC3 and synaptophysin I were labeled with TRITC and FITC at 14 d after exposure,respectively,a obvious increase in yellow punctate aggregation was observed in rat hippocampus of MW group and the Pearson's Rr was increased(P<0.01).5.Changes of the AMPK/mTOR pathway in rat hippocampus.The ratio of p-AMPK?Thr172 to AMPK?in rat hippocampus was increased at 14 d(P<0.01),and the ratio of p-mTOR Ser2448 to mTOR was decreased at 7 d(P<0.01),after microwave exposure.6.Changes in the synaptic plasticity in primary rat hippocampal neurons and neuron-like cells.(1)Structures.At 6 h and 24 h after exposure,the synaptic length of neuron-like cells in MW group was shortened and the connections between synapses were broken.(2)EPSC:The EPSC current density of primary rat hippocampal neurons in the MW group was significantly decreased at 6 h after exposure(P<0.05).(3)Glu and GABA:After microwave exposure,Glu in the culture medium of neuron-like cells was decreased at 1 h(P<0.01),GABA was increased at 6 h(P<0.01),and the ratio of Glu to GABA was decreased at 1 h and 6 h(P<0.01).7.Changes of autophagy structures,markers and related genes in neuron-like cells.(1)Structures.The autophagosomes and autolysosomes were occasionally found in neuron-like cells of the SH group,which in the MW group were increased obviously at6 h after exposure.(2)Autophagy markers and related genes.At 6 h,the number of LC3 dots in neuron-like cells of MW group was increased by co-treatment with chloroquine(P<0.01),indicating LC3 flux could be enhanced by microwave exposure.Beclin1 in neuron-like cells was up-regulated at 6 h(P<0.05),LAMP1 was increased at 12 h and24 h(P<0.01),and the expression of Atg5,Atg7 and Atg9 was increased at 6 h and 12 h(P<0.05 or P<0.01),after microwave exposure.8.Changes of the AMPK/mTOR pathway in neuron-like cells.After microwave exposure,the ratio of p-AMPK?Thr172 to AMPK?in neuron-like cells was increased at 6 h and 12 h(P<0.05 or P<0.01),the ratio of p-mTOR Ser2448 to mTOR was decreased at 6 h(P<0.05).9.Changes in the autophagy activity of neuron-like cells after AMPK/mTOR intervention.(1)Changes in AMPK phosphorylation and LC3 flux in neuron-like cells after Dorsomorphin pretreatment.Compared to microwave exposure alone,co-treatment with Dorsomorphin down-regulated phosphorylated AMPK?at Thr172(P<0.01)and reduced LC3 flux(P<0.01)in neuron-like cells,at 6 h after exposure.(2)Changes in mTOR phosphorylation and LC3 flux in neuron-like cells after Rapamycin pretreatment.Compared to microwave exposure alone,co-treatment with Rapamycin down-regulated phosphorylated mTOR at Ser2448(P<0.01)and enhanced LC3 flux(P<0.01)in neuron-like cells,at 6 h after exposure.10.Changes in the synaptic plasticity of neuron-like cells after autophagy intervention.(1)Changes in LC3 flux and synaptic plasticity in neuron-like cells after LY294002pretreatment.Compared to microwave exposure alone,co-treatment with LY294002reduced LC3 flux(P<0.01),aggravated cell structure damages and increased GABA release(P<0.01)in neuron-like cells,at 6 h after exposure.(2)Changes in LC3 flux and synaptic plasticity in neuron-like cells after Rapamycin pretreatment.Compared to microwave exposure alone,co-treatment with Rapamycin enchanced LC3 flux(P<0.01),showed no effects on cell structures and inhibited GABA release(P<0.01)in neuron-like cells,at 6 h after exposure.Conclusions1.30 mW/cm~2 microwave exposure induced cognitive impairment and synaptic plasticity damage in rats,manifested as declined spatial learning and memory ability,fluctuated brain electric activities,inhibited hippocampal LTP induction in rats,abnormal Glu and GABA metabolisms,reduced synaptic vesicles,blurred synaptic gaps and increased postsynaptic density in rat hippocampal neurons.2.30 mW/cm~2 microwave exposure led to the activation of autophagy in rat hippocampal neurons,indicated by the increased autophagosomes and autolysosomes,elevated ratio of LC3-II to LC3-I,and up-regulated Beclin1,LAMP1,Atg5 and Atg9.3.30 mW/cm~2 microwave exposure increased the colocation of autophagosomes and synaptic vesicles in rat hippocampal neurons.Autophagy-mediated degradation of synaptic vesicles was closely related to synaptic plasticity damage caused by microwave exposure.4.A cell model for synaptic plasticity damage and autophagy activation in neurons induced by microwave exposure was well established.Microwave induced synaptic plasticity damage in primary rat hippocampal neurons and neuron-like cells,shown in the shortened and broken synapses,inhibited EPSC and disordered Glu and GABA release.Microwave promoted autophagy in neuron-like cells,as demonstrated by the increased autophagosomes and autolysosomes,enhanced autophagic flux,increased expression of Beclin1,LAMP1,Atg5,Atg7 and Atg9.5.Microwave exposure activated AMPK and inhibited mTOR in neurons,an important mechanism underlying the activation of neuronal autophagy mediated by microwave radiation.6.Activated autophagy exerted a potential mechanism of self-protective response to microwave-induced synaptic plasticity damages in neurons.Inhibition of autophagy aggravated the damage of neuron-like cells caused by microwave radiation.Activation of autophagy led to a decrease in GABA release from neuron-like cells after microwave exposure. |
PDF Full Text Request