| Objective Central nervous system is sensitive to microwave radiation,among which hippocampus is an important sensitive brain region.Our previous study found that the dysfunction of hippocampal cholinergic system is involved in the pathophysiological process of neurobehavioral changes induced by microwave radiation,but the detailed mechanism has not been clarified.On the basis of previous research work,this study aims to explore the role and mechanism of hippocampal cholinergic neurons in the neurobehavioral changes induced by microwave radiation based on optical fiber recording,photogenetic and chemical genetic techniques,so as to reveal the mechanism of microwave radiation brain injury from the level of neural microcircuit.Method1.To study the effects of microwave radiation on learning and memory and anxiety in mice by neurobehavioral testing: animal model of microwave radiation was established by exposing male adult C57BL/6 mice to 2.856 GHz microwave.Morris water maze,shuttle box,Y maze,new object recognition behavior experiment were used to detect the changes of learning and memory function of mice.The behavioral experiments of open field and elevated plus maze were conducted to detect the changes of anxiety in mice.2.To observe the histopathological changes of hippocampus after microwave irradiation by light microscope and transmission electron microscope: paraffin sections of brain tissue and HE staining were used to observe the histopathological changes of hippocampus under light microscope.Ultra-thin sections of hippocampus tissue were prepared and ultrastructural changes of hippocampus were observed by transmission electron microscope3.To study the effect of microwave radiation on the calcium activity of hippocampal neurons based on genetic coding calcium imaging technology: Genetic coding calcium imaging and fiber optic recording technology were used to detect the changes in the calcium activity of neurons combined with the behavioral paradigm of new object recognition,Y maze,open field and elevated cross maze.4.To study the regulation of hippocampal cholinergic neurons on neurobehavioral changes induced by microwave radiation based on photogenetic and chemical genetic techniques: Chat-icre mice labeled with cholinergic neurons were used to study the effects of photogenetic activation and inhibition of hippocampal cholinergic neurons on neurobehavioral changes induced by microwave radiation.To investigate the effects of drug（CNO）activation and inhibition of cholinergic neurons in hippocampus on neurobehavioral changes after microwave irradiation in chat-ICRE mice.5.To detect the effects of microwave radiation on AChrelease and content in hippocampus based on genetically coded fluorescent probe and microdialysis: AChfluorescence probe combined with optical fiber recording and behavioral paradigm and microdialysis combined with mass spectrometry were used to detect AChrelease and content changes in hippocampal CA3 region.6.To investigate the mechanism of hippocampal cholinergic neurons in neurobehavioral changes induced by microwave radiation was investigated by molecular biology detection: immunofluorescence was used to detect the expression changes of VAChT and ChAT.Result1.Effects of microwave radiation on learning,memory and anxiety: Morris water maze results showed that after radiation,the number of crossing the platform in reverse space exploration experiment decreased.The shuttle-chamber experiment showed that the average active escape rate,the average active latency and the total shock time of mice were significantly decreased after radiation,suggesting that microwave radiation could reduce the ability of spatial reference memory and associative learning and memory.The results of new object recognition experiment showed that the times and time of exploring new objects were significantly reduced after irradiation,suggesting that the new object exploration behavior of mice was reduced after microwave radiation.No significant difference was observed in Y maze test.The results of open field experiment showed that the time and distance of entering the central region of mice were significantly reduced after radiation.The results of the elevated plus maze experiment further showed that the open arm distance and open arm time were significantly reduced after radiation,suggesting that radiation could induce anxious behavior in mice.2.Effects of microwave radiation on hippocampal tissue structure: At 7 days after microwave irradiation,degeneration of hippocampal neurons,mainly presenting as hyperchromatic pyrosis of nucleus,shrinkage of cell body,and slight widening of perivascular space were found by light microscopy,especially in CA3 and DG regions.Transmission electron microscopy showed that some hippocampal CA3 neurons had nuclear chromatin aggregation,edge aggregation and apoptosis.Mitochondrial swelling,focal cavitation,rough endoplasmic reticulum expansion,degranulation,synaptic bilayer structure fuzzy;Mitochondria of glial cells were swollen,focal cavitation,and perivascular space was widened.3.Effects of microwave radiation on neuronal calcium activity: Fiber optic recording combined with new object recognition experiment showed that the decreased activity of bilateral CA1 and left CA3 neurons was involved in the reduction of new object exploration behavior induced by radiation in mice.Fiber optic recording combined with Y maze experiment showed that the activity of right CA3 neurons was enhanced and involved in the new arm exploration behavior of mice after radiation.Fiber optic recording combined with open field experiment showed that the activity of CA1 and CA2 neurons on the left side decreased,and the activity of CA3 neurons on the left side increased,which was involved in the changes of anxiety in mice induced by radiation.Fiber optic recording combined with elevated plus maze experiments showed that reduced CA2 neuronal activity was involved in radiation-induced anxiety changes in mice.4.Effects of photogenetic and chemogenetic regulation of hippocampal cholinergic neurons on neurobehavioral changes after microwave radiation: The results of the open field and Y maze experiments showed that the activation of CA1 cholinergic neurons increased anxiety,while the activation of CA2 cholinergic neurons decreased anxiety,and the activation of CA3 cholinergic neurons increased anxiety and inhibited new arm exploration.Photogenetic inhibition combined with new object recognition test showed that CA3 cholinergic neuronal inhibition promoted new object exploration behavior in mice after radiation.Combined with the results of open field,Y maze and new object recognition experiments,the activation of CA3 cholinergic neurons further aggravated anxiety,inhibited the exploration of new limb,and promoted the exploration of new object in mice after radiation.5.Effects of microwave radiation on ACh,VAChT and ChAT contents in hippocampus: The results of AChrelease detected by fiber optic recording and behavioral paradigm showed that AChrelease increased in the right CA3 neuron synapse,which was involved in the novel arm exploration behavior of mice after radiation.The release of AChfrom the right CA3 synaptic decreased,and the new object exploration behavior of the mice was reduced.The results of microdialysis combined with mass spectrometry showed that the concentrations of choline and acetylcholine in hippocampal CA3 region of mice were significantly decreased and increased in 3 days and 7 days after radiation,and no significant difference was observed in the left and right sides.Immuno fluorescence results showed that VAChT and ChAT expression were both down-regulated in CA3 neurons 7 days after radiation,suggesting abnormal cholinergic transmission in hippocampus.Conclusion1.Microwave radiation caused a decline in spatial memory ability,joint learning and memory ability and new object exploration behavior,while microwave radiation also could induce anxiety like behavior and hippocampal structure damage.2.Abnormal activity of hippocampal neurons in CA1,CA2 and CA3 were involved in neurobehavioral changes induced by microwave radiation in mice,and CA3 was more sensitive.Bilateral hippocampal response was asymmetrical,and the change was more obvious on the left side.3.Activation of hippocampal cholinergic neurons after radiation can further aggravate anxiety and inhibit the new limb exploration behavior of mice,and inhibition of CA3 cholinergic neurons can promote the new object exploration behavior of mice after radiation.4.Microwave radiation can cause disturbance and abnormal transmission in CA3 region.Abnormal AChrelease from mouse CA3 neurons is one of the important mechanisms of microwave-induced behavioral changes in mice,especially in the right CA3 region. |
PDF Full Text Request