Investigating Mild Fever Induced Structural Synaptic Plasticity In Hippocampal Neuron

Objective:Fever occurs when a temperature set point is increased by more than 0.5-1.3℃.Fever is a common feature of many human and animal diseases,and plays a crucial role in the body’s response to the disease.Apart from that,many clinical disease such as stroke,traumatic brain injury and intracranial hemorrhage are accompanied by fever.And this will cause secondary damage to the brain.Therefore,there is great clinical significance to explore the effect of fever on neurons.Extreme fever of 42℃ or severe hyperthermia has been shown to irreversibly damage the structure of the brain cells.In contrast,little is known about what happens in the brain following milder forms of fever(38-39℃).In this work,we focused on investigating mild fever induced structural synaptic plasticity in hippocampal neuron.Methods:1.Prepare two carbon dioxide cell incubators,one at 37 °C and the other at 39 °C,to provide different temperature environments for neurons.2.Use trypan blue staining to stain neurons which treated at different temperatures to observe the effect of mild fever on the survival rate of hippocampal neurons.3.Using the method of Immunofluorescent staining,combined with confocal microscope,to observe the effect of mild fever on the presynaptic membrane structural protein BSN,the glutamate transporter v Glut1 which located on the surface of excitatory vesicles,the postsynaptic membrane and the location of glutamate receptor AMPARs.4.Use the Syn-PER kit to extract synaptic proteins from neurons.5.Western blot was used to observe the changes of the presynaptic membrane structural protein BSN and the glutamate transporter v Glut1 after mild fever.Results:1.The effect of mild fever on the survival rate of hippocampal neurons.The hippocampal neurons were placed in a 39℃ incubator overnight and stained with trypan blue,and the survival rate of each group was counted.The survival rate of control group was 93%,and the survival rate of fever group was 91%.So mild fever did not affect the survival rate of hippocampal neurons.2.The effect of mild fever for 2 h on the content of excitatory vesicles in synapses of hippocampal neurons.After hippocampal neurons were treated at 39℃ for 2 h,the content of v Glut1 was significantly decreased compared with the control group 37℃(P<0.01).Which indicated that the content of vesicles in synapses was significantly reduced after 2 h of mild fever.3.The effect of mild fever for 2 h on the release site of presynaptic vesicles in hippocampal neurons.After hippocampal neurons were treated at 39℃ for 2 h,the BSN content did not change significantly compared with the control group 37℃(P >0.05).Which indicated that the vesicle release function of the presynaptic membrane was not affected after 2 h of mild fever,that is,the fever did not affect the function of the presynaptic membrane to release neurotransmitters.4.The effect of mild fever for 2 h on the excitatory synaptic vesicles of hippocampal neurons.After hippocampal neurons were treated at 39℃ for 2 h,the content of excitatory synaptic vesicles which located in the AZ was decreased significantly(P<0.05).Which suggested that mild fever would affect the position of excitatory synaptic vesicles in the presynaptic membrane and reduce the content of excitatory vesicles in the released state.5.The effect of mild fever overnight on the content of excitatory vesicles in synapses.After hippocampal neurons were exposed to 39℃ overnight,compared with the control group 37℃,the v Glut1 content decreased,but the decrease trend was less than 2 h treatment experiment.6.The effect of mild fever overnight on the release site of presynaptic vesicles in hippocampal neurons.After hippocampal neurons were exposed to 39℃ overnight,there was no significant change in BSN content(P>0.05)compared with the control group 37℃.Which indicated that the vesicle release function of the presynaptic membrane was not affected with the prolongation of the heating time,that is,the heating did not affect the function of the neurotransmitter released by the presynaptic membrane.7.The effect of mild fever overnight on excitatory synaptic vesicles of hippocampal neurons.The content of synaptic vesicles of 39°C group was significantly reduced(P<0.01),and the degree of reduction was higher than that in the 2 h condition(P<0.05).The location of the presynaptic membrane was altered,and excitatory synaptic vesicles moved away from the site of vesicle release in the presynaptic membrane.In the meantime,mild fever remodeled the structure of the presynaptic membrane in hippocampal neurons.8.The effect of mild fever on post-synaptic glutamate receptors AMPARs in hippocampal neurons.After hippocampal neurons were exposed to 39 ℃ overnight,the excitatory glutamate of the post-synaptic membrane in 39℃ group was no significant change in the content of acid receptor AMPARs(P>0.05).Which indicated that mild fever did not affect the function of the postsynaptic membrane.9.The effect of mild fever on the synapses of cortical neurons.After the cortical neurons were kept at 39℃ overnight,the BSN and v Glut1 contents had no significant changes (P>0.05)compared with the control group at 37℃.Which indicated that mild fever has different effects on synapses of hippocampal neurons and cortical neurons.10.The data of TMT showed that insulin signaling pathway may take part in the remodeling activity of synapses.Conclusion:1.Mild fever remodels the structure of the presynaptic membrane of hippocampal neurons to keep the presynaptic membrane in an inhibitory state.After 2 h of mild fever,the content of synaptic vesicles which loaded with excitatory neurotransmitter decreased significantly,and the content of synaptic vesicles located in the neurotransmitter release zone of the presynaptic membrane was also significantly reduced,all of which indicated that mild fever would inhibit the activity of the presynaptic membrane.2.After hippocampal neurons were treated at 39℃ overnight,the content of synaptic vesicles in the presynaptic neurotransmitter release zone decreased more significantly than that treated at 39℃ for two hrs.This suggests that the inhibitory effect of mild fever on the presynaptic membrane increases with time,3.After the hippocampal neurons were treated at 39℃ overnight,the content of AMPARs in the postsynaptic membrane did not change,suggesting that mild fever may have no effect on the postsynaptic membrane of hippocampal neurons.4.The synaptic proteins that were significantly decreased in the synapses of hippocampal neurons during mild fever while it did not change significantly in cortical neurons,indicating that mild fever has different effect on synapses of hippocampal neurons or cortical neurons.