Underlying Mechanism Of 5-HT2AR/5-HT1AR In Hippocampus Participating In Anxiety-like Behavior Of Post Traumatic Stress Disorder Mice

BackgroundPost traumatic stress disorder is a psychiatric disorder that is caused by catastrophic events or abnormal threats,which is characterized by postponed and long-existed mental disorders.And the patients with PTSD frequently have fierce anxieties or fears.Anxiety will seriously affect people's quality of life and work efficiency,as well as interfering with the development of individuals and society.Trauma or stress can result in structural and functional changes of amygdala,hippocampus,hypothalamus-pituitary-adrenal and serotonin system,and cause anxiety etc.The hippocampus is one of the most important regions of the limbic system in the brain and plays an active role in anxiety via increasing its sensitivity to surrounding change.Serotonin is an important kind of monoamine neurotransmitters that is widely distributed in the central nervous system.It is involved in a variety of mental diseases such as anxiety and emotional disorders.Serotonin receptors have 7 types,among which 5-HT2A and 5-HT1A receptors are mainly distributed in the hippocampus,the cortex,etc.,and closely associated with anxiety.The expression of 5-HT2A receptor(5-HT2AR)can enhance anxiety-like behavior under stress conditions,whereas 5-HT1A receptor(5-HT1AR)can inhibit it.5-HT1A receptor activation decreases ERK phosphorylation.However,is there a connection between 5-HT2ARand 5-HT1AR,which will affect the anxiety-like behavior?Do these contacts influence the anxiety-like behavior by the ERK pathway?All of these challenges are exclusive and worthy to be further investigated.ObjectivesTo clarify the effect of antagonizing 5-HT2AR/5-HT1AR in hippocampuson mice's anxiety-like behavior and the regulatory mechanism of ERK pathway.Methods1.The animal model of PTSD was established by subjecting the male C57BL/6 mice and the male ePet-Cre-Ai9 mice to the process of conditioned fear stress(CF)combining with single prolonged stress(SPS).2.To explore the role of antagonizing 5-HT2AR/5-HT1 AR in hippocampusin the PTSD mice,the 5-HT2A receptor antagonist,Ketanserin,and the 5-HT1A receptor antagonist,WAY100635 was respectively applied to the control during establishing the animal model with the male C57BL/6 mice.3.When the animal model was established using the male C57BL/6 mice and the male ePet-Cre-Ai9 mice,the animal behaviors were analyzed by the open field test,the freezing behavior test and the elevated plus maze test.4.In order to illuminate the role and mechanism of antagonizing 5-HT2AR/5-HT1AR in hippocampus on the anxiety-like behavior in the PTSD mice and the regulatory mechanism of ERK pathway,the expression of the 5-HT2A and 5-HT1A receptors,ERK1,ERK2,pERKl,pERk2,c-Myc and BDNF in the male C57BL/6 mice hippocampi was separately detected by western blot,RT Q-PCR and the immunofluorescence labeling technology.5.The role of the expression of the 5-HT2A and 5-HT1A receptors of 5-HTergic neuronin the male ePet-Cre-Ai9 mice hippocampi was analyzed by the immunofluorescence labeling technology.Results1.When the animal model was established on the male C57BL/6 mice,an open-field test was applied to examine the PTSD model in question.The results indicated that compared to the sham group,times through the central grille and total distance of the PTSD group significantly decreased,middle distance/total distance of the PTSD group notably increased.In the freezing behavior test,the percentage of freezing behavior of the PTSD group was prominently added compared with that of the sham group.The freezing behavior of the 5-HT1AR antagonist group was signally changed compared to the PTSD group.The freezing behavior of the 5-HT2AR antagonist group was added compared to the PTSD group,but there is not a significant difference.In an elevated plus maze test,the results showed that after the animal model established in 7 days,OA entries and OA time of the PTSD group were dramatically decreased compared to the sham group.On day 14,OA entries and OA time of the PTSD group were memorably increased compared to the sham group.On day 21,OA entries of the PTSD group were significantly added,but OA time of the PTSD group was notbly decreased compared to the sham group.These activities of the 5-HT1AR antagonist group were obviously changed compared to the sham group."OA entries" denoted that the numbers of entries into the open arms/(the numbers of entries into the open arms +closed arms);"OA time"indicated that the time spent in the open arms/(the time spent in the open arms +closed arms).2.When the animal model was established on the male C57BL/6 mice,the relative expression of 5-HT2AR and 5-HT1AR was analyzed by Western blot.RT Q-PCR and Immunofluorescent Labeling analysis.The results demonstrated that the expression of 5-HT2AR in the mice hippocampi of the PTSD group was signally increased on day 14,and notably decreased as time passed on day 14 and 21,however,it was always dramatically added in the 5-HT1AR antagonist groups.The relatively expression of 5-HT1AR of the PTSD group in the mice hippocampi increased all the time,but did not obviously augmented in the 5-HT2AR antagonist groups.The relatively expressions of ERK1?ERK2?pERK1?pERk2?c-Myc of the PTSD group in the mice hippocampi were increased and there was time effect in all of these changes.When the animal model was established using the male C57BL/6 mice,the relative expression of BDNF of the PTSD group in the mice hippocampi was decreased,but was respectively increased in the 5-HT2AR and 5-HT1AR antagonist groups.3.When the animal model was established using the male C57BL/6 mice and the male ePet-Cre-Ai9 mice for 14 days,the expressions of 5-HT2AR and 5-HT1AR in 5-HTergic neuronwere analyzed by immunofluorescent labeling analysis.The results showed that the expressions of 5-HT2AR and 5-HT1AR of 5-HTergic neuronin the mice hippocampi were added.Conclusions1.The CF combining with SPS process can obviously change the mice behavior and make the mice obtain the fear memory,thus the CF combining with SPS process is a preferable model for PTSD.2.Antagonizing 5-HT1AR expressions can significantly promote the expressions of 5-HT2AR,aggravate the mice anxiety-like behavior and there is the time effect.There is no influence on antagonizing 5-HT2AR expressions to the expression of 5-HT1AR.3.Antagonizing 5-HT1AR expressions promoting the expressions of 5-HT2AR can aggravate the mice anxiety-like behavior by the activation of ERK pathway.There is the time effect on the activation of ERK pathway via ERK phosphorylation.There is no influence on antagonizing 5-HT2AR expressions to the activation of ERK pathway via ERK phosphorylation.4.BDNF has a resistantrole on antagonizing 5-HT1AR expressions promoting the expressions of 5-HT2ARaggravating the mice anxiety-like behavior.5.The expressions of 5-HT2AR and 5-HT1AR of 5-HTergic neuron in the PTSD mice hippocampi were added and enhanced their anxiety-like behavior.