Study On The Role Of MAOA And Contactin-5 In Regulating Abnormal Fear And Cognition In The Medial Prefrontal Cortex Caused By PTSD

Objective: Posttraumatic stress disorder(PTSD)is a long-lasting,complex psychiatric disorder that develops after exposure to a sudden traumatic event(e.g.serious traffic collisions,terrorist attacks,physical violence,unexpected death of a loved one,etc.),characterized by avoidant behavior,hyperarousal and pathologically enhanced fear.Longterm pathologically enhanced fear memory or poor fear extinction are typical features of PTSD.Currently,the exact pathogenesis of PTSD is not fully elucidated despite the presence of extensive research in animals and humans.Medial prefrontal cortex(mPFC)is a key brain area involved in the regulation of emotion,cognition and memory.The results of MRI show that the volume of mPFC in patients with PTSD is significantly smaller than that of normal people,accompanied by hypofunction of mPFC.Functional neuroimaging studies show that abnormal neurophysiological changes in the prefrontal cortex are involved in the pathogenesis of PTSD.In the studies on the medial prefrontal cortex involvement in PTSD,the previous studies of our research group has basically cleared that dysfunction of calcium signalling pathway,endoplasmic reticulum stress and autophagy leading to abnormal neuron function of the medial prefrontal cortex,which were involved in the pathogenesis of PTSD.Serotonin is involved in the pathophysiological mechanisms of mood regulation and anxiety.It has been well documented that dysfunctions in serotonergic neurotransmitter system are involved in numerous mental and behavioral disorders,including depression,anxiety and attention deficit hyperactivity disorder.According to reports in the literature,hypofunction of central serotonin is one of the pathogenesis of PTSD.The mitochondrial monoamine oxidase A subtype(MAOA)catalyzes the degradation process of various monoamine neurotransmitters and plays a key role in the metabolism of serotonin.In previous studies,a large body of evidence indicates that MAOA and its coding genes are involved in the regulation of emotions and behaviors.However,whether MAOA play a role in the pathogenesis of PTSD remains unclear.Contactin-5(Cntn 5),also referred to as neural recognition molecule(NB-2),belongs to the contactin subgroup of immunoglobulin superfamily.Cntn 5 is a glycosylphosphatidylinositol(GPI)-anchored glycoprotein and expressed in the central nervous system.The cell adhesion function of Cntn 5 is closely related to the formation and connection of brain circuits in early development and maintenance of synapse in adult.It has been reported that NB-2 deficiency can lead to insufficient formation of glutamate synapses and induce neuronal apoptosis.And it has been reported that NB-2 played an important role in promoting neurite outgrowth and confirmed that mice with NB-2 deficiency exhibited significantly reduced neural excitability.Whether Cntn 5 changes in PTSD and whether it is involved in the pathogenesis of PTSD have not been reported.Among the various animal models used to investigate the pathogenesis of PTSD,single prolonged stress(SPS)mimics the symptoms and neuroendocrine changes observed in patients with PTSD and is widely employed.In the present study,we also used SPS stimuli to create an animal model of PTSD.In this study,behavioral experiments,including elevated plus maze test(EPM),fear conditioning test(FCT),and prepulse inhibition(PPI)test were used to detect anxiety levels,fear memory and sensory gating function in PTSD-SPS rats.We also used Y maze to detect spatial working memory and spatial cognitive ability of PTSD-SPS rats through spontaneous alternation test(SAT)and novel arm exploration test(NAET).This study aims to investigate the pathophysiological role of MAOA and Cntn 5 in the pathogenesis of abnormal behavior in PTSD by locating and quantifying MAOA and Cntn 5 in the medial prefrontal cortex of rats after SPS stimulation.And we used moclobemide(Moc,MAOAinhibitor)for early intervention in SPS rats,and detected two synapse-related proteins to further explore the relationship between change of MAOA and fear abnormalities caused by SPS and the possible mechanisms by observing the changes of postsynaptic density proteins-95(PSD 95)and synapsin I(SYN 1).The present study provides experimental basis for further elucidating the pathogenesis of PTSD,and provides ideas for the development of new and more effective drugs for the treatment of PTSD.Methods: 1.Single-prolonged stress(SPS)was employed to create the animal model of PTSD.2.Sixty rats were used for behavioral tests and randomly divided into the following two groups: normal control group(Cont)and SPS group.EPM,FCT and PPI were used to detect anxiety levels,fear memory and sensory gating function in rats of Cont and SPS groups.Y maze test was employed to investigate spatial working memory and spatial cognitive ability in rats of Cont and SPS group via SAT and NAET.3.Forty-four rats were randomly divided into the following four groups: normal control group(Cont),SPS 1d,4d and 7d groups.Immunofluorescence staining,Western Blot and Real-Time PCR were used to detect the expression of MAOA in the infralimbic cortex(IL)of each group.4.Immunofluorescence staining and Western Blot were used to detect the expression of Cntn 5 in the mPFC of normal control and SPS groups.5.Western Blot was used to detect the protein expression of Glu R 1 and GluR 2 in the m PFC of normal control and SPS groups.6.N-type calcium channel currents of neurons in the mPFC were detected via whole-cell configuration of patch-clamp recording technique.7.A separate group of sixty rats were randomly divided into the following four groups: Cont rats with administration of vehicle(CON+vehicle);SPS followed by administration of vehicle(SPS+vehicle);SPS followed by administration of Moc(SPS+Moc);and Cont rats with administration of Moc(CON+Moc).EPM was used to evaluate anxiety level of rats in each group.FCT was used to observe fear memory of rats in each group.Western Blot was used to detect the protein expression of PSD 95 and SYN 1 in the IL of each group.Results:1.The behavioral changes of rats with PTSD: the results of EPM showed that rats after SPS stimuli exhibited fewer entries into the open arms(P < 0.01)and less time spent in the open arms(P < 0.01)than normal control rats.There was no significant difference in average speed between normal control and SPS group(P > 0.05).The results of FCT showed that rats of SPS group exhibited increased freezing behavior and higher freezing ratio during the conditioned stimulation than rats of Cont group(P < 0.01).The results of SAT showed that the percentage of spontaneous alternation(%SAs)and number of SAs in SPS group did not significantly differ from that in Cont group(P > 0.05).The results of NAET showed that rats of SPS group exhibited fewer entries into the novel arm(P < 0.05)and less time spent in the novel arm compared with Cont group(P < 0.01).Further,rats of SPS group exhibited longer latency period to the novel arm for the first time than rats of Cont group(P < 0.01).The results of PPI test showed that there was no significant difference in PPI between SPS group and Cont group when pre-stimulation was set to 70 dB,72 dB,and 76 dB(P > 0.05).2.Changes of MAOA in the IL of mPFC in rats of PTSD-SPS: the results of immunofluorescence staining showed that the positive expression of MAOA was mainly distributed in the cytoplasm and dentries,and the positive signal labeled with Cy3 was red fluorescence.Analysis results showed that MAOA-immunopositive cells ratio was significantly increased in SPS 1d and 4d groups compared with Cont group(P < 0.05).The results of Western Blot showed that the expression of MAOA protein increased after exposed to SPS stimulation(P < 0.05),which peaked at 1 day after SPS stimuli and was still higher at 4 days after SPS stimuli than Cont group,but decreased in SPS 7d group.The results of Real-Time PCR showed that the expression of MAOA mRNA increased significantly 1 day after SPS stimulation(P < 0.05),and then decreased.3.Changes of Cntn 5 in the mPFC in rats of PTSD-SPS: the results of immunofluorescence staining showed that the positive expression of Cntn 5 was mainly distributed in the cytoplasm and dentries of mPFC neurons,and the positive signal of FITC was green fluorescence.Analysis results showed that Cntn 5-immunopositive cells ratio was significantly decreased after exposed to SPS stimulation compared with Cont group(P < 0.05).The results of Western Blot showed that the expression of Cntn 5 protein decreased after exposed to SPS stimulation(P < 0.05).4.Western Blot was used to detect the protein expression of GluR 1 and GluR 2 in the mPFC.The results showed that the expression of GluR 1 and GluR 2 in the mPFC decreased after exposed to SPS stimulation(P < 0.05).5.The results of whole-cell patch-clamp showed that there were no significant changes in N-type calcium channel and total calcium channel current density of mPFC neurons after exposed to SPS(P > 0.05).6.Changes of behavioral and expression of synaptic-associated proteins after intervention with Moc in rats of PTSD-SPS: the results of EPM showed that the interaction between groups(Cont/SPS)and treatments(vehicle/Moc)had significant impacts on the entries into the open arms and time spent in the open arms(P < 0.01).Post hoc comparisons showed that Moc had a significant effect on SPS stimulation.SPS+Moc group showed more entries into the open arms(P < 0.01)and longer time spent in the open arms(P < 0.01)compared to SPS+vehicle group,which indicated improvement of anxiety behavior.The results of FCT showed that the interaction between groups(Cont/SPS)and treatments(vehicle/Moc)had significant impacts on the freezing ratio during the conditioned stimulation of test phase(P < 0.01).Post hoc comparisons showed that Moc had a significant effect on SPS stimulation.SPS+Moc group showed lower freezing ratio during the conditioned stimulation of test phase(P < 0.01),which indicated reduced fear memory.The results of PSD 95 and SYN 1 using Western Blot showed that the interaction between groups(Cont/SPS)and treatments(vehicle/Moc)had significant impacts on the expression of PSD 95 and SYN 1 in IL neurons(P < 0.01).Post hoc comparisons showed that Moc had a significant effect on SPS stimulation.SPS+Moc group showed increased protein levels of PSD 95 and SYN 1(P < 0.05).Conclusion: 1.Rats of PTSD-SPS exhibited increased levels of anxiety,increased fear memory and poor fear extinction,decreased spatial recognition and exploration ability,impaired spatial cognition,but spatial working memory and sensory sensorimotor gating function did not change.2.The activation of MAOA in the IL region of rats after SPS stimulation might be related to the abnormal fear caused by PTSD.3.Administration of Moc could reduce the anxiety level of PTSD-SPS rats,and might facilitate fear extinction and attenuate fear memory by increasing the level of synaptic-associated proteins PSD 95 and SYN 1 in the IL of mPFC.4.The expression of Cntn 5,GluR 1 and GluR 2 protein in the mPFC of PTSD-SPS rats decreased,leading to abnormalities in the glutamate neurotransmitter system,which might be one of important mechanisms of reduced spatial recognition and cognitive impairment in rats with PTSD.