| Part one: FGF2 Alleviates PTSD Symptoms by Regulating GLAST Function in Astrocytes via JAK/STAT PathwayObjectives: FGF2 is an important mitogen that has the ability to regulate neuronal and astrocytic activities.Previous study reported that FGF2 ameliorated SPS induced PTSD-like behaviors and astrocytic inhibition.GLAST/GLT-1 are cognition related glutamate transporters that are expressed on astrocytes and involved in regulation of the extracellular level of glutamate and neuronal survival.In the present study,we used the SPS model to study the effects of fibroblast growth factor 2(FGF2)on the expression of astrocytic GLAST and GLT-1 and on PTSD-like behaviors and its potential molecular mechanism.Methods:70 adult male SD rats(250-300 g,8 weeks old)were randomly divided into control(n=15),SPS(n=25),SPS+ FGF2(n=15)and SPS+FGF2+AG490(JAK/STAT inhibitor,n=15)groups.The rats endured SPS treatment,intraperitoneal injections of FGF2(20 mg/kg·day)and AG490(10 mg/kg·day).Rats endured fear conditioning tests from day 7 to 28 after SPS induction,and open field test and elevated plus maze test were implemented on day 7/14/28 after SPS induction.After the behavioral tests,the rats were sacrificed and the CSF were used to detect the glutamate level,and the hippocampal tissue were taken to implement Western blot and immunofluorescent tests detecting the GFAP,GLT-1,GLAST,p-JAK1,JAK1,p-JAK2,JAK2,p-STAT3 and STAT3 in the hippocampus.FGF2(30 ng/m L)and AG490(50 μM)were administered to the secondary hippocampal astrocyte for 48 h,and the cells were harvested for Western blot and immunofluorescent test.Results: SPS enhanced the contextual and cued fear conditioning responses 7 days and 14 days after SPS induction(compared with the control group,P<0.05).Fourteen days after SPS,the freezing time reflecting the contextual and cued fear conditioning of the SPS+FGF2 group were shorter than those of the SPS group(P<0.05).This effect was blocked by AG490(compared with the SPS+FGF2 group,P<0.05).In the open field test(OFT)and the elevated plus maze test(EPMT),FGF2 significantly alleviated the anxiety like behaviors induced by SPS: in the OFT,compared with the control group,the distance and time in the central area were significantly reduced both on SPS-7 day and SPS-14 day(P<0.01),compared with the SPS group,the two parameters of the SPS+FGF2 group were larger than those of the SPS group(P<0.01),compared with the SPS+FGF2 group,the two parameters were reduced again(P<0.05);in the EPMT,compared with the control group,the time in open arms(%)and entries into open arms(%)were significantly reduced both on SPS-7 day and SPS-14 day(P<0.01),compared with the SPS group,the two parameters of the SPS+FGF2 group were larger than those of the SPS group(P<0.01),compared with the SPS+FGF2 group,the two parameters were reduced again(P<0.05).In the HPLC test,Western blot test and the immunofluorescent test: compared with the control group,the glutamate level was higher in the SPS group(P<0.01),the GFAP,GLAST,GLT-1,p-JAK1,p-JAK2,and p-STAT3 were lower(P<0.01),compared with the SPS group,the glutamate level in the CSF was lower in the SPS+FGF2 group(P<0.01),the GFAP,GLAST,GLT-1,p-JAK1,p-JAK2,and p-STAT3 were higher(P<0.05);compared with the SPS+FGF2 group,the glutamate level in the SPS+FGF2+AG490 group were increased again(P<0.05),and the GFAP,GLAST,GLT-1,p-JAK1,p-JAK2,and p-STAT3 were decreased again(P<0.05).FGF2 increased the GFAP,GLAST,GLT-1,p-JAK1,p-JAK2,and p-STAT3 expressions in the astrocytes in vitro(compared with the control group,P<0.05),AG490 decreased the expressions of the aforementioned molecules in vitro(compared with the control group,P<0.05).Conclusions: SPS disturbed the glutamate equilibrium and induced PTSD like behaviors through inhibition of the astrocytic glutamate receptors via inhibition of the JAK/STAT signaling of the astrocytes;FGF2 treats the PTSD like behaviors via activation of the astrocytic JAK/STAT signaling.Part two: NGF Ameliorates PTSD Symptoms by Remodeling Neural Plasticity via Activation of the Trk/ERK/CREB PathwayObjectives: NGF is a widely used nerve growth factor.Studies implied that NGF was disturbed in PTSD patients.The neural plasticity abnormalities constitute the neurobiological basis of stress related disorders.In the present study,we established the mice SPS model to investigate whether NGF could affect SPS induced PTSD like behaviors and changes of the synaptic plasticity through activation of the ERK/CREB signaling.Methods: Experiment 1: 75 adult male C57 mice were randomly divided into control,SPS-3 day,SPS-7 day,SPS-14 day and SPS-28 day groups(n=15 for each group),and the expressions of p-Trk,p-ERK,p-CREB,PSD95 and Synaptophysin were determined after different quiescent time.Experiment 2: 105 adult male C57 mice were randomly divided into control,SPS,SPS+0.3,SPS+1.0,SPS+3.0,SPS+10.0 and SPS+30.0(μg/kg·day NGF)groups(n=15 for each group).All group mice were used to detect the contextual and cued fear conditioning responses,after which were sacrificed and detected the p-CREB expression using the Western blot test.Experiment 3: 60 adult male C57 mice were randomly divided into control,SPS-14 day,SPS-14 day+saline and SPS-14+NGF(10 μg/kg·day)groups(n=15 for each group),the expressions of p-Trk,p-ERK,p-CREB,PSD95 and Synaptophysin were determined.Experiment 4: the in vitro hippocampal neuron were treated with NGF(50 ng/m L)and PD98059(MEK1 inhibitor,20μM)for 48 h to detect the molecular and morphological changes.Results: Compared with the control group,SPS enhanced the contextual and cued fear conditioning responses(P<0.01).NGF dose dependently shortened the freezing time in the contextual(effective dose 50%=1.935 μg/kg·day)and cued(effective dose 50%=1.873 μg/kg·day)fear conditioning paradigm.Considering the clinical usage of NGF,the dose-curve effect and ED50 of our present study,we adopted the dosage of 3.0 μg/kg·day in the following experiments: in the EPMT,compared with the control group,the time in open arms(%)and entries into open arms(%)were significantly reduced by SPS(P<0.01),while the time in closed arms(%)and entries into closed arms(%)were increased by SPS(P<0.01),compared with the SPS group,the time in open arms(%)and entries into open arms(%)of the SPS+NGF group were increased(P<0.01),and the time in closed arms(%)(P<0.05)and entries into closed arms(%)(P<0.01)were decreased;in the OFT,compared with the control group,the distance and time in the central area were significantly reduced by SPS(P<0.01),compared with the SPS group,the two parameters of the SPS+NGF group were increased(P<0.05),changes of the total distance and average velocity of all groups had no statistical significance,implying that SPS and NGF had no effect on the locomotion of all groups.The Golgi staining results demonstrated that compared with the control group,the total lengths of the apical and basal dendrites were shorter in the SPS group(P<0.01),the number of dendrites,number of intersections and spine densities were reduced in the SPS group(P<0.05);NGF reversed the SPS induced changes of synaptic plasticity,compared with the SPS group,the total lengths of the apical and basal dendrites were longer in the SPS+NGF group(P<0.05),the number of dendrites,number of intersections and spine densities were increased in the SPS+NGF group(P<0.05).Western blot results illustrated that compared with the control group,the expression levels of the hippocampal p-Trk,p-ERK,p-CREB,PSD95 and synaptophysin were gradually decreased with the elongation of the quiescent time,and reached their lowest level on SPS-14 day(P<0.05).Chose the 14 day as the quiescent time,administration of NGF increased the aforementioned protein expressions(compared with the SPS group,P<0.05).In vitro study further demonstrated that NGF increased the number of dendrite and total dendrite length(compared with the control group,P<0.01),PD98059 reduced the number of dendrite and total dendrite length(compared with the control group,P<0.05),compared with the PD98059 group,the number of dendrite and total dendrite length were significantly increased(compared with the PD98059 group,P<0.05),demonstrating that NGF ameliorated PD98059 induced impairment of the synaptic plasticity via abolishing the neuronal ERK inhibition induced by PD98059;compared with the control group,NGF increased the p-Trk,p-ERK,p-CREB and MAP2 expression(P<0.05),while PD98059 decreased the expression of these proteins(P<0.05),and the reductive effects were reversed by NGF(compared with the PD98059 group,P<0.05).Conclusions: NGF alleviated SPS induced impairment of the hippocampal neuronal plasticity via activation of the neuronal ERK/CREB signaling pathway and ameliorated PTSD-like behaviors.Part three: r TMS Ameliorates PTSD Symptoms through Enhancement of the Glutamatergic Transmission and Synaptic Plasticity in ACC via the PTEN/Akt SignalingObjectives: r TMS is a novel physiological therapeutic method that is increasing adopted in handling the psychiatric disorders.We previously reported that r TMS was effective in ameliorating the anxiety like behaviors in a SPS model,but whether r TMS had beneficial effects on the fear responses in the SPS paradigm,and the molecular basis of the therapeutic effects of r TMS still needed to be further investigated.Based on our previous study and combining results from the clinical imaging studies,we investigated the effects of r TMS on the glutamatergic neuronal transmission and synaptic plasticity in the anterior cingulate cortex(ACC)and its possible molecular underpinnings.Methods: Firstly,we studied the effect of different quiescent period(7 day/14 day)of SPS and frequency(high,15 Hz or low,1Hz)of r TMS on the expression of glutamate receptors in rat ACC,60 adult male SD rats(250-300 g,8weeks old)were randomly divided into 5 groups(12 per group): control(sham r TMS stimulation),SPS-7d,SPS-14 d,SPS+Hr TMS and SPS+Lr TMS.Since a 14-day quiescent period more effectively affects the glutamate receptors expression,7 days after the SPS induction,rats of the corresponding groups were treated with r TMS once a day for 7 days.48 SD rats were randomly divided into control,si PTEN,bp V and wortmannin groups(12 per group)to study the expressions of PTEN and p-Akt in rat ACC.To study the relationship between Hr TMS and PTEN/Akt signaling on the expressions of glutamate receptors and the synaptic plasticity in the ACC,96 SD rats were randomly divided into control(sham r TMS),SPS,SPS+Hr TMS,SPS+Hr TMS+ bp V,SPS+Hr TMS+si PTEN and SPS+Hr TMS+wortmannin groups(16 pre group).After the r TMS intervention,rats were endured with the OFT,EPMT,FCT,and PPI test.Western blot and immunofluorescent staining test were adopted to investigate the expressions of glutamate receptors,PTEN,p-Akt and Akt.Results: Western blot results illustrated that compared to the control group,the expressions of the NR1,NR2 A,NR2B,Glu R1,Glu R2,Glu R3 and Glu R4 in ACC of the SPS-7d,SPS-14 d,SPS+Lr TMS and SPS+Hr TMS groups were all significantly reduced(P<0.05),compared with the SPS-14 d group,the aforementioned molecules in the SPS+Hr TMS group were significantly increased(P<0.01).Compared with the control group,the PTEN from the SPS group was significantly increased(P<0.01),while the p-Akt were significantly reduced(P<0.05).Compared with the SPS-14 d group,the PTEN in the ACC of the SPS+Hr TMS group was reduced,while the p-Akt content was significantly increased(P<0.05).The immunofluorescent staining test results confirmed the Western blot finding that the Glu R1 and NR2 B expression were decreased in the SPS-7d,SPS-14 d,SPS+Lr TMS and SPS+Hr TMS groups(compared with the control group,P<0.05),compared with the SPS-14 d group,the Glu R1 and NR2 B expression in the SPS+Hr TMS group were increased(P<0.05).Neu N staining confirmed that the p-Akt that regulated by SPS and Hr TMS located in the neuron.si PTEN reduced the PTEN content compared with the control group(P<0.05),bp V and si PTEN increased the p-Akt expression,while wortmannin decreased it(P<0.01).Compared with the SPS+Hr TMS group,the ACC expressions of the p-Akt,NR2 B and Glu R1 in the SPS+HTMS+si PTENand SPS+Hr TMS+bp V groups were significantly increased(P<0.05),and those proteins were decreased in the ACC of the SPS+Hr TMS+wortmannin group(P<0.01).The Golgi staining test illustrated that compared with the control group,the number of dendrites,number of intersections and spine densities were reduced in the SPS group(P<0.01);compared with the SPS group,the aforementioned parameters were increased in the SPS+Hr TMS group(P<0.05);compared with the SPS+Hr TMS group,the number of dendrites,number of intersections and spine densities of the SPS+Hr TMS+bp V group were further increased(P<0.05),and these parameters of the SPS+Hr TMS+wortmannin group were reduced(P<0.05).FCT demonstrated that compared with the control group,the contextual and cued fear conditioning responses in the SPS group were enhanced(P<0.01).Compared with the SPS group,the freezing time of the SPS+Hr TMS group were significantly reduced(P<0.05).Compared with the SPS+Hr TMS group,the freezing time of the SPS+Hr TMS+bp V group was reduced(P<0.05),while the freezing time of the SPS+Hr TMS+wortmannin group was increased(P<0.05).In the EPMT,compared with the control group,the time in open arms(%)and entries into open arms(%)of the SPS-7d and SPS-14 d groups were significantly reduced by SPS(P<0.01),compared with the SPS group,time in open arms(%)and entries into open arms(%)of the SPS+Hr TMS group were significantly increased(P<0.05).Compared with the SPS+Hr TMS group,the time in open arms(%)of the SPS+Hr TMS+bp V group was increased(P<0.05),while time in open arms(%)and entries into open arms(%)of the SPS+Hr TMS+wortmannin group were decreased(P<0.05).In the OFT,compared with the control group,the distance and time in the central area were reduced in the SPS group(P<0.01),compared with the SPS group,the parameters of the SPS+Hr TMS group were increased(P<0.05),compared with the SPS+r TMS group,the time in the central areaof the SPS+Hr TMS+bp V group was increased,and the distance and time in the central area of the SPS+Hr TMS+wortmannin group were decreased(P<0.05).In the PPI test,compared with the control group,the PPI indexes of the SPS group were reduced(P<0.01),compared with the SPS group,the PPI indexes of the SPS+Hr TMS group were increased(P<0.05),compared with the SPS+Hr TMS group,the PPI indexes of the SPS+Hr TMS+bp V group were further increased(P<0.05),and the indexes of the SPS+Hr TMS+wortmannin group were reduced(P<0.05).Conclusions: Our present study confirmed the previous results that high frequency of r TMS ameliorated SPS induced anxiety like behavior,and concurrently found that high frequency of r TMS alleviated SPS induced contextual and cued fear responses through enhancement of the glutamatergic neural transmission and synaptic plasticity via activation of the PTEN/Akt signaling in the ACC. |
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