| ObjectiveTo explore the role of loss of hippocampal y-aminobutyric acid(GABA)-ergic interneurons in pathological fear memory of post-traumatic stress disorder(PTSD).Taking hippocampal GABAergic interneurons as targets,the effects of Guiluerxianjiao(GLEXJ)on enhancing fear memory extinction retention in PTSD and the possible mechanisms were explored.Methods1.Establishment of a rat model of PTSDA rat model of PTSD was established using inescapable foot shock.Rats were exposed to foot shock twice a day with an interval greater than 4 hours,lasting for 6 days.Rats received 15 trials of a 10s tone co-terminating with a foot shock(intensity 2mA,duration 2s)in 30 min.The inter-trial interval was randomly varied.The rats in the control group were placed in the chamber for 30 minutes without tone or foot shock delivery.2.Grouping of animalsAnimals were randomly divided into four groups:the control group(Control),the model group(PTSD),the Guiluerxianjiao group(GLEXJ),and the paroxetine group(PRX).From the first day of modeling,rats in the GLEXJ and PRX groups were given intragastric administration for 21 consecutive days,and rats in the Control and PTSD groups were administrated with equal volumes of pure water.3.Indicator detection(1)Behavioral tests:the anxiety-like behavior of rats was detected by the elevated plus maze test,and the contextual fear memory test was performed by the conditioned fear paradigm.(2)The immediate early gene c-fos was used as a marker of neuronal activation,and the expression of c-fos in the hippocampal CA1,CA3,and dentate gyrus(DG)regions of rats in each group was observed.(3)Glutamic acid decarboxylase 67(GAD67)was used as a marker for GABAergic interneurons,and calcium-CaM-dependent protein kinase Ⅱ(CaMKⅡα)was used as a marker for glutamatergic neurons.The immunofluorescence double-labeling method was used to determine the co-expression of GAD67,CaMKⅡα and c-fos,respectively,in the hippocampal CA1,CA3,and DG regions.Western blot technique was used to detect the protein expression of GAD67 and CaMKⅡα in the hippocampus of rats.(4)The immunofluorescence double-labeling method was used to determine the coexpression of GABAergic interneuron subtype parvalbumin(PV),somatostatin(SST),calretinin(CR)and c-fos,respectively,in the hippocampal CA1,CA3,and DG regions.Western blot technique was used to detect the protein expression of PV,SST and CR in the hippocampus of rats.(5)The immunofluorescence double-labeling method was used to determine the coexpression of perineuronal nets(PNNs)and PV in the hippocampal CA1 region.Western blot technique was used to detect the protein expression of PNN components aggrecan(ACAN)and brevican(BCAN)in the hippocampus of rats in each group.ELISA was used to detect the contents of PNN proteolytic enzyme MMP-2 and MMP-9 in the hippocampus of rats.(6)The immunofluorescence double-labeling method was used to detect the expression of excitatory synaptic marker postsynaptic density protein 95(PSD95)and inhibitory synaptic marker gephyrin on the soma of PV interneurons in the hippocampal CA1 region of rats.(7)The immunofluorescence triple-labeling method was used to determine the coexpression of PNN and PSD95,and gephyrin,respectively,on the soma of PV interneurons in the hippocampal CA1 region of rats.Results1.The elevated plus maze test showed that the total distance,the percentage of openarm distance,the percentage of open-arm time,and the percentage of open-arm entries were significantly lower in the PTSD group compared with the Control group(P<0.05).Compared with the PTSD group,the rats in the GLEXJ group had significantly higher total distance,percentage of open-arm distance,percentage of open-arm time,and percentage of open-arm entries(P<0.05),and the rats in the PRX group had significantly higher percentage of openarm distance,percentage of open-arm time,and percentage of open-arm entries compared with the PTSD group(P<0.05).2.In the contextual fear memory retrieval test,the percentage of freezing time was significantly higher in the PTSD group compared with the Control group(P<0.05).In the contextual fear memory generalization test,the percentage of freezing time was significantly higher in the PTSD group compared with the Control group(P<0.05);the percentage of freezing time and discrimination index was significantly lower in the GLEXJ group compared with the PTSD group(P<0.05).In contextual fear extinction session,repeated measures ANOVA showed a significant effect of time(block)on the percentage of freezing time(P<0.05);the interaction effect of time and group was significant(P<0.05),suggesting a statistically significant effect of group on the percentage of freezing time with increasing time.Post-hoc tests showed that in the 1st,2nd,and 3rd blocks,the percentage of freezing time in PTSD rats was significantly higher than that in the Control group(P<0.05);in the 7th block,the percentage of freezing time in the GLEXJ group was significantly lower than that in PTSD group(P<0.05).In the contextual fear memory extinction retention test,the percentage of freezing time was significantly higher in the PTSD group compared with the Control group(P<0.05);the percentage of freezing time was significantly lower in the GLEXJ group compared with the PTSD group(P<0.05).3.The number of c-fos-positive cells in the hippocampal CA1 region was significantly higher in the PTSD group compared with the Control group(P<0.05).The number of c-fospositive cells in CA1 of the GLEXJ and PRX groups was not significantly different compared with the PTSD group(P>0.05).Compared with the Control group,the number of c-fospositive cells in DG was significantly higher in the PTSD group(P<0.05).Compared with the PTSD group,there was no significant difference in the number of c-fos-positive cells in DG region in the GLEXJ and PRX groups(P>0.05).No significant difference was found in the number of c-fos positive cells in CA3 between groups(P>0.05).4.No significant difference was observed in the number of CaMKⅡα-positive cells in CA1,CA3 and DG regions of hippocampus between groups(P>0.05).The number of CaMKⅡα/c-fos double-positive cells in the hippocampal CA1 region was significantly higher in the PTSD group compared with the Control group(P<0.05);the number of CaMKⅡα/cfos double-positive cells in the CA1 region was not significantly different in the GLEXJ and PRX groups compared with the PTSD group(P>0.05).There was no significant difference in the number of CaMKⅡα/c-fos double-positive cells in CA3 and DG regions between groups(P>0.05).5.The number of GAD67-positive cells in the hippocampal CA1 region was significantly lower in the PTSD group compared with the Control group(P<0.05).The number of GAD67-positive cells in CA1 was significantly higher in the GLEXJ group compared with the PTSD group(P<0.05).There was no significant difference in the number of GAD67-positive cells in the CA1 region between the PRX and PTSD groups(P>0.05).Compared with the Control group,the number of GAD67-positive cells in CA3 was significantly lower in the PTSD group(P<0.05).There was no significant difference in the number of GAD67-positive cells in CA3 in the GLEXJ and PRX groups compared with the PTSD group(P>0.05).No significant difference was observed in the number of GAD67positive cells in DG between groups(P>0.05).No significant difference was observed in the number of GAD67/c-fos double-positive cells in the hippocampal CA1 region between groups(P>0.05).6.Compared with the Control group,hippocampal GAD67 protein expression was significantly lower in the PTSD group(P<0.05).Compared with the PTSD group,hippocampal GAD67 protein expression was significantly higher in the GLEXJ group(P<0.05).There was no significant difference in hippocampal GAD67 protein expression between the PRX and PTSD groups(P>0.05).There was no significant difference in hippocampal CaMKⅡα protein expression between the groups(P>0.05).7.The number of PV-positive cells in the hippocampal CA1 region was significantly lower in the PTSD group compared with the Control group(P<0.05);there was no significant difference in the number of PV-positive cells in the CA1 region in the GLEXJ and PRX groups compared with the PTSD group(P>0.05).There was no significant difference in the number of PV-positive cells in CA3 and DG regions between groups(P>0.05).The number of PV/c-fos double-positive cells in the hippocampal CA1 region was significantly higher in the GLEXJ group compared with the PTSD group(P<0.05).No significant differences were seen in the number of SST-positive cells and the number of SST/c-fos double-positive cells in the hippocampal CA1,CA3,and DG regions between groups(P>0.05).The number of CR-positive cells in the hippocampal CA1 region was significantly higher in the PRX group compared with the PTSD group(P<0.05).No significant differences were seen in the number of CR-positive cells in the CA3 and DG regions between groups(P>0.05).Correlation analysis showed that the percentage of freezing time in the contextual fear memory extinction retention test showed a significant negative correlation with the number of PV/c-fos doublepositive cells in the CA1 region of the hippocampus(r=-0.716,P<0.05).8.Compared with the Control group,hippocampal PV protein expression was significantly lower in the PTSD group(P<0.05).Compared with the PTSD group,there was no significant difference in hippocampal PV protein expression in the GLEXJ and PRX groups(P>0.05).There was no significant difference in hippocampal SST and CR protein expression between groups(P>0.05).9.There was no significant difference in the number of PNN-positive cells in the hippocampal CA1 region between the Control,PTSD,and GLEXJ groups(P>0.05).There was no significant difference in the number of PNN/PV double-positive cells in the hippocampal CA1 region between groups(P>0.05).Compared with the Control group,the number of PV-positive cells without PNN in the hippocampal CA1 region was significantly lower in the PTSD group(P<0.05).Compared with the PTSD group,the number of PVpositive cells without PNN was significantly higher in the GLEXJ group(P<0.05).The proportion of PNN/PV double-positive cells to the number of PV-positive cells in the hippocampal CA1 region was significantly higher in the PTSD group compared with the Control group(P<0.05).The proportion of PNN/PV double-positive cells to the number of PV-positive cells was significantly lower in the GLEXJ group compared with the PTSD group(P<0.05).Correlation analysis showed a weak positive correlation between the fluorescence intensity of PV and PNN in PNN/PV double-positive cells(r=0.268,P<0.05).10.Compared with the Control group,hippocampal ACAN protein expression was significantly higher in the PTSD group(P<0.05).There was no significant difference in hippocampal ACAN protein expression between the GLEXJ and PTSD groups(P>0.05).Compared with the Control group,hippocampal BCAN protein expression was significantly higher in the PTSD group(P<0.05).Compared with the PTSD group,hippocampal BCAN protein expression was significantly lower in the GLEXJ group(P<0.05).ELISA showed that compared with the Control group,hippocampal MMP-2 content was significantly lower in the PTSD group(P<0.05).Compared with the PTSD group,the hippocampal MMP-2 content in the GLEXJ group was significantly increased(P<0.05).There was no significant difference in hippocampal MMP-9 content between groups(P>0.05).11.The number of excitatory synaptic marker PSD95 puncta on the soma of PV interneurons in the hippocampal CA1 region was significantly lower in the PTSD group compared with the Control group(P<0.05).The number of PSD95 puncta on the soma of PV interneurons in CA1 was significantly higher in the GLEXJ group compared with the PTSD group(P<0.05).There was no significant difference in the number of inhibitory synaptic markers gephyrin puncta on the soma of PV interneurons in CA1 between groups(P>0.05).Two-way ANOVA showed that PNN had a significant effect on the number of PSD95 puncta on the soma of PV interneurons(P<0.05),and the interaction between PNN and group had no significant effect on the number of PSD95 puncta(P>0.05).Post-hoc tests showed that the number of PSD95 puncta on the soma of PNN-negative PV interneuron was significantly higher than that on the soma of PNN-positive PV interneuron(P<0.05).PNN or the interaction between PNN and the group has no significant effect on the number of gephyrin puncta(P>0.05).Conclusion1.Tonifying kidney and filling essence therapy GLEXJ can reduce the anxiety behavior and fear generalization of PTSD rats,and enhance contextual fear extinction retention.2.PTSD rats had decreased GAD67 protein expression in the hippocampus and decreased number of GABAergic interneurons in the hippocampal CA1 and CA3 regions.GLEXJ can increase the GAD67 protein expression in the hippocampus of PTSD rats,reduce the loss of GABAergic interneurons in hippocampal CA1 region,and promote the restoration of "E/I" balance in the hippocampus of PTSD rats.3.Contextual fear extinction memory retrieval can produce subregional and subcellular specific activation of hippocampal GABAergic interneurons in PTSD rats.GLEXJ increased the activation of PV interneuron in the hippocampal CA1 region,by which it may promote the contextual fear extinction retention.4.GLEXJ may promote the degradation of PNN and by increasing the content of PNN proteolytic enzyme MMP-2 in the hippocampus,reduce the protein expression of BCAN,reduce the proportion of PV interneurons coated by PNN in CA1,and then increase the number of excitatory synaptic marker PSD95 puncta on the soma of PV interneurons. |
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