Investigation Of The Mechanism Of Medial Habenula And Amygdala On Cardiovascular Activity Regulation In Rats With Post-traumatic Stress Disorder

Objective: This experiment aimed to explore the central mechanism of the medial habenula(MHb)and amygdala on the regulation of cardiovascular activity in rats with post-traumatic stress disorder(PTSD).It provides effective therapeutic targets for the clinical treatment of cardiovascular dysfunction caused by PTSD.Methods: Adult male SD rats,weighing 180-220 g,were randomly divided into control group and PTSD group,and the rats were adapted to feed for one week and start modeling.The model was established using a compound stress(CS)method combining electrical stimulation and single-prolonged stress(SPS).After one week of modeling,the weight gain values were compared between the two groups,and the behavioral tests of the open-field(OF)and elevated plus maze(EPM)were performed on both groups.When each parameter was statistically significant when compared between the two groups,the validity of the model was proven and the model could be considered successful.Through the method of Fluoro Gold(FG)retrograde tracking and immunofluorescence colocalization,the retrograde labeling of MHb neurons after microinjection of FG into rostral ventrolateral medulla(RVLM)was observed,and then MHb was immunofluorescence stained with vesicular glutamate transporters 2(VGLUT2)to explore whether there was a glutamatergic pathway connection between MHb and RVLM from the morphological level.Secondly,the distribution of retrograde labeling of neurons in the amygdala region after RVLM injection of FG was observed.Through in vivo multichannel synchronous recording technology,after microinjection of artificial cerebrospinal fluid(ACSF)and kynurenic acid(KYN)solution in the RVLM region of rats in the PTSD model group,electrical stimulation(ES)in the MHb region was given to observe RVLM neuronal firing and peripheral index changes.In addition,ACSF and KYN solutions were injected into the central amygdala(Ce A)region of the PTSD model rats to observe the changes of RVLM neuron discharge and cardiovascular activities.Finally,Western blotting was used in order to analyze the NMDA and AMPA glutamate receptor content in the amygdala and RVLM of PTSD and control groups.The fibrillar connections between MHb and RVLM,and between amygdala and RVLM were analyzed by the above methods to explore the central mechanism underlying the regulation of cardiovascular activity in PTSD rats.Results:1.Identification of PTSD model rats1.1 Body weight gain After 7 days of modeling,the difference in weight gain between the two groups was examined,showing that the weight gain of PTSD group was significantly lower than that of control group(P<0.01).1.2 Behavioral test In open-field(OF)test,compared with the control group,the total distance and the distance in the central region in the PTSD group were significantly reduced(P<0.01),and the stopover time in the central region was significantly decreased(P<0.05).In elevated plus maze(EPM)test,the ratio of the open arm entry(OE%)and the ratio of open arm time(OT%)in PTSD group were lower than those in control group(P<0.01).2 Morphological observation of the glutamatergic pathway linkage between MHb and RVLM After microinjection of FG solution in the RVLM region,retrograde labeled FG neuron cells were observed in the MHb region,which was subjected to immunofluorescence staining of vesicular glutamate transporters 2(VGLUT2)and glutamate-positive neurons were observed in the MHb region.By synthesizing the MHb images observed under different excitation light,the double-labeled neurons in the MHb region can be observed.3 Morphological observation of pathway association between amygdala and RVLM After the FG solution was injected into RVLM,retrograde labeled neurons were observed in Ce A region,and their cell bodies and fibers were golden yellow.4 Functional investigation of the glutamatergic linkage between MHb and RVLM When MHb was given different intensity electrical stimulation,the firing frequency of RVLM neurons in the ACSF group was significantly increased(P<0.05),the heart rate was significantly accelerated(P<0.05),diastolic blood pressure and mean arterial pressure were significantly increased(P<0.05),while the firing frequency of RVLM neurons in the KYN group was significantly reduced(P<0.05),heart rate was significantly slowed down(P<0.05),systolic blood pressure(P<0.01),mean arterial pressure,and diastolic blood pressure were significantly reduced(P<0.05).5 Function of amygdala glutamate receptor on cardiovascular activity regulation in rats with PTSD After Ce A injection of ACSF in PTSD rats,there was no significant statistical significance in RVLM neuronal firing frequency(P>0.05),systolic blood pressure decreased significantly(P<0.05),and diastolic blood pressure and mean arterial pressure were not significantly different(P>0.05).After KYN injection,the firing frequency of neurons in the RVLM region increased significantly(P<0.05),heart rate increased significantly(P<0.05),systolic blood pressure decreased significantly(P<0.05),and there were no significant statistical differences in diastolic blood pressure and mean arterial pressure(P>0.05).6 Changes of glutamate receptor content in the RVLM Compared with the control group,the protein expressions of the NMDA receptor subunit NMDAR1 and the AMPA receptor subunit Glu R2 in the RVLM region of PTSD group were significantly increased(P<0.05).7 Changes of glutamate receptor content in the amygdala Glu R2 protein expression in the control group was significantly higher than that in the PTSD group(P<0.05),but no significant difference was found in NMDAR1(P>0.05).Conclusion: 1.There may be a glutaminergic pathway link between MHb and RVLM,and the release of Glu from MHb in PTSD rats may act on NMDA and AMPA glutamate receptors in RVLM region to regulate cardiovascular activities.2.Amygdala glutamate receptors may be involved in the regulation of cardiovascular activity in rats with PTSD,and may produce inhibitory function regulation.