Effects And Mechanism Of Electrical Vagus Nerves Stimulation On Anxiety-like Behaviors Of Rats

Objective:Vagus nerve stimulation?VNS?is mainly used in the treatment of drug-resistant and refractory depression.In recent years,many studies have shown that VNS can also be used in the treatment of pain,inflammation,drug addiction and gastrointestinal diseases.However,there are only few studies about how vagus nerve stimulation relieves anxiety.Amygdala is a key area,which is closely associated with epilepsy,depression and anxiety disorders.It is not clear whether amygdala is involved in the anxiolytic effect of VNS.In this study,we will observe whether electrical stimulation of cervical vagus nerve could effectively improve the anxiety symptoms of rats and the effects of vagus nerve stimulation on the excitability of amygdala will be discussed.Methods:According to the current intensity of vagus nerve stimulation,adult male SD rats were divided into Con group,0 mA?Sham?group,0.25 mA group,0.5 mA group and 0.75 mA group randomly.The rats were anesthetized and exposed the left cervical vagus nerve except Con group rats.After electrode was placed around the vagus nerve,the VNS group rats received stimulation repeteadly.After 6 days of left cervical vagus nerve stimulation,we used the elevated plus maze and open-field test to test anxiety-like behavior in rats.Sucrose preference test and forced swimming test were used to evaluate the depression-like behavior in rats.In vivo PET was used to investigate the effect of VNS on glucose metabolism in the amygdala of different groups.Immunofluorescence techniques were utilized to investigate the fluorescent expression of c-fos in amygdala neurons.Western blotting techiques were employed to determine the protein levels of AMPARs and NMDARs.Micro-injection techniques were employed to investigate the effect of virus infected and damaged BLA on VNS-induced antianxiety-like behavior in rats;The slice whole-cell patch clamp techiques were used to record the mEPSCs in BLA and the mEPSCs and mIPSCs of CeA;the mEPSCs of CeA were recorded after the incubation with phentolamine or propranolol.Results:?1?After continuous stimulation for 6 days,compared with the Con group,the open arm time?Con:72.08±10.44 sec,0 mA?Sham?:70.61±11.08 sec,0.25 mA:87.21±13.00 sec,0.5 mA:102.10±4.52 sec,0.75 mA:103.30±13.14 sec?and open arm distance of 0.5 mA and 0.75 mA group?Con:2.54±0.39 m,0 mA:2.48±0.36 m,0.25 mA:3.30±0.46 m,0.5 mA 4.39±0.38 m,0.75 mA:4.03±0.63 m?were increased significantly,the central time?Con:12.23±2.37 sec,0 Ma?Sham?:15.59±3.26 sec,0.25 mA:14.53±2.84 sec,0.5 mA:23.60±3.40 sec,0.75 mA:24.16±4.98sec?and center distance?Con:0.82±0.17 m,0 Ma?Sham?:0.92±0.21 m,0.25 mA:1.11±0.20 m,0.5 mA:1.92±0.30 m,0.75 mA:1.64±0.33 m?in the open-field test were significantly increased in 0.5 mA group and 0.75 mA group.?2?After continuous stimulation for 6 days,sucrose preference and the immobility in the forced swim test had no difference among the three groups.?3?After continuous stimulation for 6 days,compared with the Con group and Sham group,functional imaging of ¹⁸F-FDG PET had been documented as a hypometabolic pattern typical in the amygdala of VNS group.As expected,VNS greatly improved glucose metabolism in the amygdala from3.29±0.11?%ID/cc?and 2.95±0.15?%ID/cc?to 3.78±0.16?%ID/cc??n=4-5 p<0.05 vs Con group,p<0.05 and p<0.01 vs Sham group??4?After continuous stimulation for 6 days,compared with the Con group and the Sham group,the expression of c-fos was increased significantly in VNS group.?5?After continuous stimulation for 6 days,the amplitude of mEPSCs in the BLA neurons of VNS rats was increased from 10.57±0.38 pA to 12.66±0.71 pA in the VNS rats,the frequency of mEPSCs was increased from 2.64±0.26 Hz to 3.87±0.52 Hz?n=10-11 cells from 6rats per group,two-tailed t-test,p<0.05 vs Sham?;?6?The membrane protein levels of GluA1 in the amygdala of VNS rats was increased from 1.00±0.12 to 1.33±0.06?n=6,p<0.05 vs Sham?,but the total protein level of GluA1 was unchanged.Meanwhile,the total and membrane protein level of GluA2,GluN1,GluN2A and GluN2B receptors was unchanged.?7?After repeated stimulation,damaged BLA with pAAV-flex-taCasp3-TEVp increased the time in the open ram in elevated plus maze test from 31.00±5.33 sec to 69.11±10.89 sec in the open-field test?n=15,p<0.05vs Cre+GFP?.?8?The amplitude of mEPSCs in the CeA of VNS rats was increased from 11.21±0.46 pA to 13.65±0.92 pA,and the frequency increased from 1.68±0.30 Hz to 2.90±0.51 Hz?n=11-13 cells from 6-7 rats per group,p<0.05 vs Sham?.There was no differenc in the amplitude or frequency of mIPSCs betwee Sham and VNS group.?9?Incubation with phentolamine for 10 min did not change the amplitude and frequency of mEPSCs in CeA neurons,but incubation with propranolol for 10 min reduced the amplitude of mIPSCs of CeA neurons from 12.20±0.49 pA to10.38±0.50 pA,without alterations in frequency?n=9-10 cell from 6 rats per group,p<0.05 vs Sham?.Conclusion:After continuous stimulation for 6 days,VNS decreased the anxiety-like behavior and increased the activity of amygdala of rats.The underlying mechanisms of VNS on the anxiolytic effect were mediated through the regulation on BLA and CeA.VNS could activate BLA and CeA of amygdala.The increased activity of CeA was the underlying mechanism of VNS-induced anxiolytic effect,but increased activity of BLA blocked the VNS-induced anxiolytic effect.