| ObjectiveBased on the theory that hyper-cholinergic tone could be relate to depression disorder,we detected the effect of electroacupuncture(EA)on behaviors and cholinergic tones in the prefrontal cortex of the rats explored chronic mild unpredictable stress(CUMS)and separation.Further,we illuminated the antidepressant mechanism of EA,which might be related to the decrease in the expression of type 1 muscarinic acetylcholine receptor(MlAChR)and inhibition of hyper-cholinergic tone,and promoting synaptic plasticity to repair the synaptic damage and depressive behaviors.MethodsPart 1 Electroacupuncture ameliorates depressive-like behaviors and synaptic deficits by regulating the metabolism of ACh in rats exposed to chronic unpredictable mild stress and separation54 SPF adult male rats weighted 180 ± 20 g were used and divided into 6 groups,the normal group,model group,EA group,scopolamine(SCOP,as a positive control),physostigmine(Phys)group and EA+Phys group.Rats were exposed to CUMS and separation for 28 days except for normal group.After 28 days modeling,rats in the EA group would receive electroacupuncture treatment at GV 20(Baihui),GV 29(Yintang),LI 4(Hegu),and LR 3(Taichong)(Hans-LH202H,2/100 Hz,1～1.2 mA)for 20 minutes daily.Rats in the SCOP group received administration of scopolamine once in 48 hours.In addition,the rats in the Phys group received the intraperitoneal injection of physostigmine(0.5 mg/kg)once daily,and rats in the EA+Phys+stress group received EA stimulation and physostigmine administration as the EA and Phys group.All EA was carried out 30 minutes after the administration in the EA+Phys group.All the treatments were conducted for 2 weeks.Testing index:1)Body weight test(BWT)and behavioral tests,including sucrose preference test(SPT),novelty-suppressed feeding test(NSFT)and forced swimming test(FST)were conducted during modeling and after 2 weeks treatment.2)EILISA was conducted to test the content of ACh,AChE,ChAT in the prefrontal cortex(PFC).3)Golgi-cox Staining was conducted to test the spine density of the apical dendrites of layer V pyramidal neurons.4)Western Blot(WB)was conducted to the expression of synaptic proteins(PSD95,Synapsin I and GluR1/2)in the PFC to explore the effect of EA on synaptic plasticity.And the expression of M1AChR in the PFC.Part 2 Electroacupuncture ameliorates depressive-like behaviors and synaptic deficits by inhibiting MlAChRs in rats exposed to chronic unpredictable mild stress and separation54 SPF adult male rats weighted 180±20 g were used and divided into 6 groups,the normal group,model group,EA group,SCOP(as a positive control),arecoline(AREC)group and EA+AREC group.Rats were exposed to CUMS and separation for 28 days except for normal group.Rats in the normal,model,EA,and SCOP group received the same intervention as methods in Partl.The rats in the AREC group received the intraperitoneal injection of arecoline(2 mg/kg)once daily after 28 days chronic stress.In the EA+AREC group,rats received electroacupuncture stimulation and arecoline administration as the EA and AREC group.All electroacupuncture was carried out 30 minutes after the administration in the EA+AREC group.all the treatments were conducted for 2 weeks.Testing index.1)BWT and behavioral tests(SPT,NSFT,and open field test(OFT).2)Golgi-cox Staining was conducted to test the spine density and dendritic complex of the apical dendrites of layer V pyramidal neurons.3)WB was conducted to the expression of synaptic proteins(PSD95,Synapsin I and GluR1/2)and the BDNF/mTORCl signaling proteins in the PFC to explore the effect of EA on synaptic plasticity.4)The expression of M1AChR in the PFC to explore the effect of EA on M1AChR using WB.ResultsPart 11)The effect of EA on behavioral changes of rats with hyper-cholinergic tone induced by physostigmine during CUMS.Before modeling,there were no deference in BWT and sucrose preference rate between the normal and model group(P>0.05).After 28 days CUMS modeling,there were significant differences in BWT and sucrose preference rate between the normal and model group(P<0.05),indicating that the depressive models were built successfully.Before treatment,7 days treatment,and 2 weeks treatment,there were significant differences in the BWT and sucrose preference rate among 6 groups(all P<0.05),as well as the immobility time and latency feeding time(all P<0.05)after 2 weeks treatment.Compared with the Normal group,BWT and sucrose preference rate was decreased significantly in the model group(all P<0.05),Pronged immobility time and latency feeding time were also found in the model group(all P<0.05).Compared with the model group,the B WT and sucrose preference rate increased significantly in the EA group and SCOP group,shortened immobility time and latency feeding time were detected(all P<0.05).There were inductions of BWT and sucrose preference rate and prolonged immobility time and latency feeding time in Phys groups when compared to the model group(all P<0.05).There were significant increases in BWT and sucrose preference rate,and decreases in immobility time and latency feeding time in EA and EA+Phys group compared with the Phys group(all P<0.05).2)The effect of EA on the expression of cholinergic tones in the PFC of rats with hyper-cholinergic tone induced by physostigmine during CUMS.There were significant differences in the content of ACh,AChE and ChAT among 5 groups(all P<0.05).Compare with the normal group,there were significant increases in the content of ACh,ChAT,and M1AChR,and decreases in the content of AChE in the model group.significant decreases were found in the content of ACh,ChAT,and M1AChR,and increase in the content of AChE in the EA group.In the Phys group,there were significant increase in the content of ACh and M1 AChR,but no significant differences were found in the content of AChE and ChAT.Compared with the Phys group,there were significant decreases in the content of ACh,ChAT,and M1 AChR,and increase in the content of AChE in the EA and EA+Phys group.3)The effect of EA on spine density of rats with hyper-cholinergic tone induced by physostigmine during CUMS.There were significant differences in total spine density,as well as the mature,and immature spine density among 4 groups(all P<0.05),but filopodia spine density(P>0.05).Compare with the normal group,there were significant decreases in total,mature,and immature spine density in the model group(all P<0.05).Compared with the model group,total,mature and immature spine density decreased significantly in Phys group(P<0,05).Compared with the Phys group,there were significantly increased total,mature,and immature spine density in the EA+Phys group(all P<0.05).4)The effect of EA on the expression of synaptic proteins in the PFC of rats with hyper-cholinergic tone induced by physostigmine during CUMS.There were significant differences in the expression of BDNF,GluR1,GluR2.PSD95,and Synapsin I among 4 groups(all P<0.05).Compare with the normal group,there were significant decreases in the expression of BDNF,GluRl,GluR2,PSD95,and Synapsin I in the model group.Compared with the model group,the expression of BDNF,GluR1,GluR2,PSD95,and Synapsin I decreased significantly in Phys group(P<0.05).Compared with the Phys group,there were significantly increased expression of BDNF,GluRl,GluR2,PSD95,and Synapsin I in the EA+Phys group(all P<0.05).Part 21)The effect of EA on behavioral changes in rats with hyper-activation of M1AChRs during CUMS.After 28 days CUMS,significant differences in the BWT and sucrose preference rate was discovered among six groups(P<0.05).Compared with the normal group,there were significant decreases in BWT and sucrose preference rate in the model group(all P<0.05).However,there were no significant differences in EA,SCOP,AREC.and EA+AREC group when compared to the model group(all P>0.05).It implicated successfully modeling.After 2 weeks intervention,compared with the normal group,BWT and sucrose preference rate decreased significantly in the model group(P<0.05).The reduction of horizontal activity,vertical activity and grooming frequency was found in the model group compared to the normal group(all P<0.05).The latency feeding time also prolonged in the model group(P<0.05).Compared with the model group,the BWT and sucrose preference rate as well as horizontal activity.vertical activity and grooming frequency all increased significantly in the EA group and SCOP group(all P<0.05).However,there were significant decreases in the BWT,sucrose preference rate,and horizontal activity frequency,increases in vertical activity and grooming frequency in the AREC group compared to the model group(all P<0.05).Compared with the AREC group,there were significant increases in the BWT,sucrose preference rate,and horizontal activity.vertical activity and grooming frequency in the EA and EA+AREC group(all P<0.05).2)The effect of EA on the spine density and dendrite complex of the apical dendrites of layer V pyramidal neurons in rats with hyper-activation of MlAChRs during CUMS.There were significant differences in total spine density,as well as the mature,immature spine,and filopodia density among 6 groups(all P<0.05).Compare with the normal group,there were significant decreases in total,mature,and immature spine density(all P<0.05).but no significant difference in filopodia density in the model group(P>0.05).Compared with the model group,there were significant increases in total,immature,and filopodia density(all P<0.05),but no significant difference in mature spine(P>0.05)in the EA group.There were significant increases in total.mature.and immature density(all P<0.05),but no significant difference in filopodia spine(P>0.05)in the SCOP group compared to the model group.However.there were significant decreases in the total,mature,and immature spine density(all P<0.05),but no difference in filopodia spine(P>0.35)in the AREC group compared to the model group.When compared with the AREC group,there were significant increases total,mature,and immature spine density in the EA and EA+AREC group(all P<0.05).There were significant differences in the dendrite length and intersections among 6 groups(all P<0.05).Compare with the normal group,the apical dendrite length and intersections from diameter 100 μm to 190 μm significantly decreased in the model group(all P<0.05).Compared with the Model group,the apical dendrite length and intersections from diameter 100 μm to 190 μm increased in the EA and SCOP group(all P<0.05).No difference was found in the apical dendrite length between the EA and SCOP groups(P>0.05),while interactions at diameters from 10 μm to 90 μm and from 130 μm to 160 μm were significantly different(all P values<0.05).Compared with the model group,there was no significant difference in the apical dendrite length(P>0.05)in the AREC group,but significant decreased intersections from diameter 100 μm to 170 μm(all P<0.05)however,intersections from diameter 110 μm to 170 μm significantly increased(all P<0.05)in the EA+AREC group compared to AREC group.However,there was no significant difference noted in apical dendrite length(P>0.05).3)The effect of EA on the expression of synaptic proteins and BDNF/mTORC1 signaling proteins in the PFC of rats with hyper-activation of M1AChRs during CUMS.There were significant differences in the expression of BDNF,mTORCl,p-mTORC1,GluRl,GluR2,PSD95.and Synapsin I among six groups(all P<0.05).Compare with the normal group,there were significant decreases in the expression of BDNF,mTORCl,p-mTORC1,G1uR1,GluR2,PSD95,and Synapsin I in the model group(all P<0.05).Compared with the model group,the expression of BDNF.mTORC1,p-mTORC1 GluRl,GluR2,PSD95,and Synapsin I increased significantly in the EA and SCOP group,but decreased significantly in the AREC group(all P<0.05).Compared with the AREC group,there were significantly increased expression of BDNF,mTORCl,p-mTORC1,GluR1,GluR2,PSD95,and Synapsin I in the EA+AREC group(all P<0.05).4)The effect of EA on the expression of M1AChRs in the PFC of rats with hyper-activation of M1AChRs during CUMS.There were significant differences in the expression of M1AChRs among six groups(P<0.05).Compare with the normal group,there were significant increases in the expression of M1AChRs in the model group(P<0.05).Compared with the model group,the expression of M1AChRs decreased significantly in the EA and SCOP group,but increased significantly in the AREC group(P<0.05).Compared with the AREC group,there were significantly decreased expression of M1AChRs in the EA+AREC group(P<0.05).Conclusion1.EA could ameliorate depressive-like behaviors and synaptic deficits in rats exposed to chronic unpredictable mild stress and separation.The mechanism of its antidepressant was involved in the regulation of cholinergic tones,and related to the increase in AChE to recover the hyper-level of ACh.2.EA could ameliorate depressive symptoms by inhibiting the expression of M1AChR in the PFC,which was involved in the inhibition of hyper-cholinergic tones,following activating of BDNF/mTORC1 signaling and increasing synaptic plasticity. |
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