Regulatory Effect Of Ketamine On Depression-like Rats Through The Dopamine D2 Receptor

Depression is a common mental illness characterized by depression and lack of pleasure.About 15?30% of patients with severe depression cannot respond to two or more standard antidepressants(for example,serotonin and reuptake inhibitors)and are determined to have treatment-resistant depression(TRD),The molecular mechanism of depression is unknown.The development of new TRD therapies is driven by four major break throughs:(1)increased dopaminergic neurotransmission can improve TRD symptoms;(2)Lack of pleasure when central dopaminergic nerve transmission is low;(3)antidepressants can significantly enhance neuroplasticity;(4)Ketamine showed anti-depressant effect in TRD patients and enhanced synaptic plasticity in preclinical animal models.Therefore,the purpose of this study was to explore whether the dopamine system played a biological role in the antidepressant effect of ketamine,focusing on the second of the five dopamine receptors(dopamine D2 receptor,DRD2),and conducting experiments at the individual and cellular level.SD rats were randomly divided into 5 groups(n=10,half male and half female),the control group(group C)received intraperitoneal injection of normal saline,and the other 40 rats were fed with chronic unpredictable stimulation combined with solitary feeding for 21 days,of which 10 groups received intraperitoneal injection of normal saline as model group(group D),ketamine group(group K)was injected intraperitoneally with ketamine once every 7 days for 3 times,and ketamine group(group K)was injected intraperitoneally with ketamine once every 7 days for 3 times.Group L received intraperitoneal injection of dopamine second receptor inhibitor L-741,626,once every 7 days for 3 times,while group K+L received two kinds of drugs,and L-741,626 was given 0.5 h in advance with the same mode of administration as ketamine.Behavioral tests were carried out on rats in each group,including sugar water preference test(SPT),tail suspension test(TST)and forced swimming test(TST)to determine the behavioral changes of depression-like rats.The body weight of the rats was weighed and recorded once a week to explore the changing trend of the body weight of the rats during the experiment,and the brain weight of the rats was weighed after the rats were killed.Protein was extracted from nucleus accumbens and hippocampus of rats.Nissl staining and HE staining were used to detect the number of hippocampal neurons,immunohistochemistry was used to detect the expression of synaptic proteins PSD-95,SYN and SYP,and Golgi staining was used to detect synaptic plasticity.PC12 cells,a kind of dopaminergic cells,were selected for in vitro verification,and corticosterone was used to simulate the state of depression at the cellular level.The optimal concentrations of corticosterone,ketamine,pramipexole and L-741,626 were detected by CCK-8 method.The cells were divided into four groups: control group(group C),corticosterone injury group(group D),corticosterone injury plus ketamine group(group K),corticosterone injury plus L-741,626 group(group L),corticosterone injury plus ketamine and L-741,626 group(group K+L),corticosterone injury plus pramipexole group(group P).The morphology of the cells was observed under the microscope,and the cellular proteins were extracted.The protein expressions of DRD2,AKT,p-AKT,GSK3? and p-GSK3? were detected by Western blot.The contents of monoamine neurotransmitter DA and its metabolite HVA and glucocorticoid corticosterone in tissues,blood and cells were detected by ELISA kit.The experimental results show that:(1)Rats fed with a combination of chronic unpredictable stimulation and loneliness can simulate the depression-like state caused by human social stress;(2)Behavioral tests showed that the rats in depression-like state had a lower preference for sugar water than the control group,which proved their lack of pleasure,and showed the prolongation of immobility time in forced swimming test and tail suspension test,showing their behavior despair.ketamine could reverse this state,while L-741,626,a dopamine second receptor inhibitor,attenuated the effect of ketamine;(3)Nissl's and HE staining of rat brain tissue showed that depression-like state could change the nerve cells of rat brain,such as cell collapse,unclear or disappearance of nucleolus and incomplete structure.The use of ketamine improved the state of nerve cells in the brain,and L-741,626 partially antagonized the effect of ketamine;(4)Immunohistochemical detection of synaptic protein expression and Golgi staining showed that the synaptic plasticity of depression-like rats changed.Ketamine could increase the expression of synaptic protein and increase synaptic density.L-741,626 antagonized the effect of ketamine,indicating that ketamine may have an antidepressant effect through the second dopamine receptor;(5)Corticosterone can cause damage to dopaminergic cells-PC12 cells.From the cell morphology,it can be seen that ketamine can protect the injured cells,and the use of pramipexole can improve the cell survival rate;(6)The results of western blot showed that the protein expressions of DRD2,p-GSK3?,GSK3?,pAKT and AKT in hippocampus?nucleus accumbens and cells were statistically different except AKT,but as far as the ratio of p-AKT to AKT was concerned,the ratio was also statistically significant,and the difference between p-GSK3? and GSK3? was statistically significant;(7)The changes of the contents of DA,HVA and CORT confirmed that the depression-like state was accompanied by the changes of neurotransmitters and glucocorticoids.These results suggest that ketamine can play an antidepressant effect through the DRD2.