Antipsychotics Regulate GABA_AR With PKA-CREB Signaling Pathway

Background:Schizophrenia is one of the most common psychiatric disorders with great attentions in clinical practice for its enigmatic etiologies and pathophysiological mechanisms.?-aminobutyric acid?GABA?is the main inhibitory neurotransmitter in the central nervous system,which exerts its biological activities mainly via GABA_AR.Numerous studies suggested that GABAergic system abnormalities are tightly related to the pathophysiological process,antipsychotic efficacy,cognitive function improvement and other profiles of schizophrenia.Both typical and atypical antipsychotic drugs can modulate the expression of GABA_AR.However,the underlying mechanism for modulating of GABA_AR by antipsychotic drugs remains obscure.The D₂ receptor?D₂R?is a critical target which possibly contributes to the major downstream signaling pathway?PKA-CREB pathway?in the development and progression of schizophrenia.Therefore,antipsychotic drugs may indirectly regulate the expression of GABA_AR through the activation of PKA-CREB signaling pathway by D₂R.It is urgent to clarify the relationship between GABA_AR and D₂R-activated PKA-CREB signaling pathway and exploit novel agents to cure and prevent this disease.Objective:To explore the role of D₂R and its downstream PKA-CREB signaling pathway in the regulation of GABA_AR expression and improvement of cognition by antipsychotic drugs,and provide further theoretical basis for the improvement of the mechanism of antipsychotic drugs.Methods:1.Twenty-four male Kunming mice weighing 18-22 g were randomly divided into2 groups:control group?Saline 10 ml/kg,i.p,q.d?and MK801 model group?MK8010.5 mg/kg,i.p,q.d?.After one-week administration,behavioral tests?locomotion activity and novel object recognition?were performed to measure the effect of antipsychotic drugs on locomotor activity and cognitive functions.2.Ninty-six male Kunming mice weighing 18-22 g were randomly divided into 8groups:control group?Saline 10 ml/kg+Vehicle i.p,b.i.d?,aripiprazole group?4.0mg/kg,i.p,b.i.d?,haloperidol group?2.0 mg/kg,i.p,b.i.d?,risperidone group?1.0 mg/kg,i.p,b.i.d?,MK801 model group?MK801 0.5 mg/kg,i.p,q.d?,MK801+aripiprazole group?MK801 4.0 mg/kg+Vehicle i.p,b.i.d?,MK801+haloperidol?2.0 mg/kg,i.p,b.i.d?,and MK801+risperidone?1.0 mg/kg,i.p,b.i.d?.After one-week administration,behavioral tests?locomotion activity and novel object recognition?were performed to measure the effect of antipsychotic drugs on locomotor activity and cognitive functions.All mice were sacrificed and brains were immediately removed.Brain regions?PFC,CPu,NAc?were collected according to brain microdissection atalas.Western Blotting was carried out to detect the expression of GABA_AR and PKA-CREB pathways.Pearson correlation analysis was used to analyze the correlation between GABA_AR expression and D₂R level or PKA-CREB pathways.2.Human glioma cell line SH-SY5Y was cultured in vitro,and the cytotoxicity of the antipsychotics?aripiprazole,haloperidol,and risperidone?was analyzed by MTT assay to determine the administration concentration.3.In vitro,the changes in the expression levels of GABA_AR and PKA-CREB pathway-related proteins were tested after PKA inhibitor H89 and cAMP agonist Forskolin interfered with the PKA-CREB signaling pathway to explore the possible mechanism of PKA-CREB pathway mediating GABA_AR regulation.Results:1.Schizophrenia-like mice model establishment and evaluationAfter MK801 treatment,locomotor activity in the MK801 group increased significantly?t=2.462,df=22,P<0.05?,and the NOR Index in the MK801 group decreased significantly?t=9.21,df=22,P<0.001?.2.Effect of antipsychotic drugs on behavior test of miceAntipsychotic drugs significantly affected the locomotor activity(F_?3,88?=16.11,P<0.001)and cognitive function(F_?3,88?=13.41,P<0.001)in mice.Locomotor activity in the haloperidol?P<0.001?,risperidone?P<0.001?and aripiprazole?P<0.001?group were decreased significantly;NOR Index in the aripiprazole?P<0.001?and risperidone group?P<0.01?were significantly increased.3.Effect of antipsychotic drugs on the expression of GABA_ARGABA_AR was significantly up-regulated after one week of administration with aripiprazole?P<0.001?and risperidone?P<0.05?in the hippocampus.After one week of administration,GABA_AR in the caudate putamen and prefrontal cortex were significantly down-regulated in the risperidone?P<0.05?and the aripiprazole?P<0.01?group.4.Effect of antipsychotic drugs on expressions of D₂RAfter one week of drug administration,antipsychotic drugs had a significant effect on the expression of D₂R in the hippocampus(F_?3,40?=2.959,P<0.05)and caudate putamen(F_?3,40?=6.75,P<0.001).Correlation analysis shows that the expression of GABA_AR was positively correlated with the level of D₂R in the haloperidol group?r=0.472,P<0.01?,and the expression of GABA_AR was positively correlated with the level of D₂R in the risperidone group?r=0.345,P<0.05?.in vitro,GABA_AR expression was significantly up-regulated after aripiprazole treatment?P<0.001?.5.Effect of antipsychotics on PKA-CREB pathwayIn the hippocampus,aripiprazole increased the protein levels of PKA?P<0.05?and CREB?P<0.05?,risperidone increased the protein levels of PKA?P<0.05?and GABA_AR expression was positively correlated with the protein levels of PKA?r=0.308,P<0.05?and CREB?r=0.549,P<0.001?.In vitro,haloperidol?P<0.05?and aripiprazole?P<0.01?increased the expression of PKA?P<0.05?significantly;risperidone?P<0.001?and aripiprazole?P<0.001?increased the the protein levels of CREB significantly.6.Changes in GABA_AR expression after interfere the PKA pathwayOn a cellular level,the expression of PKA?P<0.001?,p-PKA?P<0.001?,CREB?P<0.001?,p-CREB?P<0.05?,and GABA_AR?P<0.001?was significantly up-regulated after forskolin treatment,expression of PKA,p-PKA,CREB,p-CREB,and GABA_AR were significantly up-regulated after antipsychotic drugs treatment.PKA inhibitor H89reduced the expression of p-PKA?P<0.01?and p-CREB?P<0.001?.There is no significant differnces in the expression of GABA_AR.Conclusion:The study showed that short-term administration of antipsychotic drugs significantly regulates GABA_AR expression and improves cognitive level in animals.Antipsychotic drugs may modulate GABA_AR activity and improve animal cognition t hrough the D₂R and PKA signalling pathway.