Effects Of Nicotine Exposure On Hippocampal Epac Signaling Pathway In C57 Mice

Objective:Tobacco smoking is a major public health problem in China and across the world.Past decades have witnessed a significant achievement in preventing tobacco smoking and treating addiction and other adverse events related to smoking.Due to the complexity of neurobiology in addiction,no medication can completely cure tobacco addiction,leading to smoking abstinence and repulse,as well as an increase in economic burdens to family and society.Although women account for half of all smokers,and many studies have shown that adult males and females respond differently to smoking,current neurobiological studies on the effects of men and women on smoking are limited.Previous studies have demonstrated that the exchange protein directly activated by cAMP(Epac)signaling pathway is involved in drug addiction behavior.In order to further explore the neurobiological mechanism underlying nicotine addiction,hippocampal Epac signaling pathway and behavioral changes in adult nicotine-added mice were explored.Methods:(1)Primary hippocampal neurons were cultured in vitro,and the morphology of hippocampal neurons was observed.The purity of neurons was further identified by immunofluorescence staining GFAP and MAP2.(2)Nicotine was added to mature primary hippocampal neurons cultured in vitro,and the expression of Epac protein was detected by Western blot.The effect on Epac protein expression was observed by the addition of methyllycacotine(MLA).(3)Establish a nicotine-induced adult C57BL/6 mouse(including male and female)conditioned place preference(CPP)paradigm.(4)The market experiment recorded changes in the voluntary activities of adult female and male nicotine-preferred mice.(5)Western blot analysis of protein expression of hippocampal Epac protein,p-CREB,a7 nAChR and PKA in CPP male and female nicotine-conditioned miceResults:(1)The expression of Epacl protein was up-regulated after nicotine treatment in vitro.After 8 hours,the expression of Epac1 protein was increased,and then it recovery to normal level,while the expression of Epac2 was increased at 4 h,8 h,12 h,and 24 h.Nicotine can increase the expression of Epac protein in hippocampal neurons,and the addition of MLA inhibits Epac2 protein expression.(2)Nicotine can develop CPP in adult mice at 0.2 mg/kg(male,SC)and 0.4 mg/kg(female,SC).(3)In the open field experiment,compared with the control group,the male mice spent more time staying in the central area significantly,indicating less anxiety-like behavior of male mice,while the spontaneous activity of female mice increased significantly as compared to vehicle-treated mice.(4)After CPP experiments,Western blot studies showed that hippocampal Epac2 protein was significantly increased in both male and female mice,but the change of Epacl did not present difference between vehicle and nicotine treated mice.In female mice,Rapl protein was significantly elevated and phosphorylated CREB was significantly reduced.Conclusion:Nicotine acts on hippocampal neurons in vitro to stimulate Epac expression,and MLA can inhibit Epac expression.Nicotine induced changes in the hippocampal Epac signaling pathway in CPP mice.In conclusion,nicotine exposure can increase the expression of Epac signaling in the hippocampus.This study provides a new pharmacological target for the treatment of nicotine abuse and dependence.Figure[13]table[0]reference[48]...