Role Of Epac In The Regulation Of ECB-mediated Inhibitory Synaptic Plasticity Of VTA Dopamine Neurons

Aims:The activation of phospholipase C（PLC）-βand PLCγhas been linked to the facilitation of depolarization-induced suppression of inhibition（DSI）and long-term depression of inhibitory postsynaptic currents（IPSCs）（I-LTD）in the ventral tegmental area（VTA）.So,PLCεmight contribute to the facilitation of DSI and I-LTD in the VTA.Exchange protein directly activated by cAMP（Epac）activates the PLCεpathway.Thus,we hypothesized that the activation of Epac-PLCεpathway might play an important role in the facilitation of DSI and I-LTD in the VTA.This study investigated whether Epac was involved in the endocannabinoid dependent inhibitory synaptic plasticity in the VTA,with a focus on the role of Epac in the cocaine addiction and cocaine related conditioned place preference（CPP）performance.Methods:（1）Animals:Epac^+/+,Epac^-/-,Epac1^-/-,Epac2^-/-,PLCε^+/+（C57BL/6）and PLCε^-/-mice were used in this study.（2）Brain slice electrophysiology:Midbrain slices containing VTA were made.Whole-cell patch clamp recordings of IPSCs and sIPSCs were made in VTA dopamine neurons.DSI and I-LTD were induced using an internal solution with or without 8-（4-chlorophenylthio）adenosine 3’,5’-cyclic monophosphate（8-CPT）.（3）In vivo single-unit extracellular recordings from VTA dopamine neurons were made to explore the role of Epac in the cocaine-induced increase in action potential（AP）firing.To confirm cell type,neurons were labeled with neurobiotin and brain slices were used for immunostaining of tyrosine hydroxylase（TH）after recordings.（4）Epac2^-/-,PLC?^-/-and wild type（WT）control mice were used in the cocaine CPP test to investigate whether Epac-PLC?pathway is required in cocaine-induced CPP.（5）The role of resveratrol pretreatment in improving cocaine CPP and cocaine addiction induced reduction of GABAergic inhibition in VTA dopamine neurons was investigated.Results:（1）8-CPT facilitates endocannabinoid-mediated DSI and I-LTD.（2）Epac2 knock-out blocks 8-CPT induced facilitation of DSI and I-LTD in the VTA.（3）PLC?knock-out blocks 8-CPT induced facilitation of DSI and I-LTD in the VTA.（4）Diacylglycerol lipase（DAGL）inhibitor DO34 blocks 8-CPT induced facilitation of DSI and I-LTD in the VTA.（5）Epac2 knock-out or PLC?knock-out blocks cocaine CPP.（6）Epac2 knock-out or PLC?knock-out blocks cocaine induced reduction of GABAergic inhibition in VTA dopamine neurons.（7）Epac2 knock-out blocks cocaine induced increase of VTA dopamine neurons excitability.（8）Resveratrol pretreatment improves cocaine CPP and cocaine addiction induced reduction of GABAergic inhibition in VTA dopamine neurons.Conclusions:Epac plays an important role in the regulation ofγ-aminobutyric acid（GABAergic）synaptic plasticity of VTA dopamine neurons.Epac facilitats endocannabinoid-mediatedDSIandI-LTDbyactivatingthe Epac2→PLCε→DAGL→2-AG pathway.（2）The activation of Epac might be one of the molecular mechanisms of cocaine CPP.（3）Resveratrol pretreatment restores cocaine CPP by improving the reduction of GABAergic inhibition in VTA dopamine neurons.