The Mechanism Of Altered SK Channel Activity In Nac And MPFV During Morphine Withdrawal

Background and objective:Substance dependence are "relapsing brain disorders" that develop with repeated drug exposure in those that are vulnerable because of genetic,developmental,and/or psychosocial factors.Its mechanism involves long-term and persistent dysregulation of neural circuits,particularly in executive control systems and reward systems.Morphine is a kind of opioids.Repeated morphine exposure can lead to addiction,and relapse after MW(MW)occurs easily in the majority of individuals.MW often leads to neuroadaptations in the Nucleus Accumbens(NAc)and medial prefrontal cortex(mPFC),which plays a role in the disruption of executive control-reward circuits in drug addiction.Therefore,it is desperately needed to explore the mechanism of neuroadaptations during MW.Numerous studies show that the NAc region of the ventral striatum is a main part of the reward circuitry.The prelimbic(PrL)and infralimbic(IL)cortices,the two sub-regions of the mPFC,play essential roles in the neural circuits for executive control.The excitability of neurons in NAc and mPFC can be affected by the amplitude of the action potential afterhyperpolarization(AHP),which is regulated by small conductance calcium-activated potassium(SK)channels.rodent studies suggest that SK channels in NAc and mPFC regulate drug seeking behavior,but the mechanism is still not clear.On one hand,previous studies implied that actions of the intracellular CK2 and PP2A on the SK-calmodulin(CaM)complex could regulate the Ca2+ sensitivity of SK channels.On the other hand,Rac1,a member of Rho family of small GTPases,might modulate SK channel activity and firing patterns,and contribute to structural and behavioral plasticity in NAc and mPFC in response to repeated drug exposures.However,whether MW induced enhancement of SK related proteins and Racl signaling are directly or indirectly associated with SK channel related neuroadaptations is still not clear.Therefore,the purpose of our study is to explore whether SK channels activity in the NAc or mPFC were changes during MW.And,we are also trying to figure out the mechanism that modulates SK channels during MW,which may be the potential therapeutic targets for medication development in opiate use disorders.Methods:Morphine-induced CPP rat model were established in this study.And all rats were randomly assigned to undergo one-week or three-week morphine/saline withdrawal.We performed a separate set of experiments including electrophysiology,behavior(condition place preference),immunohistochemistry,western blotting and retrograde tract-tracing to assess the effects of SK channel related neuro-adaptations in NAc and mPFC.To explore the role of Rac1 in the SK channel alterations we performed a Rac1 pull-down and detection assay and LC-MS/MS iTRAQ analysis.Results:Part one:NAcs AP firing and SK channel activity were altered after three-week MW1.NAc shell neurons from morphine-treated rats exhibited a significantly larger basal I/O slope than neurons from saline-treated rats(p<0.05).2.SK inhibition by apamin(100 nM)could greater enhance the I/O slopes of NAc shell neurons in rats after MW compared with that in saline group(p<0.05).And apamin eliminated the difference in the I/O slopes of the two groups after apamin exposure(p>0.05).3.SK related peak tail currents were significantly smaller in NAc shell neurons from the morphine-withdrawal animals than in those from saline-treated controls(p<0.05).4.SK2 and SK3 subunit expression was significantly decreased in the NAc core and shell of the MW rats compared to that of saline control(SC)rats.Part two:The research on the regional targeting of mPFC’s projections to NAc1.Retrograde tracers tetramethylrhodramine-dextran(TMR)were injected into either the NAcc and NAcs to enable visualization of ipsilateral PrL or IL neurons that projected to each target region.2.The density of NAcs-IL double labeled neurons was significantly higher than that of NAcs-PrL(p<0.05).Part three:The effects on MW induced neurophysiological alterations in mPFC-NAc1.In NAcs,MSNs exhibited enhanced AP firing frequency under one-week MW compared with SC(180 pA:p<0.05;220 pA:p<0.05).2.Layer 5 pyramidal neurons in IL from MW rats displayed a significantly smaller basal I/O slope than neurons from SC rats(p<0.05).3.SK channel related peak tail currents after hyperpolarization were extensively larger in IL neurons from one-week and three-week MW rats(p<0.05).Part four:Study on the regulatory mechanism of MW induced mPFC changes in SK channel protein level1.SK3 subunit expression was significantly increased in mPFC(both IL and PrL)of one-week MW compared to SC rats(p<0.05).And SK3 subunit channels are widely expressed in the membranes of IL neurons.2.In the mPFC,CK2β was downregulated in MW compared to SC rats(p<0.05).In both mPFC and NAc,the activity of PP2A were increased in MW compared to SC rats(p<0.05).3.After one-week MW compared with SC,six members of Small GTPases in RAS superfamily were altered in mPFC,including five upregulated(Rab2B,Rab3B,RhoA,RhoC and Rac1)and one downregulated(R-Ras)(p<0.05).4.A significant increase in the levels of Rac1-GTP(active Rac1)was detected in the mPFC after one-week MW compared with SC(p<0.05).Part five:The effect of Rac1 on excitability of pyramidal neurons in IL and morphine induced CPP behaviors.1.Bath perfusion of neurons with NSC23766 dramatically increased their firing frequency and caused them to fire irregularly(p<0.05).2.Downregulating Rac1 in the IL could significantly reduce the conditioning score of morphine induced CPP(p<0.05).3.Rac1-shRNA-expressed rats also revealed a significant decrease in the expression of SK2 or SK3 channel subunits(SK2:p<0.05;SK3:p<0.05).Conclusion:In the present study,we could conclude that:(1)the excitability of pyramidal neurons in layer 5 of IL was decreased,whereas that of MSN in the NAcs was increased;(2)in mPFC,SK currents and the expression of SK subunit SK3 were increased,whereas in NAc the expression of SK2 and SK3 were decreased;(3)the PL and IL projects to both the core and shell of NAc,while the IL projects to the shell preferentially;(4)the expression of the SK subunits,PP2A,CK2α and CK2β were differentially regulated in mPFC and NAc though the activity of PP2A was increased both in mPFC and NAc;(5)Racl expression and signaling was increased in mPFC and involved in regulation of AP firing and SK related currents;and(6)Racl plays crucial role on the process of morphine induced CPP and regulating the expression of SK channel.