Ⅰ：Experimental Research Of BK_ca Channels In The Nicotine Addiction In Mice;Ⅱ：Gentiopicroside Attenuates Morphine Rewarding Effect Through Down-regulation Of NR2B Receptors In Nucleus Accumbens

PartⅠSmoking is the important risk factor leading to severe cardiovascular andcentral nervous system （CNS） diseases, and the death rate of diseases related tosmoking increased dramatically. Therefore, how to get rid of smoking and clarifythe mechanisms involved in smoking addiction are the focuses in this field.Nicotine, the main component of tobacco, is the major substance fornicotine addiction or dependence, while the mechanisms involved remainunclear. There are lots of similarities in neurobiological changes betweenaddiction and learning memory, including similar critical brain regions,functional molecules and ion channels. Among them, large conductanceCa²⁺-activated K+-channel (BK_Ca) is widely expressed in the CNS and playsimportant roles in lots of neural activities. Activation of BK_Cachannel producesafter hyperpolarization （AHP）, subsequently decreases excitability of the neurons. At the same time, BK_Catakes part in neural networks throughmodulation of neuron excitability and neurotransmitter release. Our previousdata show that BK_Caare involved in anxiety development by affecting neuronselectricphysiological activities in amygdala, suggesting BK_Camay play a criticalrole in higher nervous activities including learning and memory. However,whether BK_Caplays roles in nicotine addiction and the underlying mechanismsare still unclear. In this study, multiple methods including molecular biology,behavior test, and electrophysiology were used to investigate the roles andmechanisms of BK_Cainvolved in nicotine addiction. It is hopeful that this studymay shed light on the possibility of potential target for nicotine addictiontreatment.ObjectiveBK_Cais widely expressed in the CNS and takes part in lots of neuralfunctions through modulation of neuron excitability and neurotransmitter release.Our previous data show that BK_Cais involved in anxiety development byaffecting neurons electricphysiological activities in amygdala, implicating BK_Camay play a critical role in higher nervous functions. In this study, we willexplore the roles of BK_Caof nucleus accumbens septum （NAc） in thedevelopment of nicotine-addiction and the mechanisms involved in the process.It may shed light on the potential target for the therapy of nicotine addiction.Methods1. Immunofluorescence double-staining were used to check the expression ofBK_Cain the excitatory and inhibitory neurons. The change of BK_Caexpression under nicotine stimulation was tested in the primary cultureneurons. 2. Nicotine-addiction mouse model was establish to investigate the alteration ofBK_Caexpression in mice NAc.3. Roles of BK_Cain nicotine-addiction were determined by subcutaneousinjection of NS1619(BK_Caagonist) and Paxilline (BK_Caantigonist) inaddicted mice and the behavioral changes accordingly.4. Lentiviurs shRNA for BK_Cawas performed to further explore the roles ofBK_Canicotine-addiction.5. Whole-cell patch clamp technique was used to investigate the effects ofBK_Cachannels on the excitability of neurons in NAc and the alteration ofBK_Cachannels in nicotine-addiciton.Results1. BK_Cachannels were observed coexsited in both VGLUT2positive andGAD67positive cells, implicating BK_Cachannels are expressed inexcitatory and inhibitory neurons.2. Expression of BK_Cain cultured neurons was found decreased after treatmentwith nicotine （50μM） for24hours.3. Conditioned place preference （CPP） test was used to test nicotine-addiction,which was induced by subcutaneous injeciton of nicotine （0.5mg/kg） forsuccessive5days. Expression of BK_Cain NAc was decreased significantlyin the addition mice.4. Subcutaneous injection of NS1619, the agonist of BK_Ca, decreaseddrug-seeking behavior in addiction mice.5. Knocked down BK_Caexpression in NAc by shRNA promoted drug-seekingbehaviors.6. Using the whole-cell patch clamp technique we found that NS1619, theBK_Caagonist, increasd the peak of the fAHP in the NAc neurons of addiction mice. The function of BK_Cachannels was decreased in thenicotine-addiction mice.Conclusions1. BK_Cachannels were expressed in both excitatory and inhibitory neurons andthe expression of BK_Cawas decreased after nicotine treatment.2. Expression of BK_Cachannels was decreased in NAc of nicotine-addictionmice. Increase of BK_Cachannel function （using agonist NS1619） coulddecrease drug-seeking behavior, however, inhibition of BK_Caexpressionby shRNA caused enhancement of drug-seeking behavior.3. Whole-cell patch-clamp recordings reveal that BK_Cachannels involve in thedevelopment of nicotine-addiction by regulating the excitability of thenetwork. Modulation of the BK_Cachannels may be the potential target forthe treatnment of drug addition.PartⅡObjective: Gentiopicroside （Gent） is one of the secoiridoid compoundisolated from Gentiana lutea. This compound exhibits analgesic activities andinhibits the expression of GluN2B-containing N-methyl-D-aspartate （NMDA）receptors in the anterior cingulate cortex in mice. Nucleus accumbens （NAc） is aforebrain structure known for its role in drug addiction. However, little is knownabout the role of Gent on morphine dependence and synaptic transmissionchanges in the NAc.Methods: Conditioned place preference （CPP） test and behavioralsensitization of locomotor activity were used to investigate drug-seeking relatedbehaviors. Brain slices containing NAc were prepared, and whole-cell patch-clamp recordings were performed to record the excitatory postsynapticcurrents （EPSCs）. Expression of proteins was detected by Western blot analysis.Results: Systemic administration of Gent attenuated the CPP effect inducedby morphine, but had no effect on morphine-induced behavioral sensitization.Gent significantly reversed overexpression of GluN2B-containing NMDAreceptors and dopamine D2receptors in NAc during the first week of morphinewithdrawal. However, the compound did not affect the overexpression ofGluN2A-containing NMDA receptors, GluA1, and dopamine D1receptors.Lastly, Gent significantly reduced NMDA receptors-mediated EPSCs in theNAc.Conclusions: Our study provides strong evidence that Gent inhibitsmorphine dependence through downregulation of GluN2B-containing NMDAreceptors in the NAc.