The Molecular Mechanism Underlying Cocaine-induced Dendrite Remodeling

Drug addiction is defined as loss of control over drug intake and compulsive drug taking despite horrendous adverse consequences.Drug addiction may become a lifelong status once in shape.It can persist for months or years after discontinuation of drug use.Drug addiction develops gradually and progressively during repeated exposure to drug.The occurrence of adaptive changes is a very complicated biological process.The behavioral abnormalities associated with cocaine addiction are extremely long-lived.Therefore,there has been considerable interest in identifying long-lasting drug-induced changes at the molecular and structural level in neuronal circuits regulated by dopamine and glutamate Notably,longterm exposure to cocaine has been found to increase the number of dendritic branch points and spines of MSNs in NAc and CPu.These structural changes have been shown to persist for at least one months after the last drug exposure and have been suggested to underlie long lasting alterations in synape.Neuron dendrite remodeling is the structural basis of cocaine addiction.The data suggest that the net growth of dendritic arbor occurs as a result of several distinct events:emergence of a new branch,selective maintenance of the new branch,and extension of branch length.Some branches give birth to dendritic spines,which are acceptable site of the excitatory synapes,and the corresponding inhibitory synapse mainly on the dendritic branches.The structure of the dendritic arbor critically determines what synaptic inputs a neuron receives and how they are integrated.It is suggested that structural plasticity associated with exposure to drugs of abuse reflects a reorganization of patterns of synaptic connectivity in these neural systems,a reorganization that alters their operation,thus contributing to some of the persistent sequela associated with drug use-including addiction.Robinson reported that over exposure to cocaine can lead to increases in dendritic branching and spine density in medium neurons of nucleus accumbens.This morphological change would maintain at least a month after drug withdrawal.The development and structural plasticity of dendritic arbors are governed by several factors,including synaptic activity, neurotrophins and other growth-regulating molecules.Persistent changes in behavior and psychological function that occur as a function of experience,such those associated with learning and memory,are thought to be due to the reorganization of synaptic connections(structural plasticity) in relevant brain circuits.Long-lasting adaptations in synapse are believed to underlie addictive behaviors associated with cocaine.There are many signaling pathways involved in neuronal structural plasticity This study aimed to exploring the molecular mechanism underlying cocaine-induced dendrite remodeling in the process of cocaine addiction.Our goal is to investigate whether the dopamine receptor signaling pathway,NMDA receptor signaling pathway,ERK signaling pathway,as well as Rac1 signal molecular is involved in the cocaine-induced dendrite remodeling.Specific inhibitors is applied to block the corresponding signaling pathway to explore whether the signaling pathways are involved in cocaine-induced dendrite remodeling,thereby revealing the molecular mechanism underlying cocaine-induced dendrite remodeling in the process of cocaine addiction.In the present work we set up an animal model of chronic cocaine,Kunming mice experienced 5 days of cocaine injection and 2 days of drug withdrawal for 4 cycles 28 days totally.Their brains were removed and then processed by Golgi-Cox staining, which is a common approach to quantify the structure of dendrites and the density of dendritic spines.The present results show that there are increases in dendritic branching and spine density in medium neurons of striatum nucleus accumbens and caudateputamen.Thus,we have applied specific antagonist of D1 and D3 dopamine receptor,NMDA receptor,ERK and Rac1 to block the signaling pathway to explore whether they were involved in cocaine-induced dendrite remodling.The main findings are as follows:1.Using D1 dopamine receptor specific inhibitor SCH23390 and the dopamine D3 receptor specific inhibitor NGB2904 to explore whether the D1 dopamine receptor and dopamine D3 receptor is involved in the cocaine-induced dendrite remodeling. Kunming mice were injected SCH23390 and NGB2904 respectively 30 minutes before the injection of cocaine to prepare animal models of chronic cocaine.Their brains were removed,processed by Golgi-Cox staining.In CPu and NAc area,the results showed that the cocaine-induced neuronal morphological changes will be significantly reversed by the application of D1 dopamine receptor inhibitor SCH23390.Dendritic branching and spine density is decreased by SCH23390.This indicates that D1 dopamine receptors played an important role in cocaine-induced dendrite remodeling.Then,dendritic branching and spine density is increased by NGB2904 significantly,which suggests that D3 dopamine receptors played significant role in cocaine-induced neuronal dendrite remodeling.Furthermore, dopamine D1 and D3 receptors play opposite roles in regulating cocaine-induced dendritic remodeling.2.Using NMDA receptor specific inhibitor MK801 to explore whether the NMDA receptor is involved in the cocaine-induced dendrite remodeling.Kunming mice were injected MK801 30 minutes before the injection of cocaine to prepare animal models of chronic cocaine.Their brains were removed and processed by Golgi-Cox staining. In CPu and NAc area,the increases in dendritic branching and spine density of cocaine-induced were attenuated significantly when mice were given MK801,which indicates NMDA receptors played an important role in regulating cocaine-induced dendrite remodeling.3.Using Rac1 specific inhibitor NSC23766 to explore whether Rac1 signaling molecule is involved in the cocaine-induced dendrite remodeling.Kunming mice were injected NSC23766 30 minutes before the injection of cocaine to prepare animal models of chronic cocaine.Their brains were removed and processed by Golgi-Cox staining.In CPu and NAc area,the increases in dendritic branching and spine density of cocaine-induced were attenuated significantly when mice were given NSC23766, which indicates Rac1 is involved in cocaine-induced dendrite remodeling.In the process of cocaine addiction,Rac1 is activated and play its positive role to promote dendritic extension and branching and spine growth.4.Using ERK specific inhibitor SL327 to explore whether ERK signaling molecule is involved in the cocaine-induced dendrite remodeling.Kunming mice were injected SL327 30 minutes before the injection of cocaine to prepare animal models of chronic cocaine.Their brains were removed and processed by Golgi-Cox staining.In CPu and NAc area,the increases in dendritic branching and spine density of cocaine-induced were attenuated significantly when mice were given SL327,which suggests ERK is involved in cocaine-induced neuronal dendrite remodeling.5.Furthermore,electron microscope was carried out to examine ultrastructure of striatum after cocaine and different inhibitors treatments.Mice were perfused and striatum were isolated for electron microscope.The results showed that synaptic density in the striatum increase about 24%after cocaine treatment,D1 dopamine receptor inhibitor SCH23390,NMDA receptor inhibitor MK801 and the Rac1 inhibitor NSC23766 cause a significant reduction in synaptic density with degree of 43%,28%and 33%respectively comparing to cocaine group.The results are coincide with the data from Golgi-cox staining.Meanwhile,there are no significant abnormalities in ultrastructure from different groups.6.At the same time,in order to study the role of Rho family in cocaine-induced dendritie remodeling,we constructed lentiviral vectors of the Rho family,including Plenti6/v5-EGFP-RhoAL63,Plenti6/v5-EGFP-RhoANA,Plenti6/v5-EGFP-Rac1NA, Plenti6/v5-EGFP-Rac1L61.Then,we tested the activity of Rac1 by using G-LISA^TM. Our data showed that the activity of Rac1 was increased in Plenti6/v5-EGFP-Rac1L61 for 1.6 times,while the activity of Rac1 is inhibited by 47%in Plenti6/v5-EGFP-Rac1NA.The reconstruction of lentiviral vectors of Rho family lay the foundation for further studying the molecular mechanism of Rho family in cocaine-induced dendritie remodeling. In summary,this study used a series of specific inhibitors to block the dopamine receptor pathway,NMDA receptor pathway,ERK signaling pathway,as well as Rac1 signaling pathway to explore molecular mechanism of cocaine-induced dendritie remodeling.The results show that dopamine D1 and D3 receptors,NMDA receptor pathway,ERK signaling pathway,as well as Rac1 signaling pathway are involved in the cocaine-induced dendritic remodeling.Our study can help us to further understand the molecular mechanism of cocaine addiction and provide inspirations for clinical treatment of cocaine addiction...