The Regulation Of Dopamine Receptor Signaling Pathway On Dendrite Remodeling In Prefrontal Cortex Neurons

Drug addiction is defined as loss of control over 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 and spines of medium spiny neuron in nucleus accumbens and caudoputamen. These structural changes have been suggested to underlie long lasting alterations in synapse.There exist many structural and functional changes in neural system during the formation of drug addiction, one of which is the neuron dendrite remodelingin some brain area. Many reports show that the dopamine signaling regulates the process of cocaine-induced dendrite remodeling. It was reported that the D1 and D3 dopamine receptors have opposite effect on neogenin and synaptotagmin gene expression, and those longterm changes in gene expression cause neuron restructuing and behavioral abnormalities. Previous 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 reorganization that alters their operation, thus contributing to some of the persistent sequela associated with drug 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. Our goal is to investigate whether the dopamine receptor signaling molecular, NMDA receptor signaling molecular, ERK signaling molecular, as well as Rho protein signal molecular is involved in the cocaine-induced dendrite remodeling. And this study aimed to exploring the molecular mechanism underlying cocaine-induced dendrite remodeling in the process of cocaine addiction. Specific inhibitors is applied to block the corresponding signaling pathway to explore whether the signaling pathways are involved in cocaine-induced dendrite remodeling in the process of cocaine addiction.In the present work we set up, Rac1-N17and RhoAN19, two dominant negative-effect mutant varieties of Rac1 and Rho A respectively, and lentivirus of Rac1L16 as well as RhoAL63, two constitute-activated mutant varieties. We detect the biological activities of Rac1 and RhoA-GTPasess through counting the infectious amount of primarily cultured prefrontal cortex neuron cell and then we fixed the cells to have immunofluorescence,4 days after the last time of being stimulated by dopamine for 30 min. The result shows that there is an obvious increase in dendritic braching, spine density and synapse. Thus, we have applied specific antagonist of D1 and D3 dopamine receptor, Rac1N17, RhoAN19, Rac1L61, RhoAL63, the antagonist of Rac1 and RhoA to block the signialing pathway to explore whether they were involved in cocaine-induced dendrite remodling. The main findings are as follows:1. We constructed lentiviral vectors of the Rho faminly, incluing Plenti6/v5-RhoAL63, Plenti6/v5-RhoAN19, Plenti6/v5-Rac1N17, Plenti6/v5-RacL61. Then, we tested the activity of Plenti6/v5-RhoAN19, Plenti6/v5-RhoAL63, Plenti6/v5-Rac1N17, Plenti6/v5-Rac1L61 by using G-LISATM. After being stimulated 2 min by epidermal growth factor or or LPS, our data showed that compared with the activity of RhoA in Plenti6/v5-EGFP, those in Plenti6/v5-RhoAlN19 was decreased by 42.8%, while the activity of RacA was increased in Plenti6/v5-RhoA1L63 for 1.7 times. Compared with the activity of Racl in Plenti6/v5-EGFP, those in Plenti6/v5-Rac1N17 was decreased by 29.3%, while the activity of Rac1 in Plenti6/v5-Rac1L61 was increased for 2.3 times. The results are of obvious difference in the statistics, which suggests that the reconstruction of lentiviral vectors have the corresponding biological activity.2. We applied dopamine to stimulate prefrontal cortex neuron repeatedly to explore the effect of dopamine on the number of endritic branching, spine and synapse density. Our data shows that there is an obvious increse of those cells and compared with PBS group, there is significant difference between groups in terms of statistics.3. Using D1 dopamine receptor specific inhibitor SCH23390 and the dopamine D3 receptor specific inhibitor NGB2904 to explore wtheter the D1 dopamine receptor and dopamine D3 receptor is involved in the dopamine-induced dendrite remodeling. Models of chronic dopamine were injected SCH23390(10μmol/L) and NGB2904(50μmol/L) 5 min before the infection of dopamine. And we fix the cells to have immunofluorescence after being stimulated by D1 dopamine receptor specific activator SKF81297(1μmol/L) for 15 min. In the prefrontal cortex area, the results showed that the dopamine-induced neuronal morphological changes will be significantly reversed by the application of D1 dopamine receptor inhibitor SCH23390. Dendritic branching, spine and synapse density is decreased by SCH23390. This indicates that D1 dopamine receptors played an important role in dopamine-induced dendrite remodeling. Then, dendritic branching, spine and synapse density is increased by NGB2904 signigicantly, which suggests that D3 dopamine receptors played significant negative role in dopamine-induced neuronal dendrite remodeling. Furthermore, dopamine D1 and D3 receptors play opposite roles in regulating dopamine-induced dendritic remodeling.4. In order to explore whether Rac1 and RhoA affect the repeated dopamine induced dentritic remodeling, we have to remove all the culture medium of the prefrontal cortex neuron cells and use dopamine to stimulate it after 7 days of being infected by the lentiviral vector of the dominant negative effect varieties and the constitute activated varieties of Rac1 and RhoA. We fixed the cells to have immunofluorescence. The results showed that in the prefrontal cortex area, the infected Rac1N17 significantly inhibit the increase of dendritic braching, spine density and synapse induced by repeated dopamine. While Rac1L61 help dopamine to cause changes of dendritic branching, spine and synapse which suggests that Rac1 play a positive role in dopamine-induced dentritic remodeling. However, the infected RhoAN19 enhance the ability of dopamine to increase dendritic branching, spine and synapse density which means RhoA play a negative role in dopamine-induced dendritic remodeling.5. Using Rac1 specific inhibitor NSC23766 and RhoA specific inhibitor Y27632 to explore whether Rac1 and RhoA signaling moleculare is involved in the cocaine-induced dendrite remodeling. The prefrontal cortex cell were cultivated NSC23766(100μmol/L) and Y27632(100μmol/L) 3 minutes before the stimulation by dopamine. We fixed the cells to have immunofluorescence. In the prefrontal cortex area, the increase in dendritic branching, spine and synapse density of dopamine-indu ce were attenuated significantly when the cells were given NSC23776, which is the same with the case of virus-infected gorup. But there is not significant difference between Y27632 group and dopamine group in terms of statistics.Though this study,we concluded:1, we successfully constructed lentiviral vectous of dominant negative-effect varieties and constitue activated varieties of Rho and Rac1 and testeified the associated activity; 2, repeated dopamine stimulation make a significant increase in dendritic branching, spine and synapse density; 3, during the dopamine-induced dendritic remodeling, D1 and D3 dopamine receptor play different roles, and D1 receptor plays an importent part in the increase of dendritic branching, spine and synapse density; 4, during the dopamine-induced dendritic remodelding, Rac1 enhance the ability of dopamine to increase dendritic branching, spine and synapse density, which means Rac1 play a positive role in dopamine-induced dendritic remodeling while RhoA play a negative role in dopamine-induced dentritic remodeling.In summary, this study set up the dominant negative-effect varieties and constitue activated varieties of Rac1 and RhoA to prepare the chronic dopamine addiction model, and used a series of specific inhibitors to block the dopamine receptor pathway, Rac1 signaling pathway to explore molecular mechanism of cocaine-induced dendritic remodeling. The results show that dopamine D1 and D3 receptors and Rho signaling pathway are involved in the dopamine-induced dentrite remodeling, which dopamine D1 receptor and Rac1 play a positive role in dopamine-induced dendritic remodeling while dopamine D3 and RhoA play a negative role in dopamine-induced dentritic remodeling. Our study can help us to further understand the molecular mechanism of cocaine addiction and provide inspirations for clinical treatment of cocaine addiction.