| Background and objectivesDrug addiction is a long-lasting brain disease.More than half of people with cocaine addiction suffer from mental disorders,depression is the most common.Negative emotional state is considered to drive the power of thirst and relieve drugs,thus alleviating the depressive symptoms of abusers is essential to drug withdrawal.Many studies have reported a series of neuroplasticity changes in the hippocampus during depression,such as molecular and morphological changes.Racl,as an important member of the small G protein family,plays an important role in the regulation of skeletal protein morphology,especially in the formation and maintenance of dendritic spines.Recently,Rac1 in the NAc region of striatum has been reported involved in synaptic remodeling and depression-related behavioral changes in mice model of social defeat.So far,the role of Racl signaling pathway in the development of depression-like behavior during cocaine withdrawal is unclear.Therefore,we focused on the hippocampus to investigate the regulation of Racl signalling pathway in the depression-like behavior after cocaine withdrawal.Methods1.We established mouse depression-model after cocaine withdrawal,that is,given mice cocaine for 14 days of intraperitoneal injection,dose with 15 mg/kg,three times a day,at nine,ten,eleven o'clock per day.Make mice intake of cocaine continuously,the body will fell crash after withdrawal from cocaine2.Track the changes of body weight in the process of modeling and after the withdrawal.Evaluate the depression-like behavior of mice by tail suspend test,forced swimming test,sucrose preference test.3.We constructed Rac1 active mutant lentivirus(Rac1CA).The activity of Racl GTPase in hippocampus was increased by stereotactic injection,and the consequent protein expression,morphological and behavioral changes were detected.4.Construct Racl specific gene knockout in hippocampus.It was injected into hippocampus,the specific knockout of Racl would be get after Cam K ?in hippocampus initiated the expression of Cre recombinase.5.The GST pull-down was performed to detect the activity of Racl GTPase after cocaine withdrawal and Rac1 CA lentivirus transfection.6.Western blotting was used to detect the activity of phosphorylated Pak,Limik,cofilin,CREB and ERK,which are downstream effctors of Racl,and the expression level of BDNF,after virus transfection and when mice showed depression-like behavior.7.DCX immunofluorescence was used to detect the regeneration of neonatal neurons,and Brdu immunohistochemistry to detect the proliferation of neurons in hippocampal DG region.8.Morris water maze to detect the spatial memory and working memory of mice.Results1.Construction the model of depression-like behavior after cocaine withdrawal Successful.Forced Swimming Test and Tail Suspend Test showed the duration of absolute immobility time increased,the decline in body weight indicated decreased appetite in mice,while sucrose preference test suggests the state of anhedonia in depressed mice,all of which proved that the depression model was constructed successfully.2.A series of changes in neural plasticity occurred in the hippocampus in depression model after cocaine withdraw:1)The phosphorylation activity of Pak,Limik,cofilin,the downstream effectors of Racl,were all decreased along with the downregulation of Racl GTPase activity.2)The expression of BDNF also declined.3)The regeneration of neonatal neurons and proliferation of neuron in the hippocampus DG region were decreased,which repeated administration of cocaine destroyed normal nerve regeneration in the hippocampus.4)The dendritic spine density of the hippocampal CA1 region was higher than that of the control group,On the one hand,the dendritic spine density in the hippocampus CA1 region in model group increased compared with the control group.On the other hand,the proportion of the three dendritic spines also changed,compared with the control group,while the proportion of mushroom spine was at the same level.The components of the three dendritic spines Changed after modeling,the ratio of immature dendritic spine greatly increased,in contrast,the ratio of mature dendritic spine was reduced.5)The spatial memory and working memory of depression mice were impaired.3.Continuous activation of Rac1 in the hippocampus region effects on the phenotypes of mice model after cocaine withdrawal1)The phosphorylation activity of Pak,Limik and cofilin,the downstream effectors of Rac1 increased following the increased activity of Rac1 GTPase.2)The expression of BDNF increased,possibly due to increased phosphorylation of CREB and ERK activity.3)The regeneration of new neurons and proliferation ability of neuron in the hippocampus the DG region turned out to be improved.4)In the hippocampus CA1 area,the dendritic spine density decreased compared with the model group,the proportion of thin spine decreased from 51%to 46%and the proportion of stubby spine increased from 31%to 40%,and the percentage of immature dendritic spines decreased compared with the depression model group,while mature spine is increased.5)Depression-like behavior after cocaine withdrawal was improved.6)The spatial memory and working memory of depression mice were improved.4.The Racl gene was specifically knocked out in the hippocampus by Cre virus,and then established a depression model.The knockout of Racl could counteract the increase of dendritic spine caused by cocaine during the modeling process,but the proportion of the three types of spines is similar to that of the modeling group,and the knockout of Rac1 exacerbates depression-like behavior.ConclusionIn this study,we proved that the Rac1 signaling pathway is involved in the regulation of depression-like behavior after cocaine withdrawal,through the Pak-Limik-cofilin signaling pathway affecting the synaptic remodeling in hippocampal CA1 region,the expression of BDNF,the regeneration of neonatal neurons and proliferation of mature neuron in the hippocampus DG region,and then participate in the regulation of related behavioral plasticity. |
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