Signalings Via Rac1 And Cdc42 Regulate Cocaine-induced Structural Remodeling Of Dendrites And Spines

Drug addiction is a major social problem in the world,it has about 10 million people died from drug addiction each year in the world.Similarly,drug addiction has become a growing social problem in our country,the registered addicts is about 1 million,but the actual number is much higher than this.It has far-reaching social significance and huge market prospects to carry out the study of the mechanisms of drug addiction,and further to find an effective treatment for drug addiction.Drug addiction is a long-lasting brain disease.Long-term exposure to cocaine produces enduring neuronal alterations in intracellular signaling pathways,structural changes in dendritic morphology,and behavioral plasticity.One of the long-lasting neural adaptations observed in several animal models of addiction is an increase in dendritic spine density on dopaminoceptive medium spiny neurons(MSNs)in striatum.Such neuroadaptations may contribute to the changes in synaptic plasticity underlying cocaine addiction.Although several groups have documented that repeated cocaine exposure increases spine density in striatum,the precise molecular mechanisms leading to these morphological changes have yet to be fully investigated.Dendritic remodeling requires architectural changes in dendrites and spines through modification of actin cytoskeleton.Previous studies have indicated that changes in spine morphology are controlled by modification of the actin cytoskeleton regulated by small GTPases.The Rho family of small GTPases,including Racl,RhoA,and Cdc42,are key regulators of the actin cytoskeleton rearrangement and play important roles in dendritic morphogenesis.It has been generally thought that RhoA and Racl/Cdc42 have antagonistic effects on dendritic spine morphology and that Racl/Cdc42 promotes the development of new spine,while RhoA inhibits their formation and maintenance.This property of cycling between GTP and GDP states hasprovided a means to identify and purify proteins that regulate the nucleotide state of GDP and GTPases.GTPase-activatingproteins(GAPs)have been characterized for many members of the Ras family.Guanine nucleotide dissociation inhibitors(GDIs)were identified based on their ability to inhibit thedissociation of GDP.It has subsequently been determined that they also bind to the GTP state,inhibiting the intrinsic and GAP-stimulated GTP hydrolysis.In general,GAPs and effectors have a high affinity for the GTP-bound state,while guanine nucleotide dissociation inhibitor proteins bind most tightly to the GDP-bound state.guanine nucleotide exchange factors(GEFs)can be distinguished from other regulatory proteins by their ability to interact preferentially with the nucleotide-depleted state of G-proteins.By stimulating the dissociation of GDP and subsequent binding of GTP,GEFs play an important role in the activation of Ras-like proteins.Dopamine receptors,including the D1-class(D1 and D5)and the D2-class(D2,D3 and D4),play important roles in cocaine-induced neurobiological changes in the NAc.The intracellular signaling cascades associated with various dopamine receptors have been suggested to play critical roles in drug-induced neuroadaptations in the brain.The MSNs,which comprise more than 90%of NAc neurons,can be divided into two classes:direct-pathway MSNs expressing the D1 receptor and indirect-pathway MSNs expressing the D2 receptor.Approximately 50%of MSNs express only D1 receptors,35-40%express only D2 receptors,and 10-15%express both D1 and D2 receptors.Our group has proved that the D1 and D3 receptors oppositely regulate cocaine-induced structural remodeling of dendrites and spines,likely via the consecutive regulation of phosphorylation of NR1,ERK activation in the NAc and CPu.A separate study found that cocaine suppresses the activity of the myocyte enhancer factor 2(MEF2)protein via the phosphorylation of MEF2 at Ser408/444 in the NAc,and this reduced activity of MEF2 contributes to the structural remodeling of dendritic spines and the sensitized response s to cocaine.Importantly,this study found that D1 re ceptor signaling regulates MEF2 activity following cocaine exposure.There are many signaling pathways involved in neuronal structural plasticity.This study was to explore the molecular mechanism underlying cocaine-induced dendrite remodeling in the process of cocaine addiction from three aspects.The main methods as following:Part one:We build a Rac1 inactive mutant lentivirus(Rac1N17).We explore the Racl activity 7 days and 28 days of cocaine treatment by using GST-pull down technology in the mouse striatum,including nucleus accumbens(NAc)and striatum caudate putamen(CPu).The activity of Racl upstream and downstream molecules,including Tiaml,RacGAP1,and Cofilin were detected by using western-blotting.By using stereotaxic injection technique,Rac1N17 was injected into the CPu area ofmouse brain and allowed to recover for 14 days,the 5 days and 28 days of cocaine addiction model were buildt to detect the dendritic branching and dendritic spines density changes in CPu neurons which expressed lentivirus positively;At the same time,behavioral sensitization experiment and conditioned place preference assay were used to detect cocaine-induced behavioral changes in the mice infected by Rac1N17 in the CPu and the mice pretreated with Rac1-specific blocking agent NSC23766.Part two:Modified T29-1 line mice which use CamK? gene to start Cre gene expression were hybridized with LoxP-Cdc42-LoxP mice to constructed forebrain-specific the Cdc42 gene knockout mice.Due to the T29-1 series of gene-modified mice expressed the cre gene mainly in neurons in the hippocampal CA1 region,whose knockout rate can reach 98%,we focused study on the CA1 region of the hippocampus.By using PCR technology,Nissl staining and immunohistochemical staining and Golgi-cox staining,the genotype and the basic morphology of the mice are detected.Chronic cocaine addiction model were built using wild-type mice,Cdc42 activity was detected by GST-pull down technology in the hippocampus.By using forebrain-specific Cdc42 gene knock out mice and wild-type mice as controls,a chronic cocaine addiction model was constructed.Using Golgi-cox staining method to detect the changes in dendritic spine density induced by cocaine in hippocampal CA1 region.Part three:By using biocytin staining method staining to reveal cocaine-induced dendritic remodeling,and using biocytin,D1 and D2 co-immunofluorescence technology to detect which type of neuron occured dendritic remodeling induced by cocaine treatment.On the basis of previous study by our group,by using Dl,D3,NMDA,ERK specific inhibitor to block the corresponding signaling pathway,we explored the signaling pathways which regulated cocaine-induced dendritic remodeling through MEF2 signaling.The main results as following:Part one:1.We first measured the Racl activity in the NAc and CPu following cocaine exposure.We found that the Rac1 activity was significantly decreased in the NAc after 7 days and 28 days repeated cocaine injections(7d:F=167.058,P<0.001;28d:F=84.001,P<0.001).Surprisingly,the Racl activity was increased in the CPu at 15 min after last cocaine administration in both 7 days and 28 days cocaine treatments(7d:F=31.188,P<0.001;28d:F=35.557,P<0.001).This altered activation lasted to 2 hr and returned to baseline 4 hr after last cocaine injection in 7 days cocaine treatments.The altered activation patterns in 28 days cocaine treatments were more lasting than that observed following 7 days cocaine injections.That is to say,the increased Racl activity in 28 days cocaine treatments lasted to 4 hr and returned to baseline 8 hr after last cocaine administration.These data suggest that Rac1 activity is differentially regulated in NAc and CPu following repeated cocaine treatment.Next,to identify the mechanism by which cocaine downregulates Rac1 activity in the NAc and upregulates Racl activity in the CPu,we examined the guanine nucleotide exchange factor(GEF)Tiaml,an upstream positive regulator of Racl,and the GTPase activating protein(GAP)RacGAP1,an upstream negative regulator of Rac1.In the NAc,7 days and 28 days cocaine administration significantly downregulated Tiaml expression(7d:F=37.263,P<0.001;28d:F=4.415,P=0.010),but increased RacGAPl expression(7d:F=13 8.118,P<0.001;28d:F=35.085,P<0.001).On the contrary,7 days and 28 days cocaine administration induced significantly increasing Tiaml expression(7d:F=65.944,P<0.001;28d:F=19.761,P<0.001),but downregulated RacGAP1 expression in the CPu(7d:F=22.861,P<0.001;28d:F=29.756,P<0.001).These data indicate that repeated cocaine exposure exerts its effect through Tiam1 and RacGAP1 to regulate Rac1 activity differentially in the NAc and CPu.Cofilin,a downstream target of Racl signaling,was recently reported to be activated,as evidenced by the decrease in the phosphorylated form of cofilin,after repeated cocaine treatment.We thus determined the expression pattern of phosphorylated cofilin in the NAc and CPu after repeated cocaine treatment.We found that in line with the opposite activation of Racl activity after repeated cocaine exposure in the NAc and CPu,the amount of phosphorylated cofilin was decreased in the NAc(7d:F=84.420,P<0.001;28d:F=56.437,P<0.001),but increased in the CPu(7d:F=5.191,P=0.007;28d:F=48.762,P<0.001),while the amount of total cofilin was not change after 7days or 28days cocaine treatment.These results suggest that Racl differentially regulates cofilin activation in the NAc and CPu following repeated cocaine treatment.2.Using two cocaine-injection protocols to induce dendrites and spines in the CPu,the mice were treated with 5 days regimenor 28 days regimen before CPu MSN dendrites and spine density were analyzed.For the dendrites analysis,the 5 days cocaine injections induced a significant increase in CPu dendrites compared to chronically saline-treated mice infected with the control EGFP lentivirus(F=73.109,P<0.001).Expression of dominant negative Racl(Rac1N17)significantly attenuated the cocaine-induced increase in dendrites of the MSNs(F=73.109,P<0.001).Expression of Rac1N17 did not affect basal CPu dendritic branching in saline-treated mice(t=0.392,P=0.703).The 28 days cocaine injections also induced a significant increase in CPu dendrites compared to chronically saline-treated mice infected with the control EGFP lentivirus(F=73.109,P<0.001).Expression of Rac1N17 significantly attenuated the cocaine-induced increase in dendrites of the MSNs(F=73.109,P<0.001).Expression of Rac1N17 did not affect basal CPu dendritic branching in saline-treated mice(t=0.652,P=0.529).The significant main effect(F=51.922,P<0.001)of day may have some association with mouse age,because 5 days and 28 days group mice have the same beginning age,but end with different age.Meanwhile,spine type analysis was carried out according to the published methods.The 5 days cocaine injections induced a significant increase in total spine density compared to saline-treated mice infected with the control EGFP lentivirus(F=54.848,P<0.001),and this increase mainly driven by an increase in thin spines(F=28.968,P<0.001),not stubby and mushroom spine.Expression of Rac1N17 significantly blocked the cocaine-induced increase in total spine density(F=54.848,P<0.001)and thin spine density(F=28.968,P<0.001)of the MSNs.The 28 days cocaine injections induced a significant increase in total spine density compared to saline-treated mice infected with the control EGFP lentivirus(F=54.848,P<0.001),and this increase driven by increases in all three type spine,including thin spine(F=28.968,P<0.001),stubby(F=8.793,P<0.001)and mushroom spine(F=20.395,P<0.001).Expression of Rac1N17 significantly blocked the cocaine-induced increase in total spine density,thin spine density,stubby spine density,and mushroom spine density of the MSNs(all P's<0.001).Meanwhile,we observed that RaclN17 itself reduced total spine density,and mushroom spine,but not thin and stubby spine.These findings demonstrate that the upregulation of Rac1 activity in the CPu contributes to repeated cocaine-induced dendritic remodeling.3.We bilaterally injected the Plenti-RaclN17 Lentivirus into the mouse Cpu,and used conditioned place preference and locomotor sensitization model.Lentivirus expressing GFP was used as a control.In the conditioned place preference test,Plenti-Rac1N17 expression in the CPu blocked the rewarding effects of cocaine(t=2.530,P=0.019).That is,mice expressing Plenti-Rac1N17 in their CPu spent significantly less time in a cocaine-paired environment compared to its control group.Conversely,in the locomotor sensitization test,mice expressing Plenti-EGFP and Plenti-Rac1N17 in the CPu exhibited similar increase in their locomotor activity after cocaine treatment(F=0.012,P=0.913).In addition,after one week of withdrawal of cocaine,the plenti-EGFP and Plenti-RaclN17-expressing mice remained similar sensitivity to a cocaine challenge dose.Pretreated with NSC23733,mice spent significantly less time in a cocaine-paired environment compared to its control group(t=2.291,P=0.044)and exhibited a decrease in their locomotor activity after cocaine treatment.These findings demonstrate that Racl is involved in cocaine-induced reward learning.Part two:1.Modified T29-1 line mice which use CamK? to start Cre gene were hybridized with LoxP-Cdc42-LoxP mice to build forebrain-specific Cdc42 knockout mice(KO).The genotyping results of PCR prove that the mice which we constructed containing both the Cre gene and the homozygous LoxP-Cdc42-LoxP genes,which indicated that we successfully constructed a forebrain-specific Cdc42 knockout mice.In addition,the hippocampus of mice were stained using Nissl staining in 3-month-old and 6-month-old forebrain-specific Cdc42 knockout mice,we found that KO mice have no significant morphological difference compared to wild-type(WT)mice with the same age.By using Cdc42 immunohistochemical staining to test the Cdc42 protein levels in the hippocampus of 3-month-old and 6-month-old forebrain-specific Cdc42 knockout mice,we found that,in 3-month-old KO mice,the gene knock-out area mainly concentrated in the CA1 region and CA3 area,while in 6-month-old forebrain-specific Cdc42 knockout mice,the knock-out region distribute to the entire hippocampus,including CA1,CA3 and DG region.By using Golgi-cox staining to reveal the neurons in hippocampus,we found that 3-month-old and 6-month-old forebrain-specific Cdc42 gene knock-out mice,compared to wild-type mice with the corresponding age,its dendritic spine density of hippocampal CA1 neurons decreased significantly(3m:t=9.848,P<0.001;6m:t=9.600,P<0.001).It indicated that Cdc42 knocked down in hippocampal CA1 region can reduce the basis level of the dendritic spines density in CA1 neurons.2.Wild-type mice were injected intraperitoneally with cocaine to construct a 28-day cocaine addiction animal model,the tissue was abstracted at 15min,2h,8h time point after the last injection of cocaine,Cdc42-GTPase activity was detected using GST-pull down technology in the hippocampus.The results showed that chronic cocaine activates the Cdc42-GTPase activity in the hippocampus,elevated at 15min,reached to the highest at 2h,and returned to the basic level at 8h(F=187.121,P<0.001),suggesting that Cdc42-GTPase activity may be involved in cocaine-induced neuronal plasticity in the hippocampus.3.We choose the 3-month-old and 6-month-old forebrain-specific Cdc42 knockout mice to produced 28-day cocaine addiction models,using Golgi-cox staining to reveal mouse the dendritic spines in CA1 subregion of hippocampus.In WT mice,cocaine can induce a significant increase in the dendritic spines density in CA1 subregion of hippocampus.In the 3-month-old and 6-month-old forebrain-specific Cdc42 knockout mice,Cdc42 gene knockout significantly inhibited the cocaine-induced increase in dendritic spine density(3m:=165.847,P<0.001;6m:F=120.434,P<0.001).The data hint that Cdc42 is involved in cocaine-induced dendritic remodeling in the CA1 regions of hippocampus.Part three:1.We analyzed the dendritic changes using biocytin staining in a separate cohort of mice from those used for Golgi-Cox.For dendritic spine density,in both male.and female mice,the biocytin staining MSNs emerged higher dendritic spine density than that in Golgi impregnation group(F=9.97,P<0.001).Meanwhile,the increasing spine density induced by cocaine treatment in female mice was higher than that in male mice(F=33.49,P<0.001),which indicated that there are sex differences on spine density in response to repeated cocaine treatment.For dendritic branching,in both male and female mice,the biocytin-filled MSNs showed higher numbers of dendrites than that in Golgi impregnation group(F=34.13,P<0.001),and the increasing number of dendrites induced by cocaine treatment was similar in female and male mice(F=0.23,P=0.635).Thus,biocytin staining appears to be useful to probe the structure and function of MSNs in the NAc.Next,we performed biocytin staining,dynorphin and D2 receptor colocalization analysis,and found that cocaine-induced dendritic changes occurred in both dynorphin-and D2 receptor-positive neurons.In order to further determine whether there are sex differences in the MSN subpopulations that display cocaine-induced dendritic changes,we evaluated these subpopulations after cocaine treatment in male versus female mice.We revealed that the subpopulations of MSNs in the NAc that display cocaine-induced dendritic changes were similar in male and female mice(?2=0.011,P=0.994).2.We treated mice with the D1 and D3 receptor antagonists prior to chronic cocaine administration.Animals were then analyzed 4,12,24,48,and 72 hr following the final injection,and the striatum was isolated for IHC analysis using an antibody that recognized phospho-Ser408 MEF2A.We found that cocaineexposure produced a time-dependent increase in MEF2 phosphorylation at Ser408,with a maximal effect observed 24 hr following the last cocaine injection,compared with saline-treated control mice and returned to baseline 72 hr the final cocaine injection(NAc:F=1351.608,P<0.001;CPu:F=922.497,P<0.001).Importantly,the degree of MEF2 phos phorylation at Ser408 was enhanced in NGB2904-pretreated miceand decreased in SCH23390-,MK801-and SL327-pretreated mice 24 hr following the final cocaine injection(NAc:F=1893.723,P<0.001;CPu:F=1088.314,P<0.001).Immunohistochemical analyses revealed that the levels of Ser408 phosphorylation in medium spiny neurons in the NAc and CPu were similar in SCH23390-,NGB2904-,MK801-and SL327-alone treated mice,compared to saline-treated mice(NAc:F=1.387,P=0.254;CPu:F=2.330,P=0.070).Using double-label immunohistochemistry,we found that phosphorylation of MEF2A at Ser408 occurs primarily in D1 receptor-expressing neurons in the striatum.Together,these findings suggest that the D1 and D3 receptors differentially regulate MEF2 phosphorylation following chronic cocaine treatment,likely through NMDA receptor activation and ERK signaling.Conclude:1.Racl signaling may play a critical role in mediating cocaine-induced neuroadaptation in CPu.2.We successfully constructed forebrain-specific Cdc42 gene knockout mice,and use it to explore that Cdc42 activity is involved in mediating cocaine induced dendritic spine density increases in the CA1 subregion in hippocampus.3.There are gender differences in cocaine-induced neuronal remodeling.Chronic cocaine-induced changes in dendritic parameters occurred in MSNs of the NAc belonging to both the direct(D1-expressing)and indirect(D2-expressing neurons)pathways.the D1 and D3 receptors oppositely regulate cocaine-induced structural remodeling of dendrites and spines,likely via the consecutive regulation of phosphorylation of NR1,ERK activation,and MEF2 activi ty in the NAc and CPu.In this study,We use a series of inactive mutant lentiviral,gene knockout technology,specific signaling pathway blockers to inhibit the Rac1 activity in CPu,block the expression Cdc42 in CA1 region of hippocampus,and block D1 and D3 signaling pathway activity,to study the role of Racl signaling pathway,Cdc42 activity and D1 and D3 signaling pathways in cocaine-induced neuronal dendritic remodeling and behavioral plasticity.The results show that,Racl signaling pathway in the CPu,Cdc42 activity in CA1 region of hippocampus,and D1 and D3 signaling pathways are involved in the cocaine-induced morphological plasticity,suggesting that multiple signaling pathways regulate cocaine-induced neuronal plasticity together.These data provide further insights into the signaling pathways that control the cocaine-induced structural and behavioral plasticity that has been implicated in the persistence of drug addiction,and provides a lot of inspiration for the clinical treatment of cocaine addiction.