Microinjection Of CART (Cocaine-and Amphetamine-regulated Transcript) Peptide Into The Nucleus Accumbens Inhibits The Cocaine-induced Upregulation Of Behavior Sensitivity

Objcetive:Repeated exposure to addictive drugs enhances dopamine receptor(DR)signaling and the ultimate phosphorylation of the cyclic adenosine5′monophosphate(c AMP)-response element-binding protein(CREB)expression in the nucleus accumbens(NAcc).These effects are known to contribute to the expression of behavioral sensitization.The CART peptide is a key regulatory factor of addictive drug reward factor.Research reports confirm CART peptide in the nucleus accumbens(NAcc)can not change activity in rats,and inhibited rewarding effects of addictive drugs.In this study,a series of experiments inject the CART peptide into the nucleus accumbens region by stereotaxic technology,and observed(1)the phosphorylation of CREB,(2)dopamine receptor signaling activity and(3)extracellular signal-regulated kinase(ERK)phosphorylated kinase signaling.Methods:Adult male healthy SD(Sprague Dawley)mice(260-280g)were randomly divided into:(1)Saline group: Saline(intra-NAc)+Saline(i.p.);(2)Cocaine group:Saline(intra-NAc)+Cocaine(15mg/kg,i.p.);(3)CART(1.0μg,intra-NAc)+Cocaine(15mg/kg,i.p.);(4)CART(2.5μg,intra-NAc)+Cocaine(15mg/kg,i.p.);A bilateral stainless steel guide cannula assembly was implanted above the nucleus accumbens using a motorized stereotaxic stereodrive.Target coordinates relative to bregma were A/P + 1.7,L/M ± 1.6,and D/V – 7.5.Detection indicators are as follows:(1)Measurement of locomotor activity;(2)Nissl staining to determine the injection site as the location of the nucleus accumbens;(3)Western Blot detect the level of D1 R,D2R;(4)To detect the phosphorylation level of D3 R in the nucleus accumbens by Co-immunoprecipitation;(5)Western Blot detect the level of ERK、P-ERK、CREB、P-CREB;Results:1、Measurement of locomotor activity showed that rats microinjected into the NAcc of CART 55–102 peptide inhibits the expression of locomotor and sensitizationeffects by cocaine.Groups indicate microinjection of either saline or CART 55–102(1.0 μg or 2.5μg)followed by either saline or i.p.injection of cocaine.Day-course data are shown as the group mean(+SEM)locomotor activity counts obtained during the 1 h test period following the treatment for 7 days.Rats microinjected with saline showed a significantly greater locomotor response to cocaine compared to saline groups(***p < 0.001)for 7 days and behavioral sensitization responses to cocaine for6 days compared to the first day(+++p < 0.001).The sensitized response was absent when CART 55–102 was microinjected followed by cocaine injection(##p < 0.01 and###p < 0.001).2、The Western blot analysis revealed that the 5-day-injection of cocaine(15mg/kg)increased the protein levels of the D1Rs(n =4,p < 0.001)and D2Rs(n =4,p< 0.01,)in the NAcc on the 5th day when compared with the control(saline,n = 4)group.However,cocaine induced-significant increases in the protein level of the D1 Rs and D2 Rs was inhibited by the repeated-microinjection into the NAcc of CART55–102(1.0 or 2.5μg/side)on the 5th day(n =4,p < 0.01 and p < 0.05).3、A series of coimmunoprecipitation and Western blot analysis experiments were carried out to measure the D3 R phosphorylation in the NAcc.Cocaine was found to increase serine phosphorylation,as detected by an antibody selective for phosphoserine(n =4,p > 0.001)in basal D3 R precipitates.This increase did not occurred after CART 55–102(1.0 or 2.5 μg/side)microinjection into the NAcc of on the 5th day(n =4,p < 0.01),indicating that the CART peptide promotes the dephosphorylation of D3 Rs.4、Western blot analysis revealed that the repeated injection of cocaine following the microinjection of saline increased the levels of CREB and ERK phosphorylation in the NAcc to approximately 1.3 and 2.0 times those obtained by saline treatment.However,the injection of CART 55–102(1.0 or 2.5 μg/side)completely blocked the CREB and ERK phosphorylation effects of cocaine(n =4,p <0.01 or p < 0.001,)in this site.Moreover,the levels of CREB and ERK in the NAcc showed no significant changes after administration of cocaine with CART 55–102peptides,and it appeared almost the same as control levels(n =4).Conclusion:Microinjection of CART(cocaine-and amphetamine-regulated transcript)peptide into the nucleus accumbens in a dose-dependent inhibits the cocaine-induced upregulation of behavior sensitivity,its mechanism is related to the cocaine-induced blocked rat brain dopamine signaling pathway(DR-ERK/CREB).