Activin A Attenuates Cocaine Withdrawal-induced Anxiety And Depression Of Mice Through Increasing Neurogenesis

Drug abuse threats the physical and psychological health of human and secuirity of society.Drug addiction is the feeling of dependence on drug physically and psychologically so that it will urge addict to obtain drug without thinking about harmful consequences. Once an addict ceases taking drugs, he will generally appear a series of withdrawal symptoms such as anxiety and depression. The degree of affective disorder reflects drug abstinence and craving, which also force the addicts on obtaining drug with extreme means, or harming themselves, even commit suicide. So the investigation of affective disorder after drug abstinence is very important.Activin A is an important member of transforming growth factor β superfamily,it has been reported that ActivinA plays important role in many biological events, not noly improve the synthesis and secretion of Follicle-Stimulating Hormone (FSH), but also participate in cell differentiation, proliferation, metabolism, immune response, tissue repair, neuroprotection and endocrine. Interestingly, some recent investigation found that Activin A could inhibit the anxiety and depression behavior in animal model (chronic electroconvulsive seizures rat and Knock mice) and the relative mechnism maybe associate with the neurogenesis of Activin A in dentate gyrus of hippocampus.The neuroplasticity in hippocampus is associated with the anxiety and depression. Hippocampus is a central nevrous system of learning and memory, participating in many emotional changes like anxiety and depression.Meanwhile,the development of anxiety and depression is accompanied with the dendritic remolding of neuron.It has been demonstated that the nerve cycle and connection is a lifelong procedure which including recombination and rearrangement. Neuron can make some adaptive response to various stress,and make some morphological changes which are called neuroplasticity. More specifically,neuron transfer and process messages by synaptic connection, and synapse make biological response to these stimulation, which is so called plasticity. It is very important for the body to keep nomal function in a changeful environment. Meanwhile, it make the brain get the self-repair like skin and liver tissue. However, neuroplasticity is not beneficial under any circumstance. The abnormal plasticity may lead to a harmful response to the stimulation. For example,harmful or long-time stimulation will make hippocampus dendrite atrophic and lose excitatory synapses,consequently which cause affective disorder.Neurogenesis is proliferation,migration,differentiation and integration of neural stem cells and neural precursor cells,which is one form of neuroplasticity. Neurogenesis is consider that it can relieve anxiety and depression.Many anxiolytic and antidepressive drug can enhance neurogenesis of the dentate gyrus which can blocking harmful neuroplasticity and relieve anxiety and depression.MAPK(Mitogen-activated protein kinase) pathway is important signal pathway of eukaryon,transmit signal from outside to inside of cell. MAPK regulate cell proliferation,differentiation,migration,survival,apoptosis,stress response and inflammation as co-component in signal transduction. ERK,JNK,P38 are important member of MAPK pathway, ERK1/2 signal transduction pathway regulate cell proliferation,growth,migration,survival.It has been reported that cocaine withdrawal can induce anxiety and depression 24 hours to 7days after cocaine withdrawal. There have been reported that ActivinA could relieve the symptom of anxiety and depression in animal model, and the mechanism is Activin A could increase the neurogenesis in hippocamous. But the further mechanism is still unclear, and it is also unknown about the function of ActivinA in modulate emotional changes, and also the mechanism of enhancement of neurogenesis. Therefore, our experiment will do the following study:1. Whether Activin A could decrease the anxiety and depression induced by the cocaine withdrawal?2.Do ActivinA increase the neurogenesis in hippocampus thus regulate the emotional changes?3.Do these biological effect have any relationship wih MAPK signaling pathway?In our experiment,the Kunming mice were divided into four groups:① Saline+PBS;②Saline+ActivinA;③Cocaine+PBS;④Cocaine+Activin A.The group③ and④ were accepted a consecutive 14 days’i.p. injection of Cocaine, 20mg/kg,whereas the group① and②injected the same amount Saline. In the 8th day, sterotaxic technology were adapt to palte two infusion cannulaes in bilateral hippocampus,the coordinate is:bregma-2.0mm,midline±1.5mm,sub-dura 1.8mm.We conduct consecutive injection for 14 day and withdrawal after 48h.Behavioral test:①Saline+PBS 9;②Saline+ActivinA 9;③Cocaine+PBS 10;④Cocaine+Activin A 10; withdrawal for 48h, group①③ is injected with lul PBS in bilateral hippocampus while group②④ is injected with Activin A (50ug/ml,1ul). Open field test observe center-distance, center-entries, center-time. Elevate plus maze observe open arm-entries,open arm-time. Tail suspension test and Forced swimming test observe immobility-time.For the immunohistochemisty and immunofluorescence histochemistry, the mice were divided into 4 groups:①Saline+PBS;②Saline+ActivinA;③Cocaine +PBS; ④Cocaine+Activin A, five animals each group.Withdrawal for 48h, group① ③is injected with lul PBS in bilateral hippocampus while group②④ is injected with Activin A(50ug/ml,lul).Once for one day for consecutive two days.At the same time,all mice are injected with BrdU(200mg/kg,per 12h,i.p) for consecutive three days.For the immunohistochemisty and immunofluorescence histochemistry, the mice were divided into 4 groups:①Saline+PBS;②Saline+ActivinA;③Cocaine+PBS; ④Cocaine+Activin A, five animals each group.For western blot, the mice were divided into 4 groups: ①Saline+PBS;② Saline+ActivinA; ③ Cocaine+PBS; ④ Cocaine+Activin A, four animals each group.Withdrawal for 48h, group ①③ is injected with lul PBS in unilateral hippocampus while group②③ is injected with Activin A(50ug/ml,lul).30min after injection,mice are dissected,and get hippocampus tissue.Western blot observe the phospho-ERK.Results:1.Open field test:As the center-time, compared with group Saline+PBS, the group Cocaine+PBS make a significant difference(P<0.05),and group Cocaine+Activin A is longer than group Cocaine+PBS(P<0.05). As the center-distance and center-entries, group Cocaine+PBS has no significant difference with Saline+PBS(p>0.05), and group Cocaine+Activin A has no significant difference with Cocaine+PBS (p>0.05)2. Elevate plus maze:the open arm-entries, group Cocaine+PBS is less than group Saline+PBS,and group Cocaine+Activin A is more than group Cocaine+PBS (P<0.05).The open arm-time in group Cocaine+PBS is shorter than group Saline+PBS,and group Cocaine+Activin A is longer than group Cocaine+PBS(P<0.05).3. Tail suspension test:As immobility-time, group Cocaine+PBS is longer than Saline+PBS,and group Cocaine+Activin A is longer than group Saline+PBS(P<0.05).There is no significant difference between group Saline+Activin A and group Saline+PBS(P>0.05)4. Force swimming test:As immobility-time,group Cocaine+PBS is longer than Saline+PBS,and group Cocaine+Activin A is longer than group Saline+PBS(P<0.05).There is no significant difference between group Saline+Activin A and group Saline+PBS(P>0.05)5.BrdU labeled immunohistochemistry:the number of BrdU+ cell in group Cocaine+PBS is more than Saline+PBS,and group Cocaine+Activin A is more than group Saline+PBS(P<0.05).There is no significant difference between group Saline+Activin A and group Saline+PBS(P>0.05)6. BrdU and Dcx double- labeled immunofluorescence:the number of BrdU+/Dcx+ cell in group Cocaine+PBS is more than Saline+PBS,and group Cocaine+Activin A is more than group Saline+PBS(P<0.05).There is no significant difference between group Saline+Activin A and group Saline+PBS(P>0.05)7. Western blot:after 30min of the stereotaxic surgey of injection of ActivinA and PBS,phospho-ERK in group Cocaine+Activin A is higher than group Saline+PBS(P<0.05).Group Saline+PBS VS group Saline+PBS, Group Cocaine (?) +PBS VS Group Saline+PBS,There is no significant difference (P>0.05)Conclusions:1.Actinvin A could decrease the emotional effects induced by Cocaine withdrawl,especially the anxiety and depression;2. Cocaine withdrawal could inhibit the neurogenesis of DG, while ActivinA could reverse it and increase the neurogenesis;3. ActivinA can increase the neurogenesis in DG after cocaine withdrawal by activating the phosphorylation of ERK.