Effects Of Urocortin3 On The Anxiety-like Behavior Of Rats And Serotonergic Neurons In Dorsal Raphe Nucleus And Hippocampal CA3

PrefaceWith animal anxiety models developing, the studies become more in mechanism about animal anxiety-like behavior and stress behavior. Researches about CRF peptide family in anxiety models were keeping profound. CRF peptide family includes CRF, urocortin 1 (Ucn 1), urocortin 2(Ucn 2), urocortin 3(Ucn 3). UCN3 is a new member of CRF peptide family, and binds with high affinity to CRF2 receptor. There were not concordant conclusions about the effects and mechanism of UCN3 on stress-like behavior. Elevated plus maze is a classical animal anxiety model, and we used it to invest the effects of UCN3 on anxiety-like behavior. Some studies showed that CRF peptide family not only bind with self receptor, but also impacted with serotonergic system. The aim of this research was to study the effects of UCN3 on stress behavior and serotonergic neurons in both dorsal raphe nucleus and hippocampal CA3. The result would provide theory about the effects of UCN3, CRF2 receptor on stress behavior and exploitation of new anti-anxiety drugs.Materials1,Experimental animalMale Wister rats (n=36) weighting about 190-210g were purchased from the center of experimental animals in China Medical University.2,Experimental reagents UCN3 (Sigma Co. LTD), Rabbit anti-5-HT and SABC kit (Boster Biotechnology Co. LTD)3,Experimental instrumentsElevated plus maze, Stereotaxic frame (Japan Narishigi), Meta Morph/Cool Snap_fx /Ax70 image analysis systemMethods1,SurgeryRats were anesthetized with hydral, mounted in a stereotaxic frame and unilaterally implanted with an indwelling cannula. Rats were allowed 1 week to recover prior to testing.2,Animal groupingRats were divided into 3 groups, UCN3 group (n=12), control group (n=12),blank control group(n=12). 10ug UCN3 was intracerebroventricularly (i.c.v) microinjected to UCN3 group. 5ul saline was microinjected i.c.v. to control group and blank control group.3,Examine behaviorRats were microinjected i.c.v. 10 minutes prior to testing and were placed onto the center of elevated plus maze.4,MeasureAfter 2 hours when UCN3 group and control group were tested behavior, all rats were anesthetized and perfused by paraformaldhyde. Middle brains were removed and made into frozen slices. Immunohistochemistry method was used to measure the expression of 5-HT in DRN and hippocampal CA3.Results1,Anxiety-like behavior in EPM Rats centrally injected with UCN3, displayed a significantly increased time spent in the open arms(t=2.45, P<0.05), higher percentage of time in the open time(t=2.75, P＜0.05), higher open arm entries(t=0.78, P<0.05),compared to vehicle-treated rats.2,The effects of UCN3 on 5-HT expression in DRN and hippocampalCA3Compared with control grpup, OD scores and positive cell of DRN 5-HT in UCN3 group significantly increased (t=2.29,P<0.05),(t=2.23,P<0.05), and OD scores and positive cell of hippocampal CA3 of UCN3 group significantly increased(t=4.46, P<0.01),(t=16.97,P<0.01).3,The effect of EPM on 5-HT expression in DRN and hippocampalCA3Compared with blank control group, OD scores and positive cell of DRN 5-HT in control group decreased unsignificantly(P>0.05).DiscussionThe principal of EPM is rodents are not only curious of new environment, but also afraid of high and open place. Open arm preference in EPM, as measured by preferential time and entries directed at the open arms, has been proposed to relate inversely to anxiety. After microinjected i.c.v. 10ug UCN3 10 minuets, the preferential time and entries of open arm are more than control group. This indicated an anxiolytic-like function for UCN3.Serotonergic neurons of the brainstem dorsal raphe nucleus are known to be important mediators of the endocrine and behavioral responses to exposure to stressors. Amydala and hippocampus is one of projections regions of 5-HT neurons. 5-HT is a significant neurotransmitter in mental disorders. The present experiment discover UCN3 increased 5-HT expression in DRN, which stimulated 5-HT neurons in DRN,and induced 5-HT expression increased in hippocampal CA3,suggesting that UCN3 increases 5-HT projection to hippocampus from DRN.The present experiment further supports that CRF2 receptor which UCN3 specially binds lead to anxiolytic-like effects in regulation of behavioral response to stress. It would provide a new way to study and cure anxiety disorders and stress related disorder.Conclusion1,After UCN3 was centrally injected , rats showed a significant increase in the time spent in the open arms, higher percentage of time in the open time and higher open arm entries. It demonstrated UCN3 produces anxiolytic-like effects.2,Lev injections of UCN3 significantly increased 5-HT expression in DRN and hippocampal CA3, suggesting that UCN3 increases 5-HT projection to hippocampus from DRN.3,Increased expression of 5-HT in DRN and hippocampal CA3 , would strengthen anxiolytic-like effects of UCN34,The present experiments demonstrated anxiolytic-like effects of UCN3 and CRF2r. It would provide a new way to study and cure anxiety disorders and stress related disorder.