Silencing Relaxin-3 in Nucleus Incertus of Adult Rodents A Viral Vector-based Approach to Investigate Neuropeptide Function 英文参考文献.docVIP

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Silencing Relaxin-3 in Nucleus Incertus of Adult Rodents A Viral Vector-based Approach to Investigate Neuropeptide Function 英文参考文献.doc

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Silencing Relaxin-3 in Nucleus Incertus of Adult Rodents A Viral Vector-based Approach to Investigate Neuropeptide Function 英文参考文献

SilencingRelaxin-3inNucleusIncertusofAdultRodents: AViralVector-basedApproachtoInvestigate NeuropeptideFunction GabrielleE.Callander1,2*,SherieMa2,3,DespinaE.Ganella1,2,VerenaC.Wimmer2,AndrewL.Gundlach2,4, WalterG.Thomas5,RossA.D.Bathgate1,2* 1Department of Biochemistry and Molecular Biology, The University of Melbourne, Victoria, Australia, 2Florey Neuroscience Institutes, The University of Melbourne, Victoria,Australia,3DepartmentofMedicine(AustinHealth),TheUniversityofMelbourne,Victoria,Australia,4DepartmentofAnatomyandNeuroscience,TheUniversity ofMelbourne,Victoria,Australia,5SchoolofBiomedicalSciences,UniversityofQueensland,Queensland,Australia Abstract Relaxin-3,themostrecentlyidentifiedmemberoftherelaxinpeptidefamily,isproducedbyGABAergicprojectionneurons inthenucleusincertus(NI),inthepontineperiventriculargray.Previousstudiessuggestrelaxin-3isamodulatorofstress responses,metabolism,arousalandbehaviouralactivation.Knockoutmiceandpeptideinfusionsinvivohavesignificantly contributed to understanding the function of this conserved neuropeptide. Yet, a definitive role remains elusive due to discrepancies between models and a propensity to investigate pharmacological effects over endogenous function. To investigate the endogenous function of relaxin-3, we generated a recombinant adeno-associated viral (rAAV) vector expressing microRNA against relaxin-3 and validated its use to knock down relaxin-3 in adult rats. Bilateral stereotaxic infusionofrAAV1/2EmGFPmiR499intotheNIresultedinsignificantreductionsinrelaxin-3expressionasdemonstratedby ablationofrelaxin-3-likeimmunoreactivityat3,6and9weeksandbyqRT-PCRat12weeks.Neuronalhealthwasunaffected as transduced neurons in all groups retained expression of NeuN and stained for Nissl bodies. Importantly, qRT-PCR confirmed that relaxin-3 receptor expression levels were not altered to compensate for reduced relaxin-3. Behavioural experimentsconfirmednodetrimentaleffectsongeneralhealthorwell-beingandthereforeseveralbehaviouralm