title: Silencing relaxin-3 in nucleus incertus of adult rodents: a viral vector-based approach to investigate neuropeptide function.
abstract: Relaxin-3, the most recently identified member of the relaxin peptide family, is produced by GABAergic projection neurons in the nucleus incertus (NI), in the pontine periventricular gray. Previous studies suggest relaxin-3 is a modulator of stress responses, metabolism, arousal and behavioural activation. Knockout mice and peptide infusions in vivo have significantly 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 infusion of rAAV1/2 EmGFP miR499 into the NI resulted in significant reductions in relaxin-3 expression as demonstrated by ablation of relaxin-3-like immunoreactivity at 3, 6 and 9 weeks and by qRT-PCR at 12 weeks. Neuronal health was unaffected 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 experiments confirmed no detrimental effects on general health or well-being and therefore several behavioural modalities previously associated with relaxin-3 function were investigated. The validation of this viral vector-based model provides a valuable alternative to existing in vivo approaches and promotes a shift towards more physiologically relevant investigations of endogenous neuropeptide function.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Melbourne, Victoria, Australia. Gabrielle.Callander@florey.edu.au
authors.forename: Gabrielle E
authors.initials: GE
authors.lastname: Callander
authors.forename: Sherie
authors.initials: S
authors.lastname: Ma
authors.forename: Despina E
authors.initials: DE
authors.lastname: Ganella
authors.forename: Verena C
authors.initials: VC
authors.lastname: Wimmer
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Gundlach
authors.forename: Walter G
authors.initials: WG
authors.lastname: Thomas
authors.forename: Ross A D
authors.initials: RA
authors.lastname: Bathgate
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics; D015870:Gene Expression; D005786:Gene Expression Regulation; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism*; D034622:RNA Interference; D051381:Rats; D012065:Relaxin / Q000235:genetics*; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D035683:MicroRNAs; D009479:Neuropeptides; D012065:Relaxin