Neurobiology of relaxin-3 systems: studies in peptide and receptor knockout mice

© 2014 Dr. Ihaia Toby HoskenThe relaxin-3 neuropeptide system in the brain forms an ascending circuit which promotes arousal-associated behaviours. Evidence contributing to this hypothesis includes anatomical mapping studies and rat behavioural assays that have tested the consequences of administering relaxin-3-like peptides. More recently, novel mouse models have become available to study the relaxin-3 system, whereby the genes encoding relaxin-3 or its cognate receptor, relaxin family peptide-3 receptor (RXFP3), have been deleted from the mouse genome. Examining how the behaviour of these relaxin-3 or Rxfp3 ‘knockout’ (KO) mice differs from wild-type (WT) controls, can provide insights into the endogenous role of relaxin-3/RXFP3 signalling. Using relaxin-3 and Rxfp3 KO mice, the studies described in this thesis aimed to investigate the involvement of endogenous relaxin-3/RXFP3 signalling in key behaviours. These studies had three main aims: (1) Assess the behavioural phenotype of the novel Rxfp3 KO mouse strain, and compare this with phenotypes previously reported in relaxin-3 KO mice; (2) Analyse and challenge the circadian activity of relaxin-3 KO mice using home-cage running wheels; (3) Measure and analyse the sleep architecture of relaxin-3 KO mice using electroencephalography (EEG) and electromyography (EMG) telemetry under basal conditions, after provision of a home-cage running wheel, and in a model of stress-induced insomnia. Firstly, Rxfp3 KO mice were subjected to a battery of behavioural paradigms, and their behaviour was compared to WT littermate controls. Rxfp3 KO mice did not display any overt differences to WT controls in general health, motor coordination, spatial or recognition memory, and sensorimotor gating. However, Rxfp3 KO mice were hypoactive on voluntary home-cage running wheels and displayed some subtle anxiolytic traits. Importantly, these phenotypes were similar to those reported in relaxin-3 KO mice, which provides further evidence that relaxin-3/RXFP3 form a tight cognate ligand/receptor pair, and that the observed common phenotypes are due to a lack of relaxin-3/RXFP3 signalling. Furthermore, these studies validate this Rxfp3 KO mouse strain, which can be used in future investigations. Secondly, in-depth analysis of the running wheel activity of relaxin-3 KO mice demonstrated fragmented running wheel use, consisting of shorter and more frequent bouts of activity compared to WT controls. Assessment of the circadian running wheel properties of relaxin-3 KO mice under constant dark conditions identified they had an endogenous circadian period similar to WT controls. However, compared to WT mice their circadian rhythms were less stable over time, they exhibited a larger phase delay in their activity onset following a light pulse under constant dark conditions, they re-entrained more rapidly to a new light schedule, and they displayed increased food anticipatory activity when food availability was restricted to the inactive/light phase. Furthermore, the masking effect of light on the running wheel activity of relaxin-3 KO mice was slightly yet significantly less than WT controls. These studies suggest relaxin-3 signalling is involved in promoting the strength of circadian rhythms, and in providing non-photic circadian timing cues. RXFP3 expression within the intergeniculate leaflet may be particularly important in mediating these effects, as in a pilot study, chronic viral-mediated secretion of an RXFP3 antagonist within this area largely recapitulated the running wheel hypoactivity phenotype observed in whole-of-body relaxin-3/Rxfp3 KO mice. Attempts to rescue the hypoactive running wheel phenotype of relaxin-3 KO mice by administering a viral vector into the cerebral ventricles that encodes and secretes an RXFP3 agonist were not successful, suggesting the phenotype could be due to developmental issues in these whole-of-life KO mice, or that the agonist peptide did not reach RXFP3 located in deep brain structures that may underlie the phenotype in sufficient concentrations. Finally, based on the hypothesis that relaxin-3 signalling promotes arousal, its contribution to sleep and wake states was assessed by recording EEG/EMG activity via telemetry. Compared to WTs, relaxin-3 KO mice did not demonstrate any differences in sleep architecture under basal conditions. However, relaxin-3 KO mice fell asleep earlier after the onset of the dark phase than WTs when mice were provided with running wheels. Further studies investigated the contribution of relaxin-3 signalling to primary insomnia, which is caused by hyper-arousal due to factors such as stress and results in decreased and poor quality (hyper-aroused) sleep. This involved developing a novel mouse model of primary insomnia, by inducing hyper-arousal by exposing mice to the ‘stimulation’ (stress/arousal) of a dirty cage change and comparing their response to that after a clean cage change. WT mice responded to the dirty cage change with increased wakefulness and disturbed sleep, consisting of increased cortical activity; both characteristics of primary insomnia in humans. Relaxin-3 KO mice demonstrated an increased anxiety-like response of increased wakefulness to the clean cage, but did not demonstrate increased cortical activity above control values following the dirty cage change. These studies suggest that endogenous relaxin-3 signalling both inhibits anxiety responses, and promotes cortical arousal. Collectively, the studies described in this thesis demonstrate that the relaxin-3 and Rxfp3 KO mouse lines can be successfully used to study relaxin-3 systems biology. Furthermore, these studies of KO mice support the hypothesis that relaxin-3/RXFP3 signaling is involved in behavioural arousal, as deficits in various aspects of arousal were observed in KO mice. These studies provide novel mechanistic insights into how relaxin-3 signalling may promote arousal, which identify promising avenues for future research. The contribution of the relaxin-3 system to arousal suggests it may be a promising therapeutic target in the treatment of mental disorders, in which dysfunctions in arousal and related motivational behaviours are commonplace