Role of the relaxin-3/RXFP3 system in the mouse: focus on septohippocampal function and memory

© 2017 Dr. Mouna HaidarAnatomical and functional studies have suggested that the highly conserved neuropeptide relaxin-3 plays an important role in a diverse range of functions, including behavioural arousal, stress responses and cognitive processes. Within rodent brain, relaxin-3 is primarily expressed by γ-aminobutyric acid (GABA) neurons in the pontine nucleus incertus (NI) that project to a number of forebrain areas containing its cognate G-protein-coupled receptor, relaxin-family peptide 3 receptor, RXFP3. Importantly, the relaxin-3/RXFP3 system performs synergistic and complementary roles to monoamine signalling (i.e. noradrenaline (NA)/locus coeruleus (LC) and serotonin (5-HT)/dorsal raphe (DR) systems) within shared downstream target regions. It has previously been established that relaxin-3-containing nerve fibres and RXFP3 mRNA and binding sites are strongly expressed in the main nodes of the septohippocampal system (SHS), including the medial septum/diagonal band of Broca (MS/DB) and hippocampal formation. Indeed, anatomical studies in the rat have demonstrated that relaxin-3-positive nerve fibres terminate on MS/DB GABAergic and cholinergic neurons that project to hippocampus, and functional studies have demonstrated that the relaxin-3/ RXFP3 system within the MS/DB modulates hippocampal theta rhythm and spatial working memory. However, the precise neurochemical and physiological mechanism(s) by which the relaxin-3/RXFP3 system modulates hippocampal activity are largely unknown and there are no studies of this system in mice. Therefore, to address this gap in knowledge, the studies described in this thesis investigated the role of the relaxin-3/RXFP3 system in the mouse in three key areas: (1) hippocampal function and memory; (2) progression of neurodegenerative disease; and (3) affective states and stress responses. Initial anatomical studies revealed that relaxin-3-positive nerve fibres/boutons make close contacts with multiple GABA neuron populations in the hippocampus, including a large population of somatostatin (SST)-positive neurons in the dentate gyrus hilus (DG hilus), and a smaller population of neurons expressing the calcium-binding proteins, parvalbumin (PV) and calretinin (CR). In subsequent functional studies, a ‘floxed’ RXFP3 mouse strain was validated and Cre-recombinase-mediated depletion of RXFP3 from the DG hilus in adult mice produced impairments in spatial reference and working memory. Together, these findings suggest endogenous relaxin-3/RXFP3 signalling is important for normal cognitive function via modulation of inhibitory GABA networks, including effects on the SST neurons that regulate the activity of principal cells and hippocampal oscillatory activity. Determining the role that relaxin-3/RXFP3 signalling plays in hippocampal function and cognition, and any synergistic actions with ascending monoamine systems, is important in relation to potential causative and/or therapeutic roles that relaxin-3/RXFP3 signalling play in disease. Dysfunction of ascending monoamine systems is a feature of several neurological diseases, including tauopathies and related neurodegenerative dementias, but it is not known if damage to the NI relaxin-3 system might contribute to cognitive decline in a similar way to the disruption of brainstem monoamine transmission in the hippocampus and frontal cortex. Therefore, using quantitative histochemical methods, the number and viability of relaxin-3 neurons in the NI was assessed in a cohort of transgenic tau-P301L mice and age- and strain-matched controls. The tau-P301L mouse is a model of human tauopathy and carries the P301L mutation (tau-4R/2N-P301L) in the tau protein, which is critical for binding to microtubules to aid their stabilization, essential for axonal transport, whereas under pathological conditions, phosphorylated tau results in aggregates that are toxic to neurons. A reduced number of relaxin-3-immunoreactive neurons was detected in the NI of 7 – 8 month old tau-P301L mice, relative to age matched controls, suggesting that reduced activity of the relaxin-3/RXFP3 system may contribute to cognitive decline in tauopathies and related disorders. This initial study of relaxin-3 neurons in a preclinical model of neurodegenerative disease indicates that further research in this and other models is warranted, including correlative studies of relaxin-3 mRNA levels and measures of relaxin-3 fibre and RXFP3 densities in key nodes of the septohippocampal system. In a series of functional studies, the relationship between the relaxin-3/RXFP3 system and monoamine signalling in controlling stress responses and affective states was explored by examining the impact of monoamine deficiency on the behavioural profile of normal C57BL/6J mice and mice with a constitutive whole-of-life deletion of the relaxin-3 or RXFP3 genes. Monoamine deficiency was achieved by withdrawal from chronic methamphetamine treatment. It was hypothesized that endogenous relaxin-3/RXFP3 signalling may compensate for the temporary reduction in monoamine signalling and reduce the depressive- and anxiety-like behaviours associated with methamphetamine withdrawal. Depressive- and anxiety-like behaviours were assessed in relaxin-3 and RXFP3 knockout (KO) mice and their wildtype (WT) littermates following withdrawal from escalating doses of methamphetamine. All groups of mice (relaxin-3 and RXFP3 KO, and WT) displayed similar sensitivity to chronic methamphetamine withdrawal in measures of body weight change, behavioural despair and anxiety-like behaviours. These data indicate that a global deficiency in endogenous brain relaxin-3/RXFP3 signalling does not exacerbate depressive- and affective-like behaviours observed during chronic methamphetamine withdrawal. In summary, these studies have demonstrated that relaxin-3/RXFP3 signalling can modulate hippocampal-dependent spatial reference and working memory most likely via modulation of key GABAergic neuron populations in the hippocampus. Furthermore, evidence was obtained that relaxin-3 neurons are dysregulated in a mouse model of tauopathy, further highlighting the importance of this system for healthy cognition. The identification of the contribution of the relaxin-3/RXFP3 system to hippocampal function and cognition encourages further research to identify the potential therapeutic value of RXFP3 targeted drugs to enhance cognitive functioning and improved mental health in a range of neurodegenerative and psychiatric diseases