Relaxin‐3 innervation of the intergeniculate leaflet of the rat thalamus – neuronal tract‐tracing and in vitro electrophysiological studies

Abstract

Behavioural state is controlled by a range of neural systems that are sensitive to internal and external stimuli. The relaxin-3 and relaxin family peptide receptor 3 (RXFP3) system has emerged as a putative ascending arousal network with putative involvement in regulation of stress responses, neuroendocrine control, feeding and metabolism, circadian activity and cognition. Relaxin-3/γ-aminobutyric acid neuron populations have been identified in the nucleus incertus, pontine raphe nucleus, periaqueductal grey (PAG) and an area dorsal to the substantia nigra. Relaxin-3-positive fibres/terminals densely innervate arousal-related structures in the brainstem, hypothalamus and limbic forebrain, but the functional significance of the heterogeneous relaxin-3 neuron distribution and its inputs to specific brain areas are unclear. Therefore, in this study, we used neuronal tract-tracing and immunofluorescence staining to explore the source of the dense relaxin-3 innervation of the intergeniculate leaflet (IGL) of the thalamus, a component of the neural circadian timing system. Confocal microscopy analysis revealed that relaxin-3-positive neurons retrogradely labelled from the IGL were predominantly present in the PAG and these neurons expressed corticotropin-releasing factor receptor-like immunoreactivity. Subsequently, whole-cell patch-clamp recordings revealed heterogeneous effects of RXFP3 activation in the IGL by the RXFP3 agonist, relaxin-3 B-chain/insulin-like peptide-5 A-chain (R3/I5). Identified, neuropeptide Y-positive IGL neurons, known to influence suprachiasmatic nucleus activity, were excited by R3/I5, whereas neurons of unidentified neurotransmitter content were either depolarized or displayed a decrease in action potential firing and/or membrane potential hyperpolarization. Our data identify a PAG to IGL relaxin-3/RXFP3 pathway that might convey stress-related information to key elements of the circadian system and influence behavioural state rhythmicity.

Central relaxin-3 receptor (RXFP3) activation reduces elevated, but not basal, anxiety-like behaviour in C57BL/6J mice

“…Relaxin-3 is primarily expressed in GABA neurons, as reflected by the co-expression of relaxin-3 and glutamate decarboxylase immunoreactivity in the rat nucleus incertus [18], which suggests RXFP3 activation may act to reinforce inhibitory signalling on target neurons. In line with such a role, in vitro studies have revealed activation of the G i/oprotein-coupled RXFP3 by relaxin-3 and truncated analogues produces a dose-dependent inhibition of cellular cAMP levels [14,53], and selective RXFP3 agonist peptides directly hyperpolarize neurons recorded in brain slices [54].…”

Sensitivity to Chronic Methamphetamine Administration and Withdrawal in Mice with Relaxin-3/RXFP3 Deficiency

“…Additionally, although evidence is accumulating that RLN3/RXFP3 signalling is able to modulate the same downstream brain areas as monoamine systems, such as the septum [46,47] and extended amygdala [23], it is not known whether RLN3/RXFP3 signalling targets the same or different/neighbouring neurons and circuits to those directly innervated by 5-HT, NA and DA networks. In this regard, the direct effects of RXFP3 activation on individual and populations of brain neurons is largely unknown (but see Blasiak et al [48]). Hence, determining the neurochemical phenotype of RXFP3-positive neurons will be an important advance, but a fully characterised, specific RXFP3 antibody is currently unavailable, preventing traditional double-label immunohistochemical approaches to neurochemically 'phenotype' RXFP3-positive neurons.…”

International Union of Basic and Clinical Pharmacology. XCV. Recent Advances in the Understanding of the Pharmacology and Biological Roles of Relaxin Family Peptide Receptors 1–4, the Receptors for Relaxin Family Peptides

“…However, the RXFP3 agonist R3/I5 shows diverse effects in cells of the intergeniculate leaflet, with both depolarization and hyperpolarization recorded in different cells from the same preparation (Blasiak et al, 2013). Thus the phenotype of the relaxin-3 recipient neuron and its connections could be crucial to the mode of RXFP3 signaling.…”