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

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.