Peptidergic control of pain and emotions: relaxin-3/RXFP3 modulation of descending pathways – RELAX

The project will implement behavioral, electrophysiological, and functional optogenetic circuit tracing approaches. It will use mouse models of pain, and transgenic mice to express regulators of neuronal activity in relaxin-3 neurons.

1. Aim 1. The project started with a verification of preliminary results, including pharmacological effects of an intracerebral injection of relaxin-3 analogues. In particular, we performed dose-response curves of the effects of A2 and A5 relaxin-3 analogs on pain behavior in the lateral ventricle. We showed that their effects were suppressed by antagonist injection.
We then extended the initial objectives of the project by also demonstrating behavioral effects obtained after injection in the basolateral amygdala (BLA). We validated the relaxin-3-Cre mouse.
2. Aim 2. Patch-clamp experiments were started in the BLA. Initial tests had shown no effect due to the lack of efficacy of the A5 agonist in patch-clamp, and the lack of labeling of somatostatin neurons in the GAD-GFP mice used. These problems have been corrected and the initial results show a clear excitatory effect of the A2 agonist on BLA neurons.
3. Aim 4. We developed the conditioned place preference test. We have shown that injection of the relaxin-3 analog A5 induces place preference for the chamber associated with this analog. We thus show that the activation of the relaxin-3/RXFP3 system has an effect not only on pain reflexes, but also on pain perception by the animal.
We studied the effects of RXFP3 agonists on depressive behavior associated with persistent pain. In this context, we used the «cuff« model of neuropathic pain. The depressive-like phenotype was assessed using the splash test and the forced swim test (FST). Although the effects are still not statistically significant, we observed that the application of A5 and A2 in painful animals decreased depressive symptoms.

The work will continue by characterizing the effects of endogenous relaxin-3 on pain behavior. We will also characterize the electrophysiological effects according to the nature of the neurons studied in the BLA, interneurons or projection neurons. We will start the study of the descending pathways between the ACC and the spinal cord. We will continue to study the effects of relaxin-3 on depressive symptoms and we will test these effects under conditions of increased pain.

Abboud C, Duveau A, Bouali-Benazzouz R, Massé K, Mattar J, Fssat P, Boué-Grabot E, Hleihel W, Landry M. (2021) Animal models of pain: diversity and benefits. J Neurosci Methods, 15;348:108997. doi: 10.1016/j.jneumeth.2020.108997.

Chronic pain is a global health problem with a huge economic burden. Pain and anxiety/depression are the most common symptoms in primary care and they are often associated. Despite their prevalence and broad impact, chronic pain disorders and their major comorbid symptoms remain poorly managed clinically.
The RELAX project focuses on modulation of chronic pain and comorbid anxiety/depression by the relaxin-3/RXFP3 peptidergic system. It proposes a multi-scale study of pain, comprising sensory and comorbid affective aspects, with complementary expertise in Bordeaux, Strasbourg and Melbourne, to study relevant complex neural microcircuits and larger networks in mouse preclinical models of persistent pain.
Relaxin-3/RXFP3 signalling is known to regulate affective processes in cortical and subcortical brain areas; and our pilot data demonstrated a central analgesic effect of a relaxin-3 agonist in rats and mice, which inhibits the activity of the anterior cingulate cortex (ACC) and deep dorsal horn neurons. Furthermore, this effect appears specific to mechanical and not thermal sensitivity. Our hypothesis is that relaxin-3 modulates neural circuits in the ACC that control spinal cord through specific descending inputs.
In Aim 1 studies, we will further document the effects of intra-ACC injection of relaxin-3 agonist or antagonist. We will also evaluate the effects of a novel agonist single-chain with a high translational potential. We will then investigate the role of endogenous release of relaxin-3 from fibres originating in the nucleus incertus, the major locus of relaxin-3 neurons. The purpose of Aim 2 studies will be to characterize the modulation of local ACC microcircuits by relaxin-3/RXFP3 signalling in mice with inflammatory pain. Aim 3 studies will identify the descending pathways that are modulated by relaxin-3 in the ACC, and characterize the spinal mechanisms involved. As parvalbumin spinal interneurons are modality-specific filters that gate mechanical, but not thermal, hypersensitivity, particular attention will be paid to their possible involvement in spinal circuits activated by relaxin-3-dependent descending pathways. Finally, Aim 4 studies will investigate the dual effect of relaxin-3 on the sensory and affective components of pain and the interactions between pain and comorbid anxiety/depression in a mouse model with inflammatory pain or pharmacologically-elevated anxiety.
Our technical approach will be to combine pharmacological approaches that switch relaxin-3/RXFP3 signalling on and off with behavioural assessment of pain. We will document the plasticity of local ACC and spinal cord microcircuits with cutting-edge calcium imaging and electrophysiological methods. We will apply viral-based functional tracing to identify and regulate descending-pain modulatory pathways. We will also perform behavioural studies to determine the mutual influence of anxiety/depression and chronic pain - both the sensory and affective components.
By alleviating both pain and elevated anxiety or depression, we predict relaxin-3/RXFP3 system-based treatments will offer a novel strategy to prevent mutual worsening of pain and anxiety/depression in chronic pain conditions. The combined mechanism should potentiate the analgesic effects of RXFP3 agonists and other pain killers.
The RELAX project will also have a major impact on the broader understanding of pain circuits, as we expect to differentiate subpopulations of ACC neurons responsible for the potentiation of spinal cord activity. We will search for specific circuits, possibly utilising parvalbumin spinal interneurons, which mediate the mechanical specificity of relaxin-3 action. Thus, our studies may identify the relaxin-3/RXFP3 peptidergic system as a key modulatory network that fine-tunes the transmission and central integration of sensory information and the emotional comorbidities associated with pathological states.