Abstract
RFamide neuropeptide VF (NPVF) is expressed by neurons in the hypothalamus and has been implicated in nociception, but the circuit mechanisms remain unexplored. Here, we studied the structural and functional connections from NPVF neurons to downstream targets in the context of nociception, using novel transgenic lines, optogenetics, and calcium imaging in behaving larval zebrafish. We found a specific projection from NPVF neurons to serotonergic neurons in the ventral raphe nucleus (vRN). We showed NPVF neurons and vRN are suppressed and excited by noxious stimuli, respectively. We combined optogenetics with calcium imaging and pharmacology to demonstrate that stimulation of NPVF cells suppresses neuronal activity in vRN. During noxious stimuli, serotonergic neurons activation was due to a suppression of an inhibitory NPVF-ventral raphe peptidergic projection. This study reveals a novel NPVF-vRN functional circuit modulated by noxious stimuli in vertebrates.
Highlights
RFamide neuropeptide VF (NPVF) is expressed by neurons in the hypothalamus and has been implicated in nociception, but the circuit mechanisms remain unexplored
Many neuropeptides remain poorly characterized, with respect to their behavioral function in vertebrates. Among these are the RFamide peptides, RFRP-1/NPSV and RFRP-3/NPVF, cleaved from a preproprotein encoded by the npvf gene
We demonstrated a causative effect between the stimulation of NPVF neurons and the inhibition of ventral raphe neurons, providing evidence for a novel neural circuit involved in pain response in vertebrates
Summary
NPSF and NPVF neuropeptides from the NPVF precursor share a strong sequence conservation between human, mouse and zebrafish (Fig. 1A), suggesting that their functions are conserved. To record neural activity during these stimuli, we used 2-photon calcium imaging of NPVF neurons in Tg(npvf:GCaMP6s) larval zebrafish (Fig. 2C). We recorded from downstream vRN neurons in Tg(elavl3:h2b-GCaMP6s) zebrafish (Fig. 2F), and found that thermal stimuli increased the activity of these cells at high levels of heat (Fig. 2G,H). These activity patterns were present in trials without tail movement (Fig. sup 3), indicating that this response is independent of locomotor activity. Optical activation of NPVF neurons (Fig. sup 4B) induced a slow suppression of vRN neurons (Fig. 3B,C) This inhibition was strongly reduced in the presence of the RFamide receptor/gpr[147] antagonist As pain in humans is often treated by opioids, whose addictive properties have a profound negative societal impact[19], the NPVF-serotonin circuit may be a novel target for development of alternative therapeutics for treatment of acute or chronic pain
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