The dorsal horn of the spinal cord is critical to processing distinct modalities of noxious and innocuous sensation, but little is known of the neuronal subtypes involved, hampering efforts to deduce principles governing somatic sensation. Here we used single-cell RNA sequencing to classify sensory neurons in the mouse dorsal horn. We identified 15 inhibitory and 15 excitatory molecular subtypes of neurons, equaling the complexity in cerebral cortex. Validating our classification scheme in vivo and matching cell types to anatomy of the dorsal horn by spatial transcriptomics reveals laminar enrichment for each of the cell types. Neuron types, when combined, define a multilayered organization with like neurons layered together. Employing our scheme, we find that heat and cold stimuli activate discrete sets of both excitatory and inhibitory neuron types. This work provides a systematic and comprehensive molecular classification of spinal cord sensory neurons, enabling functional interrogation of sensory processing.

Itch stimuli are detected by specialized primary afferents that convey the signal to the spinal cord, but how itch transmission is regulated is still not completely known. Here, we investigated the roles of the neuropeptide Y (NPY)/Y 2 receptor system on scratch behavior. The inhibitory Y 2 receptor is expressed on mouse primary afferents, and intrathecal administration of the Y 2 agonist peptide YY (PYY) 3-36 reduced scratch episode frequency and duration induced by compound 48/80, an effect that could be reversed by intrathecal preadministration of the Y 2 antagonist BIIE0246. Also, scratch episode duration induced by histamine could be reduced by PYY 3-36. In contrast, scratch behavior induced by a-methyl-5HT, protease-activated receptor-2-activating peptide SLIGRL, chloroquine, topical dust mite extract, or mechanical itch induced by von Frey filaments was unaffected by stimulation of Y 2. Primary afferent neurons expressing the Npy2r gene were found to coexpress itch-associated markers such as natriuretic peptide precursor b, oncostatin M receptor, and interleukin (IL) 31 receptor A. Accordingly, intrathecal PYY 3-36 reduced the scratch behavior induced by IL-31. Our findings imply that the NPY/Y 2 system reduces histaminergic and IL-31-associated itch through presynaptic inhibition of a subpopulation of itch-associated primary afferents. SIGNIFICANCE STATEMENT The spinal neuropeptide Y system dampens scratching behavior induced by histaminergic compounds and interleukin 31, a cytokine involved in atopic dermatitis, through interactions with the Y 2 receptor. The Y 2 receptor is expressed by primary afferent neurons that are rich in itch-associated neurotransmitters and receptors such as somatostatin, natriuretic peptide precursor b, and interleukin 31 receptors.

The role of calcitonin gene-related peptide (CGRP) in visceral and somatic nociception is incompletely understood. CGRPα is highly expressed in sensory neurons of dorsal root ganglia and particularly in neurons that also express the transient receptor potential cation channel subfamily V member 1 (Trpv1). Therefore, we investigated changes in visceral and somatic nociception following deletion of CGRPα from the Trpv1-Cre population using the Cre/lox system. In control mice, acetic acid injection (0.6%, ip) caused significant immobility (time stationary), an established indicator of visceral pain. In CGRPα-mCherry;Trpv1-Cre mice, the duration of immobility was significantly less than controls, and the distance CGRPα-mCherry;Trpv1-Cre mice traveled over 20 min following acetic acid was significantly greater than controls. However, following acetic acid injection, there was no difference between genotypes in the writhing reflex, number of abdominal licks, or forepaw wipes of the cheek. CGRPα-mCherry;Trpv1-Cre mice developed more pronounced inflammation-induced heat hypersensitivity above baseline values compared with controls. However, analyses of noxious acute heat or cold transmission revealed no difference between genotypes. Also, odor avoidance test, odor preference test, and buried food test for olfaction revealed no differences between genotypes. Our findings suggest that CGRPα-mediated transmission within the Trpv1-Cre population plays a significant role in visceral nociceptive pathways underlying voluntary movement. Monitoring changes in movement over time is a sensitive parameter to identify differences in visceral nociception, compared with writhing reflexes, abdominal licks, or forepaw wipes of the cheek that were unaffected by deletion of CGRPα- from Trpv1-Cre population and likely utilize different mechanisms. NEW & NOTEWORTHY The neuropeptide calcitonin gene-related peptide (CGRP) is highly colocalized with transient receptor potential cation channel subfamily V member 1 (TRPV1)-expressing primary afferent neurons, but the functional role of CGRPα specifically in these neurons is unknown in pain processing from visceral and somatic afferents. We used cre-lox recombination to conditionally delete CGRPα from TRPV1-expressing neurons in mice. We show that CGRPα from within TRPV1-cre population plays an important role in visceral nociception but less so in somatic nociception.

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Dorsal horn gastrin-releasing peptide receptor (GRPR) neurons have a central role in itch transmission. Itch signaling has been suggested to be controlled by an inhibitory network in the spinal dorsal horn, as increased scratching behavior can be induced by pharmacological disinhibition or ablation of inhibitory interneurons, but the direct influence of the inhibitory tone on the GRPR neurons in the itch pathway have not been explored. Here we have investigated spinal GRPR neurons through in vitro and bioinformatical analysis. Electrophysiological recordings revealed that GRPR neurons receive local spontaneous excitatory inputs transmitted by glutamate and inhibitory inputs by glycine and GABA, which were transmitted either by separate glycinergic and GABAergic synapses or by glycine and GABA co-releasing synapses. Additionally, all GRPR neurons received both glycine- and GABA-induced tonic currents. The findings show a complex inhibitory network, composed of synaptic and tonic currents that gates the excitability of GRPR neurons, which provides direct evidence for the existence of an inhibitory tone controlling spontaneous discharge in an itch-related neuronal network in the spinal cord. Finally, calcium imaging revealed increased levels of neuronal activity in Grpr-Cre neurons upon application of somatostatin, which provides direct in vitro evidence for disinhibition of these dorsal horn interneurons.