UCSF

Abstract

Almost one in five American adults suffers from chronic pain, and millions suffer from chronic itch, yet our understanding of the circuits that underlie pain and itch remain elusive. Specifically, it is unclear whether pain and itch are transmitted along distinct, so-called labeled line neuronal pathways (“specificity”) or if algogenic (pain- provoking) and pruritogenic (itch-provoking) inputs converge on a single circuit (“convergence”). There is unquestionably complex molecular and functional heterogeneity at the level of the primary sensory neurons—as well as second order, spinal cord dorsal horn interneurons— with discrete populations of neurons transmitting modality-specific pain or itch signals. That organization suggests that specificity predominates. For example, gastrin-releasing peptide-expressing interneurons have been exclusively implicated in the transmission of itch. However, whether there is specificity or convergence at the level of the dorsal horn projection neurons, which carry the message to the brain, where the pain or itch percept is eventually established, is unclear. To date the great majority of studies have relied almost exclusively on expression of the neurokinin 1 receptor (NK1R), which responds to the neuropeptide, substance P, to define the neurochemistry of projection neurons. However, as the NK1R is expressed by the majority of projection neurons, it is not a suitable molecular marker to determine whether there are functionally specific subpopulations.

Our objective was to more comprehensively profile the molecular complexity of projection neurons and to determine if subpopulations are more relevant to the transmission of pain or itch messages. To this end, in the mouse we isolated populations of projection neurons that target the parabrachial nucleus of the brainstem, a major relay in the transmission of pain and itch messages to the brain. From these projection neurons, we generated two RNA sequencing datasets of differentially expressed genes that are enriched in projection neurons compared to all neurons in the dorsal horn of the spinal cord and in the trigeminal nucleus caudalis, which processes information from the face. Among many genes enriched in the projection neurons, we focused our analysis on several that have already been implicated in pain and/or itch processing, including Cck, Nptx2, Nmb, and Crh. Importantly, these genes have not previously been associated with projection neurons. By multiple labeling in situ hybridization studies of the expression of these enriched genes, combined with retrograde labeling of projection neurons and pain- and itch-stimulus induced Fos expression, we have demonstrated that there are molecularly distinct subpopulations of projection neurons, based on their expression of the enriched genes, their spatial location, and their responsiveness to pain and itch-provoking stimulation. Corroborating electrophysiological and morphological data in the literature showing the heterogeneity of dorsal horn projection neurons, we conclude that projection neurons are diverse, and as such the NK1R is not the ideal marker to interrogate subpopulation function. The database of enriched genes identified and characterized in this study should permit more precise dissection of pain and itch circuits in the central nervous system going forward.