Exploring the molecular underpinnings of neuropathic pain in primary afferent subtypes

Dorsal root ganglia (DRG) neurons have been well described for their role in driving both acute and chronic pain. With cell bodies clustered in the spinal column, these neurons project from the periphery into the dorsal horn. Subtypes convert a range of stimuli into electrical signals, and have been traditionally defined by size, histological markers, projection pathways, and electrophysiological phenotypes. With the advance of sequencing technologies, DRG transcriptomes have been examined through whole ganglia, single cell, and more recently, deep subpopulation sequencing in naïve lines. Here, we study the deep transcriptional profiles of multiple murine DRG populations in acute and chronic pain states while considering sex interactions. We have exploited currently available transgenics to label and purify subtypes after injury for high throughput sequencing. Using bulk tissue samples, we are able to circumvent the issues of low transcript coverage and drop-outs seen with single cell datasets, increasing our power to detect changes in gene expression. In one experiment, we have studied the molecular changes in five populations, classified by their functional and molecular signatures in naive states. These include general nociceptors, peptidergic nociceptors, non-peptidergic nociceptors, C-LTMRs, and Ad + AB-RA-LTMRs. We see both stereotyped and unique subtype signatures in injured states after nerve injury at both an acute and chronic timepoint. While all populations contribution to a general injury signature, subtype enrichment changes can also be seen, possibly driven by the loss of transected NP neurons by 4 weeks. Within populations, there is not a strong intersection of sex and injury, but sex differences in na\"ive states - particularly in Ad + AB-RA-LTMRs - still contribute to differences in injured neurons. Using an SNI model of neuropathic pain, ipsilateral DRG contain both injured and intact neurons. The contributions of these populations to pain have yet to be delineated. Mirroring the techniques above, we next used an Atf3creERT2 line to label and sort fluorescent neurons prior to RNA-seq. We have complemented this with a pilot snATAC-seq study, showing that neurons cluster by subtype in naive and injured states. We have curated this data into an accessible, online platform. Together, this contributes a resource to the pain community to probe subpopulation and sex-specific differences after nerve injury at multiple timepoints.