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Abstract Unmyelinated non-peptidergic nociceptors (NP afferents) arborise in lamina II of the spinal cord and receive GABAergic axoaxonic synapses, which mediate presynaptic inhibition. However, until now the source of this axoaxonic synaptic input was not known. Here we provide evidence that it originates from a population of inhibitory calretinin-expressing interneurons (iCRs), which correspond to lamina II islet cells. The NP afferents can be assigned to 3 functionally distinct classes (NP1–3). NP1 afferents have been implicated in pathological pain states, while NP2 and NP3 afferents also function as pruritoceptors. Our findings suggest that all 3 of these afferent types innervate iCRs and receive axoaxonic synapses from them, providing feedback inhibition of NP input. The iCRs also form axodendritic synapses, and their targets include cells that are themselves innervated by the NP afferents, thus allowing for feedforward inhibition. The iCRs are therefore ideally placed to control the input from non-peptidergic nociceptors and pruritoceptors to other dorsal horn neurons, and thus represent a potential therapeutic target for the treatment of chronic pain and itch.

The extracellular matrix (ECM) is a dynamic structure of molecules that can be divided into six different categories and are collectively called the matrisome. The ECM plays pivotal roles in physiological processes in many tissues, including the nervous system. Intriguingly, alterations in ECM molecules/pathways are associated with painful human conditions and murine pain models. Nevertheless, mechanistic insight into the interplay of normal or defective ECM and pain is largely lacking. The goal of this study was to integrate bulk, single-cell, and spatial RNA sequencing (RNAseq) datasets to investigate the expression and cellular origin of matrisome genes in male and female murine and human dorsal root ganglia (DRG). Bulk RNAseq showed that about 65% of all matrisome genes were expressed in both murine and human DRG, with proportionally more core matrisome genes (glycoproteins, collagens, and proteoglycans) expressed compared to matrisome-associated genes (ECM-affiliated genes, ECM regulators, and secreted factors). Single cell RNAseq on male murine DRG revealed the cellular origin of matrisome expression. Core matrisome genes, especially collagens, were expressed by fibroblasts whereas matrisome-associated genes were primarily expressed by neurons. Cell–cell communication network analysis with CellChat software predicted an important role for collagen signaling pathways in connecting vascular cell types and nociceptors in murine tissue, which we confirmed by analysis of spatial transcriptomic data from human DRG. RNAscope in situ hybridization and immunohistochemistry demonstrated expression of collagens in fibroblasts surrounding nociceptors in male and female human DRG. Finally, comparing human neuropathic pain samples with non-pain samples also showed differential expression of matrisome genes produced by both fibroblasts and by nociceptors. This study supports the idea that the DRG matrisome may contribute to neuronal signaling in both mouse and human, and that dysregulation of matrisome genes is associated with neuropathic pain.

Neurons located in dorsal root ganglia (DRG) are crucial for transmitting peripheral sensations such as proprioception, touch, temperature, and nociception to the spinal cord before propagating these signals to higher brain structures. To date, difficulty in identifying modality-specific DRG neurons has limited our ability to study specific populations in detail. As the calcium-binding protein parvalbumin (PV) is a neurochemical marker for proprioceptive DRG cells we used a transgenic mouse line expressing green fluorescent protein (GFP) in PV positive DRGs, to study the functional and molecular properties of putative proprioceptive neurons. Immunolabeled DRGs showed a 100% overlap between GFP positive (GFP+) and PV positive cells, confirming the PVeGFP mouse accurately labeled PV neurons. Targeted patch-clamp recording from isolated GFP+ and GFP negative (GFP−) neurons showed the passive membrane properties of the two groups were similar, however, their active properties differed markedly. All GFP+ neurons fired a single spike in response to sustained current injection and their action potentials (APs) had faster rise times, lower thresholds and shorter half widths. A hyperpolarization-activated current (Ih) was observed in all GFP+ neurons but was infrequently noted in the GFP− population (100% vs. 11%). For GFP+ neurons, Ih activation rates varied markedly, suggesting differences in the underlying hyperpolarization-activated cyclic nucleotide-gated channel (HCN) subunit expression responsible for the current kinetics. Furthermore, quantitative polymerase chain reaction (qPCR) showed the HCN subunits 2, 1, and 4 mRNA (in that order) was more abundant in GFP+ neurons, while HCN 3 was more highly expressed in GFP− neurons. Likewise, immunolabeling confirmed HCN 1, 2, and 4 protein expression in GFP+ neurons. In summary, certain functional properties of GFP+ and GFP− cells differ markedly, providing evidence for modality-specific signaling between the two groups. However, the GFP+ DRG population demonstrates considerable internal heterogeneity when hyperpolarization-activated cyclic nucleotide-gated channel (HCN channel) properties and subunit expression are considered. We propose this heterogeneity reflects the existence of different peripheral receptors such as tendon organs, muscle spindles or mechanoreceptors in the putative proprioceptive neuron population.

Nociceptive information is relayed through the spinal cord dorsal horn, a critical area in sensory processing. The neuronal circuits in this region that underpin sensory perception must be clarified to better understand how dysfunction can lead to pathological pain. This study used an optogenetic approach to selectively activate spinal interneurons that express the calcium-binding protein calretinin (CR). We show that these interneurons form an interconnected network that can initiate and sustain enhanced excitatory signaling, and directly relay signals to lamina I projection neurons. Photoactivation of CR interneurons in vivo resulted in a significant nocifensive behavior that was morphine sensitive, caused a conditioned place aversion, and was enhanced by spared nerve injury. Furthermore, halorhodopsin-mediated inhibition of these interneurons elevated sensory thresholds. Our results suggest that dorsal horn circuits that involve excitatory CR neurons are important for the generation and amplification of pain and identify these interneurons as a future analgesic target.