Extended Data Fig. 8: Calca⁺ HTMRs do not project directly to the DCN but activation of their endings in the skin drives long latency responses.

Calca is expressed in a broad set of sensory neurons. These include mechanically sensitive, polymodal receptors, and thermoreceptors that have A or C-fiber conduction velocity; Calca⁺ HTMRs have higher mechanical thresholds than Aβ-LTMRs^33,34,35,36. Calca⁺ DRG neurons typically do not project to the DCN, although previous studies have shown ~10% of DRG neurons retrogradely labeled from the DCN express Calca³². Others have observed sparse Calca⁺ fibers within the DCN^37,38. a, Schematic representation of a coronal section of brainstem containing the gracile and cuneate nucleus of the DCN (Gr, Cu), and the neighboring solitary nuclei (Sol) and spinal trigeminal nucleus (spV). The rostro-caudal area of the brainstem is shown at bottom. b, Immunofluorescence of Vglut1 (magenta) and ReaChR-YFP (green) in a brainstem section of a Calca-FlpE; Rosa26^FSF-ReaChR animal showing the dorsal brainstem section corresponding to a (top). Insets of the gracile (bottom left) and spinal trigeminal (bottom right) are shown for comparison. YFP⁺ Fibers are densely labeled in the dorsal spinal trigeminal, but very few were detected in the gracile. Histology shown in a-b was performed in one animal. c, DCN neurons (random) were juxtacellularly recorded in the gracile and the latencies of responses to various stimuli were measured. DCN units had short latency responses to strong mechanical indentation beginning at time 0, in wildtype animals (top). In Avil^FlpO; Rosa26^FSF-ReaChR randomly recorded DCN units also had short latency responses to optical activation of sensory neurons in the skin (middle). In Calca-FlpE; Rosa26^FSF-ReaChR animals optical activation of Calca⁺ endings in the skin evoked longer latency responses in randomly recorded DCN units (bottom). d, Random DCN units and their average response to light activation of Calca⁺ endings in the skin in Calca-FlpE; Rosa26^FSF-ReaChR animals (top). In the same units, light was also delivered directly over the DCN (bottom). Light over the DCN evoked weak responses in 2/19 units. e, Random DCN units for Avil^FlpO; Rosa26^FSF-ReaChR animals in which all sensory neurons express ReaChR. Robust short-latency firing could be evoked in all recorded DCN units when light was delivered over the DCN (bottom). f, Random DCN neurons were recorded and light was delivered to the skin in Calca-FlpE; Rosa26^FSF-ReaChR animals. The same spot on the skin was also stimulated using a gentle (10–20 mN) vibration (50 Hz, 100 ms). Synaptic blockers were then applied to the lumbar spinal cord to measure their effect on optically- and mechanically-evoked responses. g, Raster of a random DCN unit and its response to 50 Hz vibration and optical activation of Calca⁺ endings in the skin. After a baseline period, the NMDAR antagonist MK-801 was applied to the lumbar spinal cord (10 mM, 10 µL). After 5 min, the MK-801 was washed away with saline, and NBQX was added to the spinal cord (10 mM, 10 µL). h, Average histograms of vibration responses of random DCN units in which synaptic blockers were applied to the spinal cord. i, Same units shown in h and their response to optical activation of Calca⁺ endings in the skin following application of synaptic blockers to the spinal cord. j, Random units in the DCN were recorded in a Calca-FlpE; Rosa26^FSF-ReaChR animal. The area of the gracile responding to the hindlimb was located and the DC at T12 was lesioned. Responses were then measured to optical activation of Calca⁺ endings in the skin. k, Average responses in random DCN units in hindlimb region of the gracile to optical activation of Calca⁺ endings in the skin after lesioning the DC at T12. The entire hindpaw was sampled for each unit to ensure the receptive field was not missed. Data shown as n units in N animals (n/N).

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