Fig. 7: Substance P release is required for corneal cold nociception.

a The expression of neuropeptides in mouse TRPM8⁺ trigeminal ganglionic (TG) neurons based on single-cell RNA-seq data. Each dot represents a single TRPM8⁺ neuron. Full dataset and methods are available in Nguyen et al.²⁵. b Single-cell RT-PCR using intron-spanning primers was performed on individual TRPM8-EGFPf sensory neurons that project to the cornea. All TRPM8⁺/TRPV1⁺ neurons express substance P (Tac1), while fewer TRPM8⁺/ TRPV1^- neurons express Tac1. Negative control (−): No reverse transcription reaction on RNA sample from whole TG. Positive control (TG): cDNA from whole TG. c Cold treatments (bath temperature drops from 32 to15 °C) promote the release of substance P from dissociated trigeminal neurons, as revealed by ELISA assays. TRPV1 antagonist AMG9810 (AMG, 300 nM, 3 min pre-treatment before cold stimulations) suppressed the release of substance P, compared with the vehicle control (0.0006% DMSO). Cold-associated release of substance P was also suppressed by pre-desensitization of TRPM8⁺ neurons using TRPM8 agonist cryosim-3 (10 µM). d Both the reflex blinking and eye closing responses to air flow at 13 °C were significantly reduced in Tac1^−/− mice, compared with WT controls (n = 5 mice/group). e–f NK1 antagonist alleviates cold nociception. The reflex blinking and eye closing responses to cold (air flow at 13 °C) and cryosim-3 (0.1 nmol in 1 μL) were significantly reduced 30 min after intracisternal injection of NK1 antagonist L733,060 hydrochloride (10 µg in 5 µL, n = 5 mice), compared with the vehicle-treated group (0.9% NaCl, n = 5 mice). g Model for neural pathways encoding corneal cold nociception and allodynia. SP: substance P. Data are expressed as mean ± s.e.m. Statistical analysis by two tailed Student’s t-test. * P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.