TRPV1 activity and substance P release are required for corneal cold nociception

Abstract

As a protective mechanism, the cornea is sensitive to noxious stimuli. Here, we show that in mice, a high proportion of corneal TRPM8⁺ cold-sensing fibers express the heat-sensitive TRPV1 channel. Despite its insensitivity to cold, TRPV1 enhances membrane potential changes and electrical firing of TRPM8⁺ neurons in response to cold stimulation. This elevated neuronal excitability leads to augmented ocular cold nociception in mice. In a model of dry eye disease, the expression of TRPV1 in TRPM8⁺ cold-sensing fibers is increased, and results in severe cold allodynia. Overexpression of TRPV1 in TRPM8⁺ sensory neurons leads to cold allodynia in both corneal and non-corneal tissues without affecting their thermal sensitivity. TRPV1-dependent neuronal sensitization facilitates the release of the neuropeptide substance P from TRPM8⁺ cold-sensing neurons to signal nociception in response to cold. Our study identifies a mechanism underlying corneal cold nociception and suggests a potential target for the treatment of ocular pain.

Fig. 1. TRPM8 mediates cold-induced ocular nociception.

a Representative image showing that TRPM8-expressing sensory fibers (green) densely innervate the cornea as revealed by TRPM8-EGFPf in the whole-mount cornea from Trpm8^EGFPf/+ mice. b, c The reflex blinking and eye closing responses to air flow (0.5 L/min) at different temperature in WT (n = 7–10) and Trpm8^−/− (n = 10–13) mice. d Representative video frames showing the opening and closing of the mouse eye (outlined by white dash lines). Red dash lines indicate the width and length of the palpebral fissure. e The representative width/length ratio changes of WT and Trpm8^−/− mice in response to cold air (13 °C). Black arrows indicate reflex blinking responses. f The average width/length ratio of WT mice (n = 7) is significantly lower than that of Trpm8^−/− mice (n = 6) in response to cold air (13 °C). Data are expressed as mean ± s.e.m. Statistical analysis by two tailed Student’s t-test. **P < 0.01; ***P < 0.001. All images shown are representative of three independent experiments using tissues from at least three different mice. Scale bar: 100 µm. Source data are provided as a Source Data file.

Fig. 2. TRPV1 is co-expressed with TRPM8 in corneal sensory neurons.

a Representative images showing a higher overlap between TRPM8-EGFPf and TRPV1-immunoreactivity (IR) in cornea-projecting neurons (retrogradely labeled by fluorogold from the cornea) than in skin-projecting neurons (retrogradely labeled by fluorogold from the ear skin). White arrows indicate fluorogold⁺/TRPM8⁺/TRPV1⁺ neurons, whereas hollow arrows indicate fluorogold⁺/TRPM8⁺/TRPV1⁻ neurons. b Quantification of the co-expression of TRPV1 with TRPM8 in sensory neurons innervating the cornea and ear skin, respectively (n = 3 mice/group). c Representative neuronal calcium responses to cold, capsaicin (10 µM), and KCl (100 mM) within the cornea-projection area in the ophthalmic division of the trigeminal ganglion explants from Pirt^GCaMP3/+ mice in which a calcium indicator GCaMP3 was expressed in primary sensory neurons. d Quantification of capsaicin responses in 56 cold-sensitive neurons located within the cornea-projection area of four trigeminal ganglia from three Pirt^GCaMP3/+ mice. Cap: capsaicin. Data are expressed as mean ± s.e.m. Statistical analysis by two tailed Student’s t-test. **P < 0.01. All images shown are representative of three independent experiments using tissues from at least three different mice. Scale bars in a 100 μm. Source data are provided as a Source Data file.

Fig. 3. TRPV1 enhances the responsiveness of TRPM8-expressing cells to cold.

a, b TRPV1 antagonist AMG9810 (AMG, 300 nM, 3 min pre-treatment before cold stimulation) attenuated calcium responses to cold in transfected KNRK cells that co-express TRPM8 and TRPV1. As a control, the vehicle treatment did not change their response to cold or capsaicin (Cap, 1 µM). Cold stimulation was generated by bath temperature drop from 32 °C to 15 °C. Each dot in b represents a KNRK cell that co-expresses TRPM8 and TRPV1. c Representative calcium transients of sensory neurons to cold stimuli after 10 min pre-treatment of vehicle (0.0006% DMSO in calcium imaging buffer) or AMG9810 (300 nM). Imaged neurons were located within the cornea-projection area in the ophthalmic division of the whole trigeminal ganglion explants (n = 7) from Pirt^GCaMP3/+ transgenic mice, in which a calcium indicator GCaMP3 was expressed in primary sensory neurons. The expression of TRPV1 in tested neurons was determined by their responses to capsaicin (10 µM). d Quantification of calcium responses of trigeminal neurons to cold. Each dot represents one cold-sensing neuron. e–g Cold treatments elicited greater membrane potential changes (ΔMP) and more action potentials (APs) in a subset of TRPM8^EGFPf/+ sensory neurons that are sensitive to capsaicin (1 µM, identified by calcium imaging) than those insensitive to capsaicin. h–j Repeated cold treatments did not desensitize TRPM8^EGFPf/+; TRPV1⁺ sensory neurons in the vehicle control group, as shown by stable membrane potential changes and neuronal firing. k–m Application of TRPV1 antagonist AMG9810 (300 nM, 10 min pre-treatment before cold stimulation) effectively suppressed depolarization and neuronal firing in capsaicin-sensitive TRPM8^EGFPf/+ neurons upon subsequent cold treatment. Data are expressed as mean ± s.e.m. Statistical analysis by two tailed Student’s t-test. **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file.

Fig. 4. TRPV1 is required for ocular cold nociception.

a, b Although the reflex blinking response to cold was not altered in Trpv1^−/− mice (n = 8), their eye closing responses were eliminated, compared with WT controls (n = 7). c The ocular width/length ratio of Trpv1^−/− mice (n = 8) in response to cold (13 °C) was significantly greater than that of WT mice (n = 7), but lower than that of Trpm8^−/− mice (n = 6). d The ocular width/length ratio in response to cold (13 °C) was increased in WT mice pre-treated with TRPV1 antagonist AMG9810 (0.2 nmol in 2 μL, 10 min before cold challenge; n = 5) compared with controls pre-treated with the vehicle (0.1% Tween-80 in saline; n = 5). e, f Representative images and group analysis show that TRPV1-deficiency did not impair the survival of corneal TRPM8-EGFPf neurons (retrogradely labeled by fluorogold from the cornea) in Trpv1^−/−; Trpm8^EGFPf/+ mice (n = 3), compared with control Trpv1^+/+; Trpm8^EGFPf/+ mice (n = 3). g Representative image showing that TRPM8-EGFPf sensory fibers (green) densely innervate the whole-mount cornea from Trpv1^-/-; Trpm8^EGFPf/+ mice. h Representative image showing the central projection of TRPM8-EGFPf fibers within the cornea-projection region in the spinal trigeminal nucleus of Trpv1^−/−; Trpm8^EGFPf/+ mice. Data are expressed as mean ± s.e.m. Statistical analysis by one-way ANOVA and two tailed Student’s t-test. n.s. not significant; *P < 0.05; **P < 0.01; ***P < 0.001. All images shown are representative of three independent experiments using tissues from at least three different mice. Scale bars in e, g, h 50, 100, and 250 μm, respectively. Source data are provided as a Source Data file.

Fig. 5. TRPV1 is required for dry eye-induced cold allodynia.

a Representative images showing dry eye-associated corneal abrasion as revealed under a cobalt blue light after fluorescein staining. b, c Trpv1^−/− mice (n = 7) display normal basal tear secretion (D0) and similar extents of reduced tear secretion and intensified corneal epithelial erosions as control WT mice (n = 12) after surgical removal of exorbital lacrimal glands. Sham-operated WT (n = 11) or Trpv1^−/− mice (n = 6) do not show reduced tear secretion or corneal abrasion. d Representative images and group analysis show that the expression of TRPV1 was significantly increased among corneal TRPM8-EGFPf neurons (retrogradely labeled by fluorogold) in dry eye Trpm8^EGFPf/+ mice (n = 4), compared with sham-operated mice (n = 4). White arrows indicate fluorogold⁺/TRPM8⁺/TRPV1⁺ neurons. e The proportion of cold-sensitive neurons that display capsaicin sensitivity was significantly greater in the dry eye group (n = 5), compared with the sham group (n = 3). f TRPM8-agonist cryosim-3 (0.025 nmol in 1 μL) elicited ocular nociception in dry eye WT mice (n = 6), but not in sham-operated WT mice (n = 5). Trpv1^−/− mice (n = 5) displayed significantly attenuated ocular nociception induced by cryosim-3 under dry eye conditions. g Innocuous cold (19 °C) elicited ocular nociception-associated eye closing in dry eye WT mice (n = 9), but not in sham-operated WT mice (n = 5). The eye closing response was significantly reduced in dry eye Trpv1^−/− mice (n = 7). h The effects of the TRPV1 antagonist AMG9810 (0.2 nmol in 2 μL) on ocular cold allodynia of dry eye WT mice (n = 7) at different post-treatment time points, compared with vehicle-treated control mice (n = 6). All the experiments in d–h were done four weeks after dry eye surgery. Data are expressed as mean ± s.e.m. Statistical analysis by one-way ANOVA and two tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. All images shown are representative of three independent experiments using tissues from at least three different mice. Scale bar: 100 μm. Source data are provided as a Source Data file.

Fig. 6. TRPV1 overexpression in TRPM8⁺ neurons results in cold allodynia.

a Schematic diagram for the generation of Trpm8^CreER/+; ROSA26^Trpv1/+ mice. b Representative calcium transients of TRPM8⁺ neurons in response to the TRPM8 agonist cryosim-3 (10 µM) and TRPV1 agonist capsaicin (Cap, 1 µM) in Trpm8^CreER/+; ROSA26^Trpv1/+ mice and control ROSA26^Trpv1/+ mice. c Quantification indicates that a significantly increased fraction of TRPM8⁺ neurons respond to capsaicin in Trpm8^CreER/+; ROSA26^Trpv1/+ mice (n = 5), compared with control ROSA26^Trpv1/+ mice (n = 3). d Representative images showing TRPV1-immunoreactivity (TRPV1-IR, green) in tdTomato-labeled TRPM8⁺ neurons (Tdt, red) from Trpm8^CreER/+; ROSA26^{Trpv1/tdTomato} and control Trpm8^CreER/+; ROSA26^tdTomato/+ mice. Arrows indicate TRPV1⁺/TRPM8⁺ neurons. e Quantification showing upregulated TRPV1-IR in TRPM8⁺ neurons from Trpm8^CreER/+; ROSA26^{Trpv1/tdTomato} mice (n = 3), compared with control Trpm8^CreER/+; ROSA26^tdTomato/+ mice (n = 3). f TRPM8-agonist cryosim-3 (0.025 nmol in 1 μL) evoked significantly more reflex blinking in Trpm8^CreER/+; ROSA26^Trpv1/+ mice (n = 7) than in control ROSA26^Trpv1/+ mice (n = 6). The vehicle (0.9% NaCl) did not induce significant reflex blinking in either Trpm8^CreER/+; ROSA26^Trpv1/+ (n = 7) or ROSA26^Trpv1/+ mice (n = 6). g Innocuous cold (19 °C) evoked significantly more reflex blinking in Trpm8^CreER/+; ROSA26^Trpv1/+ mice (n = 7) than in control ROSA26^Trpv1/+ mice (n = 6). h Noxious heat (45 °C) elicited similar ocular nociception-associated reflex blinking responses in both Trpm8^CreER/+; ROSA26^Trpv1/+ (n = 7) and control ROSA26^Trpv1/+ mice (n = 6). i Evaporative cooling evoked by acetone applied onto the paw skin elicited more significant nociception-associated flicking and licking responses in Trpm8^CreER/+; ROSA26^Trpv1/+ mice (n = 6) than in control ROSA26^Trpv1/+ mice (n = 6). j Trpm8^CreER/+; ROSA26^Trpv1/+ (n = 7) and control ROSA26^Trpv1/+ mice (n = 6) display similar sensitivity to heat in the Hargreaves test. Data are expressed as mean ± s.e.m. Statistical analysis by two tailed Student’s t-test. n.s. not significant; *P < 0.05; ***P < 0.001. All images shown are representative of three independent experiments using tissues from at least three different mice. Scale bar: 100 μm. Source data are provided as a Source Data file.

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.. 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.