Transient receptor potential cation channel subfamily V member 1 expressing corneal sensory neurons can be subdivided into at least three subpopulations

Abstract

The cornea is innervated by three main functional classes of sensory neurons: polymodal nociceptors, pure mechano-nociceptors and cold-sensing neurons. Here we explored transient receptor potential cation channel subfamily V member 1 (TRPV1) expression in guinea pig corneal sensory neurons, a widely used molecular marker of polymodal nociceptors. We used retrograde tracing to identify corneal afferent neurons in the trigeminal ganglion (TG) and double label in situ hybridization and/or immunohistochemistry to determine their molecular profile. In addition, we used immunohistochemistry to reveal the neurochemistry and structure of TRPV1 expressing nerve endings in the corneal epithelium. Approximately 45% of corneal afferent neurons expressed TRPV1, 28% expressed Piezo2 (a marker of putative pure mechano-nociceptors) and 8% expressed the transient receptor potential cation channel subfamily M member 8 (TRPM8; a marker of cold-sensing neurons). There was no co-expression of TRPV1 and Piezo2 in corneal afferent neurons, but 6% of TRPV1 neurons co-expressed TRPM8. The TRPV1 expressing corneal afferent neurons could be divided into three subpopulations on the basis of calcitonin gene-related peptide (CGRP) and/or or glial cell line-derived neurotrophic factor family receptor alpha3 (GFRα3) co-expression. In the corneal epithelium, the TRPV1 axons that co-expressed CGRP and GFRα3 ended as simple unbranched endings in the wing cell layer. In contrast, those that only co-expressed GFRα3 had ramifying endings that branched and terminated in the squamous cell layer, whereas those that only co-expressed CGRP had simple endings in the basal epithelium. This study shows that the majority of TRPV1 expressing corneal afferent neurons (>90%) are likely to be polymodal nociceptors. Furthermore, TRPV1 expressing corneal afferent neurons can be subdivided into specific subpopulations based on their molecular phenotype, nerve terminal morphology and distribution in the corneal epithelium.

Keywords: TRPV1; cornea; polymodal nociceptor; primary afferent neurons; sensory neurons.

Figure 1

TRPV1-IR, TRPM8 and Piezo2 expression in retrogradely labeled corneal afferent neurons. The images in all panels are of the same field of a single section through the ophthalmic division of the trigeminal ganglion. (A) Retrograde labeled corneal afferent neurons (blue) imaged through a DAPI filter. (B) TRPV1-IR (green) imaged through a FITC filter. (C) TRPM8 expression (red) imaged through a TRITC filter. (D) Piezo2 expression (white) imaged using brightfield and inverted for ease of visualization. (E) Merged. Arrowheads highlight the same corneal afferent neurons throughout. Plus signs (+) highlight TRPV1-IR corneal afferent neurons. Hashes (#) highlight TRPM8 expressing corneal afferent neurons. Asterisks (*) highlight corneal afferent neurons that express Piezo2. Scale bars = 100 μm.

Figure 2

Size/frequency distributions of subpopulations of Fast Blue (FB) labeled corneal afferent neurons identified in this study. (A) TRPV1 expressing corneal afferent neurons (gray) were small. (B) Piezo2 expressing corneal afferent neurons (gray) were medium to large sized. (C) TRPM8 expressing corneal afferent neurons (gray) were small.

Figure 3

TRPV1-IR, CGRP-IR, and NF200-IR in corneal afferent neurons. The images in all panels are of the same field of a single section through the ophthalmic division of the trigeminal ganglion. (A) Retrograde labeled corneal afferent neurons (blue) imaged through a DAPI filter. (B) TRPV1-IR (red) imaged through a TRITC filter. (C) CGRP-IR (green) imaged through a FITC filter. (D) NF200-IR (magenta) imaged through a Cy5 filter. (E) Merged. Arrowheads highlight the same corneal afferent neurons throughout. Asterisk (*) highlights a corneal afferent neuron that expresses TRPV1 and CGRP but not NF200. Plus sign (+) highlights a corneal afferent neuron that expresses NF200 but not TRPV1 or CGRP. Scale bars = 50 μm.

Figure 4

TRPV1-IR, GFRα3-IR, and NF200-IR in corneal afferent neurons. The images in all panels are of the same field of a single section through the ophthalmic division of the trigeminal ganglion. (A) Retrograde labeled corneal afferent neurons (blue) imaged through a DAPI filter. (B) TRPV1-IR (red) imaged through a TRITC filter. (C) GFRα3-IR (green) imaged through a FITC filter. (D) NF200-IR (magenta) imaged through a Cy5 filter. (E) Merged. Arrowheads highlight the same corneal afferent neurons throughout. Asterisk (*) highlights a corneal afferent neuron that expresses TRPV1 and GFRα3 but not NF200. Plus sign (+) highlights a corneal afferent neuron that expresses NF200 but not TRPV1 or GFRα3. Scale bars = 50 μm.

Figure 5

CGRP-IR, GFRα3-IR, and NF200-IR in corneal afferent neurons. The images in all panels are of the same field of a single section through the ophthalmic division of the trigeminal ganglion. (A) Retrograde labeled corneal afferent neurons (blue) imaged through a DAPI filter. (B) CGRP-IR (red) imaged through a TRITC filter. (C) GFRα3-IR (green) imaged through a FITC filter. (D) NF200-IR (magenta) imaged through a Cy5 filter. (E) Merged. Arrowheads highlight the same corneal afferent neurons throughout. Asterisk (*) highlights a corneal afferent neuron that expresses CGRP and GFRα3 but not NF200. Plus sign (+) highlights a corneal afferent neuron that expresses NF200 but not CGRP or GFRα3. Scale bars = 50 μm.

Figure 6

Confocal images of nerve terminals in two different corneal whole-mount preparations labeled with antibodies directed against β tubulin III (A) and TRPV1 (B). Images are projected from z-series through the corneal epithelium. The small panels under (A) and (B) show orthogonal views generated by projecting the z-series above it in the x-plane. Arrowheads in (A) and (B) point to simple (#) nerve terminals in the wing cell layers and ramifying (*) or complex (+) nerve terminals in the squamous cell layer. Colored bars in the orthogonal projections indicate the approximate locations of the basal epithelium (green), wing cell layers (blue) and the squamous cell layer (red). Scale bar in A = 100 μm. Scale bar in B = 50 μm.

Figure 7

Confocal images of TRPV1-IR (A) and CGRP-IR (B) nerve terminals in the same field of view in the corneal epithelium. Images are projected from z-series through the entire corneal epithelium. The small panels under each image show orthogonal views generated by projecting the z-series above it in the x-plane. Arrowheads point to TRPV1-IR ramifying nerve terminals in the squamous cell layer and arrows point to simple endings in the wing cell layer that are both TRPV1- and CGRP-IR. The TRPV1-IR and CGRP-IR images are merged in (C) to show the extent of double labeling in simple, but not ramifying nerve terminals. Colored bars in the merged orthogonal projection indicate the approximate locations of the basal epithelium (green), wing cell layers (blue) and the squamous cell layer (red). Scale bars = 50 μm.

Figure 8

Confocal images of TRPV1-IR (A) and CGRP-IR (B) nerve terminals in the same field of view restricted to deep in the corneal epithelium. Images are projected from z-series through the basal layer of the corneal epithelium. The small panels under each image show orthogonal views generated by projecting the z-series above it in the x-plane. Arrowheads point to TRPV1-IR simple nerve terminals that end in the basal epithelium. These were always CGRP-IR. The TRPV1-IR and CGRP-IR images are merged in (C) to show the extent of double labeling. Colored bars in the merged orthogonal projection indicate the approximate locations of the basal epithelium (green) and wing cell layers (blue). Scale bars = 30 μm.

Figure 9

Confocal images of TRPV1-IR (A) and GFRα3-IR (B) nerve terminals in the same field of view in the corneal epithelium. Images are projected from z-series through the entire corneal epithelium. The small panels under each image show orthogonal views generated by projecting the z-series above it in the x-plane. Arrows point to TRPV1-IR ramifying nerve terminals in the squamous cell layer and arrowheads point to simple endings that are both TRPV1 and GFRα3-IR. The TRPV1-IR and GFRα3-IR images are merged in (C) to show the extent of double labeling in both simple and ramifying nerve terminals. Colored bars in the merged orthogonal projection indicate the approximate locations of the basal epithelium (green), wing cell layers (blue) and the squamous cell layer (red). Scale bars = 30 μm.

Figure 10

Schematic of the three different subpopulations of TRPV1 expressing corneal afferent neurons we reported in the trigeminal ganglion and corneal epithelium.