CGRP and the Calcitonin Receptor are Co-Expressed in Mouse, Rat and Human Trigeminal Ganglia Neurons

The neuropeptide calcitonin gene-related peptide (CGRP) is expressed in the trigeminal ganglia, a key site in craniofacial pain and migraine. CGRP potently activates two receptors: the CGRP receptor and the AMY1 receptor. These receptors are heterodimers consisting of receptor activity-modifying protein 1 (RAMP1) with either the calcitonin receptor-like receptor (CLR) to form the CGRP receptor or the calcitonin receptor (CTR) to form the AMY1 receptor. The expression of the CGRP receptor in trigeminal ganglia has been described in several studies; however, there is comparatively limited data available describing AMY1 receptor expression and in which cellular subtypes it is found. This research aimed to determine the relative distributions of the AMY1 receptor subunit, CTR, and CGRP in neurons or glia in rat, mouse and human trigeminal ganglia. Antibodies against CTR, CGRP and neuronal/glial cell markers were applied to trigeminal ganglia sections to investigate their distribution. CTR-like and CGRP-like immunoreactivity were observed in both discrete and overlapping populations of neurons. In rats and mice, 30–40% of trigeminal ganglia neurons displayed CTR-like immunoreactivity in their cell bodies, with approximately 78–80% of these also containing CGRP-like immunoreactivity. Although human cases were more variable, a similar overall pattern of CTR-like immunoreactivity to rodents was observed in the human trigeminal ganglia. CTR and CGRP appeared to be primarily colocalized in small to medium sized neurons, suggesting that colocalization of CTR and CGRP may occur in C-fiber neurons. CGRP-like or CTR-like immunoreactivity were not typically observed in glial cells. Western blotting confirmed that CTR was expressed in the trigeminal ganglia of all three species. These results confirm that CTR is expressed in trigeminal ganglia neurons. The identification of populations of neurons that express both CGRP and CTR suggests that CGRP could act in an autocrine manner through a CTR-based receptor, such as the AMY1 receptor. Overall, this suggests that a trigeminal ganglia CTR-based receptor may be activated during migraine and could therefore represent a potential target to develop treatments for craniofacial pain and migraine.

Images are from one experiment. Apparent molecular weights (kDa) are labelled above the bands. Previous analysis of the Precision Plus ladder indicates that the apparent molecular weights run accurately under these conditions (Neris et al., 2020).

Supplementary Figure 2. Workflow of image analysis and size distribution (diameter) of neuronal cell bodies expressing the pan-neuronal marker β tubulin III in rat and mouse TG. (A)
Image analysis workflow using Prism and FIJI. Example images are from rat TG. (B) The distribution of neuronal size was analyzed relative to the total neuronal population for rats and mice. Data are the mean ± s.e.m, combined from six individual rats or mice (three male and three female).

Supplementary Figure 3. Characterization of four anti-CGRP antibodies.
(A) Dot blots testing specificity using human and rat CGRP and amylin on nitrocellulose membrane. The image is representative of 3-5 independent experiments. (B) Dot blots comparing the detection of 100 µg of human amylin (hAmy) dissolved in 100% DMSO and 100% DMSO alone on nitrocellulose and PVDF membrane. All four CGRP antibodies appear to interact with DMSO at varying levels on nitrocellulose membrane. Conversely, neither non-specific interactions with DMSO, nor cross-reactivity with 100 µg of hAmy were observed on PVDF membrane, suggesting that an interaction between the antibodies, DMSO and the nitrocellulose membrane is causing non-specific immunoreactivity. The images are representative of 1-3 independent experiments. (C) Immunoreactive staining (Ab36001, 10 µg/ml; Ab81887, 10 µg/ml; C9198, 1:4000; ABS 026-05-02, 2.65 µg/ml) in rat pancreatic islets and no primary control to further examine cross-reactivity in a tissue which highly expresses amylin. Empty arrowheads indicate examples of unstained islets and the filled arrowhead indicates an example of a stained islet. Scale bar = 500 μm. The brightness and contrast of these images was uniformly enhanced for presentation purposes. Image brightness and contrast were adjusted (contrast stretching) for presentation purposes and merged in FIJI. Images are representative of six mice (three male and three female). Figure 6. Additional non-specific pAb188 bands. (A) Immunoblots using lysate preparations from adult mouse TG (20 µg) and HEK293S cells (10 µg) transfected with mouse CT(a) or vector alone (pcDNA). (B) Immunoblots using lysate preparations from adult rat TG (20 µg) and HEK293S cells (10 µg) transfected with rat CT(a) or vector alone. Blots probed with pAb188 (4 µg/ml). Red arrows indicate likely non-specific bands which appear in the vector control sample or the ladder. MW markers are shown on the left of the blots, with apparent MW in kDa. Pink indicates overexposure. This image is representative of two western blots using individual mice or rats (one male and one female). Images were adjusted uniformly for brightness and contrast for presentation purposes.

Supplementary Figure 7. Immunocytochemical staining of transfected HEK293S cells using anti-HA and anti-myc antibodies.
(A) anti-HA (1 µg/ml) or (B) anti-myc (0.8 µg/ml). Immunoreactive staining is shown in greyscale and nuclear DAPI as blue. h = human; u, untagged. Scale bar = 100 μm. Images are representative of two to eight independent experiments in duplicate wells. For (A) and (B) brightness and contrast of these images have been enhanced uniformly within the image set for presentation purposes.

Supplementary Figure 8. Immunocytochemical staining of transfected HEK293S cells using the anti-CTR pAbPA1-84457 antibody (10 µg/ml).
Immunoreactive staining is shown in greyscale, and nuclear DAPI staining is shown in blue. h = human; r = rat. Scale bar = 100 μm. Images are representative of three independent experiments in duplicate wells. The brightness and contrast of these images has been enhanced uniformly for presentation purposes.

Supplementary Material
Supplementary Figure 9. Immunocytochemical staining of transfected HEK293S cells using anti-CTR pAb230500 antibody (5 µg/ml). Immunoreactive staining is shown in greyscale, and nuclear DAPI staining is shown in blue. h = human; r = rat. Scale bar = 100 μm. Images are representative of three independent experiments in duplicate wells. The brightness and contrast of these images has been enhanced uniformly for presentation purposes.

Supplementary Material
Supplementary Figure 10. Immunohistochemical localization of CGRP (pAb36001, 10 µg/ml) with β-tubulin III (1.2 µg/ml) and S100 (5 µg/ml) in human TG (Case 3A). Distribution of CGRP-LI in (A) puncta or (B, C) fibers relative to β tubulin III or S100. Filled arrowheads indicate examples of positive staining. *indicates examples of autofluorescence due to lipofuscin. Scale bar = 50 μm. Image brightness and contrast were adjusted for presentation purposes and merged in FIJI. Images are representative of four human cases.

Supplementary Material
Supplementary Figure 11. Immunohistochemical localization of CGRP (pAb36001, 10 µg/ml) and CTR (mAb31-01, 4 µg/ml) with S100 (5 µg/ml) in human TG. Filled arrowheads indicate examples of positive staining; empty arrowheads indicate examples of an absence of staining. Magenta arrows indicate expression of S100 in glia surrounding neurons expressing, but not colocalizing with, CGRP or CTR; white arrowheads indicate colocalization; yellow arrowheads indicate expression in adjacent neurons. *indicates examples of autofluorescence due to lipofuscin. Scale bar = 100 μm. Image brightness and contrast were adjusted for presentation purposes and merged in FIJI. Images are representative of each of the four individual human cases.  N, no signal in immunoblot or staining in histology. n.d. not determined. Y, yes staining was observed. *For immunoblots, the amounts of peptide refer to the lowest quantity that was reliably detected, with the value in parentheses being the highest quantity tested for that antibody/peptide combination in cases where there was no reliable detection. **Conservative estimate of selectivity is based on the ratio between the lowest amount of rat amylin and rat CGRP detected in immunoblots. ***Pearl-like staining resembling neuronal fibers observed.