Calcitonin receptor antibody validation and expression in the rodent brain

Abstract

Background and aim

Therapeutics that reduce calcitonin gene-related peptide activity are effective migraine treatments. However, gaps remain in our understanding of the molecular mechanisms that link calcitonin gene-related peptide to migraine. The amylin 1 receptor responds potently to calcitonin gene-related peptide, and to the related peptide amylin, but its role in relation to either peptide or to migraine is unclear. We sought to better understand the expression of the amylin 1 receptor protein subunit, the calcitonin receptor, in the rodent brain.

Methods

We profiled three antibodies for immunodetection of calcitonin receptor, using immunocytochemistry, western blotting, and calcitonin receptor conditional knockout mouse tissue. Selected migraine-relevant rat brain regions were then examined for calcitonin receptor-like immunoreactivity.

Results

All three antibodies detected calcitonin receptor protein but only one (188/10) produced robust immunostaining in rodent brain, under the conditions used. Calcitonin receptor-like immunoreactivity was apparent in the rat brainstem and midbrain including the locus coeruleus, periaqueductal grey and spinal trigeminal nucleus.

Conclusions

Anti-calcitonin receptor antibodies require comprehensive profiling to ensure confidence in the detection of calcitonin receptor. Using a validated antibody, calcitonin receptor-like immunoreactivity was detected in several brain regions relevant to migraine. Further research is needed to understand the functional consequences of calcitonin receptor expression for calcitonin gene-related peptide or amylin physiology and pathophysiology.

Keywords

CGRP amylin migraine pain G protein-coupled receptor

Introduction

There are now clear links between the neuropeptide calcitonin gene-related peptide (CGRP) and migraine pathophysiology. In particular, therapeutics that reduce CGRP activity are effective for acute and preventative treatment of migraine (1). Multiple strategies are effective, either antibodies to the peptide, or interfering with interactions between CGRP and its cellular receptors through small molecules, or an antibody.

The canonical CGRP receptor comprises a G protein-coupled receptor (GPCR), the calcitonin receptor-like receptor (CLR) and the single transmembrane receptor activity-modifying protein 1 (RAMP1). However, there is another CGRP-responsive receptor. The AMY₁ receptor is made up of the calcitonin receptor (CTR) with RAMP1. This receptor is potently activated by both CGRP and a related peptide, amylin, in vitro. Furthermore, evidence suggests that both CGRP and an amylin analogue, pramlintide, can induce migraine-like attacks (2). Therefore, the AMY₁ receptor may be relevant for migraine biology. CGRP receptor antagonist therapeutics are not highly selective for the CGRP receptor, with several gepants and erenumab showing some interactions with AMY₁ in vitro (3). Understanding the relationship between the AMY₁ receptor, CGRP, and migraine biology is important for more precisely understanding the role of CGRP in migraine.

An important step in understanding the role of the AMY₁ receptor is to clearly identify its expression patterns in the central nervous system in relation to its potential ligands. Radioligand binding experiments have been used to identify potential sites of ligand action. In rodent and primate brain, both CGRP and amylin show widespread binding to important migraine-relevant brain regions such as the periaqueductal grey (PAG), locus coeruleus (LC) and brainstem regions (4 –7). Varying ligand binding profiles suggest heterogeneity of binding sites, which could mean that both canonical CGRP receptors and AMY receptors are present (4,7). However, clearly identifying the molecular correlates of these binding sites is challenging. For the AMY₁ receptor, the relative patterns of both CTR and RAMP1 need to be assessed. Immunohistochemical studies suggest that CTR is widely expressed in the rat brain and human brainstem. However, there has been little emphasis on understanding where CTR is expressed in regions important for migraine (6,11). Studies measuring mRNA suggest CTR expression in several of these regions (12,13). However, these studies do not measure protein expression, which can differ from mRNA expression (6). Receptor protein can be measured with antibodies, provided these are sufficiently validated to allow conclusions to be drawn (14).

In this study, we focussed on CTR protein expression in rodent brain. Guided by carefully considered antibody validation standards (14), we profiled three CTR antibodies, which had varying levels of previous validation. We used multiple methods to build up profiles of each antibody’s strengths and weaknesses. Both rat and mouse tissues were used as they are important model animals in migraine research. We used immunocytochemistry (ICC), western blotting, as well as immunohistochemistry (IHC) in knockout mice. IHC in rats was then used to generate snapshots of CTR-like immunoreactivity in rat brain.

Methods

Antibodies

Three anti-CTR antibodies were chosen for validation as detailed in Table S1. The antibodies are referenced in the text as mAb4614 (mouse monoclonal), pAb188 (188/10; rabbit polyclonal) and mAb8B9 (10/13-8B9-1-1; mouse monoclonal). Comparative target antigens between species for pAb188 and mAb8B9 are shown in Figure S1. Sequences and amino acid numbering from UniProt were aligned using Clustal Omega (15,16)_. Where antigens were known, “blocking” peptides corresponding to the antigen sequence were synthesised (Supplemental Information) and antibodies pre-incubated with 50 µM peptide for 1 h at 20°C prior to immunohistochemistry. An anti-haemagglutinin (HA) tag antibody with the HA-tagged human CTR was used as a control for some experiments (Figure S2–3).

Cell culture and transfection

HEK293S cells were cultured and transfected as previously described (17). Cells were plated into 96-well poly-D-lysine-coated Cell-Carrier Ultra plates (PerkinElmer, MA, USA) at 10,000–15,000 cells per well or ∼5 million cells into 150 mm dishes (Corning, NY, USA). The N-terminally HA-tagged human CLR and CTR (CT_(a) splice variant, leucine polymorphic variant), myc- or un-tagged human RAMP1 constructs in pcDNA3.1 were as previously described (8,18,19). Untagged rat CLR, CTR_(a) and RAMP1 constructs in pCMV6 were from Origene (Rockville, MD, USA). The untagged mouse CLR, CTR_(a) and RAMP1 in pCMV6 plasmids were as previously described (20). In all cases, CLR or CTR were transfected in a 1:1 ratio with RAMP1 except where CTR was transfected alone with pcDNA3.1. The CT_(a) variant was chosen as the more abundantly expressed isoform, at least in rat (21). In addition, the antigenic sequence for pAb188 and mAb8B9 is present within both the CT_(a) and CT_(b) isoforms so it is likely that both isoforms could be detected.

Immunocytochemistry

Transfected HEK293S cells were fixed with 4% paraformaldehyde (PFA) and washed with tris-buffered saline (TBS) containing 0.1% Tween20 (TBS-T). Cells were blocked with 10% goat or donkey serum in TBS-T for 1 h at room temperature (RT). Cells were then incubated with primary antibody (pAb188, 10 µg/mL; mAb8B9, 10 µg/mL; mAb4614, 2.5 µg/mL) in 1% serum/TBS-T for 30 min at 37°C or overnight at 4°C. Cells were washed with TBS-T then incubated with secondary antibody (1:200, Table S2) and 4′,6-diamidino-2-phenylindole (DAPI) in 1% serum/TBS-T for 1 h at RT. Cells were then washed in TBS-T and imaged.

Animals and tissue collection

All procedures involving transgenic mice and their care were approved by the Veterinary Office of the Canton Zurich, Switzerland, and in accordance with the EU Directive on the protection of animals used for scientific purposes. CTR-floxed mice (Calcr<tm1(fl)>; MGI:5751436; frozen sperm was kindly provided by Dr Jean-Pierre David and Dr Thorsten Schinke, University Medical Center Hamburg) (22 –24) were crossed to Nestin-cre mice (Jax 003771; B6.Cg-Tg(Nes-cre)1Kln/J; MGI:2176173) to generate: CTR^flox/flox:Nestin^cre/wt (i.e. Nestin-Calcr^flox/cre, CTR knockout) and CTR^wt/wt:Nestin^wt/wt (i.e. Calcr^WT/WT, wildtype) mice. Animals were genotyped as previously published (22 –24). Mice were housed in a controlled environment and maintained at 21 ± 2°C, under a 12/12 hour light-dark cycle (lights off at 10.00 h). Mice were fed ad libitum standard chow diet (n°3436 Provimi Kliba, Kaiseraugst, Switzerland, 3.14 kcal/g of food) and had access to water. Each cage was equipped with a red plastic house, nest-building material and wood shavings. Prior to sacrifice, mice were handled daily to reduce stress. Male and female mice used were between 20–30 weeks of age. The transcardial perfusion procedure used is detailed in the supplemental information. Transgenic mouse tissue was used for IHC.

Wild-type tissue was collected from male and female adult Sprague-Dawley rats and C57BL/6 mice (25), in accordance with the New Zealand Animal Welfare Act (1999), and approved by the University of Auckland Animal Ethics Committee. Male and female SD rats (14–24 weeks, 272–609 g) were housed in standard open cages and male and female C57BL/6 mice (10–16 weeks, 21–29 g) in an enriched environment with 12/12 hour light-dark cycle at a temperature of 22 ± 2°C. Animals had ad libitum access to standard chow (Teklad TB 2018; Harlan, Madison, WI) and water. Rats and mice were euthanised by CO₂ inhalation followed by cervical dislocation. Rodent brain and kidney were immediately frozen in liquid nitrogen for western blotting or fixed in 4% PFA (4 or 21 h), cryoprotected in 20% sucrose and sectioned at 10 µm for brain IHC. We used the ARRIVE2 reporting guidelines when writing our report (26).

Western blotting

Full western blotting methodology is described in the supplemental information. Briefly, protein lysates were prepared from transfected HEK293S cells, or mouse and rat brain or kidney tissue in radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail. Protein lysates (0.1–20 µg, Table S3) were loaded alongside the protein ladder onto 4–12% SurePage Bis-Tris gels (GenScript, NJ, USA). Western blotting was performed as described previously (17) with the following modifications: Blots were incubated with primary antibody overnight at 4°C and 1:2,000 or 1:10,000 secondary antibody.

Immunohistochemistry

IHC was performed as previously described (2) with the following modification: Mouse brain sections were permeabilised with 0.1% Triton X-100 in TBS before immunohistochemistry was performed. The Allen Mouse Brain Atlas (27) was used for identification of mouse brain regions and for brain region nomenclature. BrainMaps 4.0 was used for rat brain regions (28).

Image preparation and analysis

ICC and IHC images were acquired using an Operetta (PerkinElmer) or ImageXpress (Molecular Devices, CA, USA) high-content imager with a 20× objective and processed as detailed in the supplemental information. Images were minimally processed using ImageJ to adjust colour and brightness for presentation purposes. When required, images were stitched using the grid/collection FIJI algorithm and 5–20% overlap as previously recommended (29). Any processing was uniformly applied across each image, and across all conditions for an antibody. Care was taken to avoid misrepresentation or loss of data, such as losing the darkest or brightest pixels in a given image by clipping the dynamic range (30). Representative ICC, western blotting and IHC images are presented from at least three independent experiments, performed using separate antibody dilutions. ICC experiments are defined as the immunoreactivity detected in cells from independent transfection and staining experiments, performed with two technical replicates. Western blotting experiments are defined as independent experiments generated using one transfected cell lysate preparation or different tissue lysates prepared from three individual rodents. IHC experiments are defined as immunoreactivity detected in brain sections from at least three different rodents unless stated. No image quantification was conducted.

Results

Immunocytochemistry

Antibodies were first screened using a transfected cell model, in line with one of the antibody validation “pillars” (14). Anti-CTR mAb4614 displayed strong detection of human, rat, and mouse CTR (Figure 1(a)). Reduced immunofluorescence was observed for human and rat CTR:RAMP1, compared to CTR alone. This reduction was not evident using an HA antibody to detect CTR, and therefore is unlikely to be due to transfection efficiency (Figure S2). Anti-CTR antibodies pAb188 and mAb8B9 detected rodent CTR, but not human CTR, with no differences in immunofluorescence observed in the presence of RAMP1 (Figure 1(b),(c)). mAb4614, pAb188 and mAb8B9 did not appear to display any immunofluorescence in cells transfected with CLR (in combination with RAMP1) or vector (Figure 1(a),(b),(c)).

Figure 1.

Immunofluorescent staining of transfected HEK293S cells using anti-CTR antibodies. (a) mAb4614 (2.5 µg/mL), (b) pAb188 (10 µg/mL) and (c) mAb8B9 (10 µg/mL) immunoreactive staining is shown in greyscale and nuclear DAPI staining in blue. Scale bars represent 200 µm. Insets display immunoreactivity for each transfection with greater magnification. In each of ((a)–(c)), mCLR:mRAMP1 has two insets: The higher magnification inset for mCLR:mRAMP1 is above and an example of vector control for each antibody is below. Images are representative of three independent experiments.

Western blotting

To further profile the anti-CTR antibodies, western blotting using cell and tissue lysates was performed. The predicted molecular weight of human, rat, and mouse CTR is approximately 52–53 kDa (31), although it also exists as other molecular forms due to alternative splicing and post-translational modification. The anti-CTR mAb4614 antibody was unsuitable for western blotting, showing no detectable bands in protein prepared from human CTR-transfected HEK293S cells (results not shown). Human HA-CTR was used as a positive control showing that the samples contained receptor (Figure S3). The pAb188 antibody detected multiple bands of varying intensity in rCTR and mCTR protein samples, the most prominent being a large immunoreactive smear between ∼50–75 kDa and a narrower band at ∼100 kDa (Figure 2(a)). Similarly, the mAb8B9 antibody detected a pair of narrower bands at ∼50 and ∼100 kDa (Figure 2(b)). Several non-specific immunoreactive bands were visible in protein from the vector control for both the pAb188 and mAb8B9 antibodies, particularly an ≥170 kDa band detected by mAb8B9 (Figure 2).

Figure 2.

Immunoblotting of cell and tissue lysates using anti-CTR antibodies pAb188 and mAb8B9. Lysates prepared from HEK293S cells, transfected with HA-hCTR, rCTR, mCTR or vector, or rat brain, rat kidney, mouse brain or mouse kidney (h: human; r: rat; m: mouse). The (a) PrecisionPlus protein ladder was used with pAb188 and the (b) Abcam protein ladder with mAb8B9. Blots were probed with primary antibody (pAb188, 4 µg/mL; mAb8B9, 2 µg/mL) and exposed for (a) 1.7 sec and 13.7 sec (pAb188) or (b) 19.7 sec and 46.3 sec (mAb8B9). Blots are representative of three independent experiments.

In protein prepared from rat and mouse brain and kidney, the pAb188 anti-CTR antibody detected multiple bands of varying molecular weights and intensities between ∼15–130 kDa (Figure 2(a), Figure S4(a)). mAb8B9 immunoreactivity was typically observed as an immunoreactive smear between ∼50–65 kDa and a second band at ∼95 or ∼120 kDa (Figure 2(b), Figure S4(b)). Overall, the pAb188 and mAb8B9 antibodies likely detect at least one form of CTR in rat and mouse brain at a size broadly consistent with species-matched CTR present in transfected cell lysate controls.

CTR-like immunoreactivity in a Calcr knockout mouse model

Continuing our validation of anti-CTR antibodies, we used a knockout mouse model which does not express Calcr in nervous tissue where Nestin is expressed (Nestin-Calcr^flox/cre). These Nestin-Calcr knockout mice were compared with wildtype Calcr^WT/WT littermates. Anti-CTR antibodies were used to stain sections containing the vascular organ of the lamina terminalis (OVLT). This region was chosen as there is strong evidence for the presence of CTR (9,13). For all three anti-CTR antibodies, strong, discrete staining was observed in the OVLT of the Calcr^WT/WT mice. This staining was absent in the Nestin-Calcr^flox/cre mice, suggesting that immunoreactivity was linked to the CTR as the Calcr gene product (Figure 3, Figure S5).

Figure 3.

Immunoreactivity of anti-CTR antibodies in the OVLT of Calcr wildtype and Nestin-Calcr knockout mice. Staining with (a) pAb188 (20 µg/mL), (b) mAb8B9 (20 µg/mL) and (c) mAb4614 (10 µg/mL) was observed in the OVLT of wildtype Calcr^WT/WT mice and absent in Nestin-Calcr^flox/cre mice. Immunoreactivity is shown in greyscale and nuclear staining in blue. Dashed lines indicate the location of the OVLT. White arrows indicate positively stained cell bodies within the region indicated by the dashed line. Absence of expected staining is indicated with an empty white arrowhead. An asterisk (*) indicates a tissue artifact. Scale bars represent 100 µm. Images are representative of results in three individual WT and KO mice.

To further confirm whether the antibodies were suitable for use in histology and able to detect CTR in multiple structures, we also looked at the nucleus accumbens (ACB), another site reported to express CTR (9,13). Using the anti-CTR mAb8B9 or mAb4614 antibodies, immunoreactive staining was not observed in the ACB of wildtype mice (Figure 4A,B) or Nestin-Calcr^flox/cre mice (not shown). Anti-CTR pAb188 produced immunoreactivity in the ACB of wildtype mice, with both neurons and fibres stained around the anterior commissure (Figure 4(c),(d)). This pattern of staining was absent in the Nestin-Calcr^flox/cre mice (Figure 4(d)).

Figure 4.

Immunoreactivity of anti-CTR antibodies in the ACB of Calcr wildtype and Nestin-Calcr knockout mice. No immunoreactive staining was observed with (a) anti-CTR mAb8B9 (20 µg/mL) or (b) anti-CTR mAb4614 (10 µg/mL) in the ACB of Calcr^WT/WT mice. Immunoreactive staining with (c) and (d) anti-CTR pAb188 (20 µg/mL) was observed surrounding the anterior commissure (ac) in wildtype Calcr^WT/WT mice and absent in Nestin-Calcr^flox/cre mice. Immunoreactivity is shown in greyscale and nuclear staining in blue. White arrows indicate positively stained cell bodies and white arrowheads indicate positive fibre staining. Absence of expected staining is indicated with empty white arrowheads. Scale bars for ((a)–(c)) represent 100 µm; scale bar for (d) is 200 µm. Images are representative of results in three individual WT and KO mice.

CTR-like immunoreactivity in rat brain

The anti-CTR antibodies were then used to probe regions of the adult rat brain, with the goal of improving our understanding of CTR rat brain expression. Initially we looked at the ACB, to compare with the results from mice. The anti-CTR pAb188 antibody generated CTR-like immunoreactivity in the rat ACB, as well as the OVLT (Figure S6(a),(b)). Pilot experiments using anti-CTR mAb8B9 and mAb4614 produced a similar pattern of ACB staining albeit with a lower intensity (Figure S6(c),(d)). Given that pAb188 produced the most robust staining in ACB and OVLT of mouse and rat, regions that are expected to contain CTR, subsequent work focussed on this antibody only. Some additional experiments were conducted with mAb8B9 to confirm the pAb188 results. Results were consistent between male and female rats, unless otherwise stated.

To improve our understanding of the pattern of epitope-specific staining for anti-CTR pAb188 in rat brain, we also used a blocking peptide control. Pre-adsorption with a peptide matching the antibody antigen reduced CTR-like immunoreactivity produced by anti-CTR pAb188 (Figure S7). This suggests that the anti-CTR pAb188 ACB immunoreactivity is driven by antibody-epitope binding rather than non-specific binding.

Anti-CTR pAb188 was then used to stain other regions of the adult rat brainstem and midbrain. The brainstem includes regions that are activated in migraine and contains multiple structures that are implicated in the transmission of pain or other migraine symptoms (32).

Sections of the caudal brainstem were stained using two locations (−14 mm from bregma (Figure 5(a)) and −11.75 mm from bregma (Figure 5(b)). In the caudal sections, strong CTR-like immunoreactivity was observed in the area postrema (AP) (Figure 5(c)). Faint AP staining was also observed with mAb8B9 (Figure S8(a),(b)). Some fibre-like structures were observed in the nucleus of the solitary tract (NTS), between the AP and the central canal. A fibre-like or “pearl-like” pattern of staining was observed in the spinal trigeminal nucleus (Sp5) (Figure 5(d)). This distinctive pattern was present in the three female, but not the two male, rat sections stained. Rostrally, the gigantocellular reticular nucleus showed positive CTR-like immunoreactivity in a network pattern of fibres and cell bodies (Figure 5(e)). This staining was also observed with mAb8B9 (Figure S8(c)). In contrast to these positively stained regions, the cerebellum was consistently free of immunoreactivity across all rat brain sections stained; an example is shown in Figure 5(f).

Figure 5.

CTR-like immunoreactivity in regions of the rat brainstem with pAb188 (20 µg/mL). The approximate locations of the brain stem regions are marked on diagrams of the coronal rat brain (modified from (27)) and correspond to approximately (a) −14 mm and (b) −11.75 mm from bregma (28). Immunoreactive staining was observed in the (c) AP (circled in white), (d) Sp5 as bead-like fibres in most rats and (e) reticular nucleus as punctate staining. No immunoreactive staining was observed with pAb188 in the cerebellum (f). Immunoreactivity is shown in greyscale and nuclear staining in blue. White arrows indicate positively stained cell bodies and white arrowheads indicate positive fibre staining. The scale bars for ((c),(d),(e)) represent 200 µm or 2 mm for (f). Images are representative of results in five rats, except for Sp5 fibres, which were observed in three of five rats.

The midbrain was stained at approximately −9.8 mm from bregma (Figure 6(a)). CTR-like immunoreactivity was observed in the LC as dense positive immunoreactivity (Figure 6(b), Figure S9). Faint staining was also observed in the LC with anti-CTR mAb8B9 (Figure S8). This contrasts with the lack of specific staining observed in other midbrain regions, such as the midbrain trigeminal nucleus (mesencephalic trigeminal nucleus, MeV). The nucleus raphe magnus (NRM) showed strong positive CTR-like immunoreactivity as a network of fibres and cell bodies, (Figure 6(c),(d)). This staining was also observed with mAb8B9 (Figure S8(d)). Immunopositive fibres and cell bodies were also observed in the parabrachial nucleus (PB) (Figure 6(e),(f)).

Figure 6.

CTR-like immunoreactivity in regions of the rat midbrain with pAb188 (20 µg/mL). The approximate locations of the midbrain regions are marked on a diagram of the coronal rat brain (modified from (27)) and correspond to approximately (a) −9.8 mm from bregma (28). Image (b) shows the positive immunoreactivity of the LC. Image (c) shows an overview of immunoreactive staining in the NRM, with higher magnification images (d) showing stained fibres and cell bodies. Image (e) shows an overview with higher magnification of immunostaining in the parabrachial nucleus (f). Immunoreactivity is shown in greyscale and nuclear staining in blue. White arrows indicate positively stained cell bodies and white arrowheads indicate positive fibre staining. The scale bars for (b), (d) and (f) represent 200 µm or 2 mm in (c) and (e). Images are representative of results in three to four rats.

CTR-like immunoreactivity was visible in the PAG, in cell bodies and fibres (Figure 7). This immunoreactivity was present along the rostro-caudal axis of the PAG; two examples are shown in Figure 7. In the process of generating this dataset, CTR-like immunoreactivity was observed in structures outside of those initially selected as regions of interest (Figures S10–12). Although full sets of images with multiple rats were not generated for these additional regions and all regions of the PAG, these data are presented with the intent of highlighting regions of interest for further investigation (Figure 7, Figures S10–12). CTR-like immunoreactivity was observed in the amygdala, lateral striatal regions, and some parts of the hypothalamus. Some variability was observed; in the raphe obscurus (RO), CTR-like immunoreactivity was present in the RO of two of four rats examined for this region.

Figure 7.

CTR-like immunoreactivity in the rat PAG with pAb188 (20 µg/mL). The approximate locations of the PAG region shown are marked on diagrams of the coronal rat brain (modified from (27)) and correspond to approximately (a) −7.6 mm and (b) −8.3 mm from bregma (28). Images (c)–(d) show an overview of the positive immunoreactivity at two different regions throughout the PAG. A higher magnification image showing cellular staining is shown in image (e) and (f). Immunoreactivity is shown in greyscale and nuclear staining in blue. White arrows indicate positively stained cell bodies and white arrowheads indicate positive fibre staining. The scale bar for ((c),(d)) is 2 mm and for ((e),(f)) is 200 µm. Images are shown from two of three rats.

Discussion and conclusions

In this study, we report comprehensive profiles of three anti-CTR antibodies using multiple techniques, which led to the use of anti-CTR pAb188 to probe migraine-relevant regions in the adult rat brain. Understanding the expression of CTR within the rat brain is important for interpreting studies related to the function and distribution of CGRP, given that the AMY₁ receptor (CTR:RAMP1) complex responds potently to CGRP in vitro. CTR is also relevant for the related peptide amylin, although this is much less abundant in nervous system tissues compared to CGRP.

Antibody validation

Antibodies are powerful experimental tools for exploring protein expression but are commonly under-validated for use in IHC. Antibodies are rarely suitable for all applications. This is demonstrated for the three anti-CTR antibodies profiled. The results of our investigations are summarised in Table 1. All antibodies showed robust immunoreactivity with rCTR and mCTR in an overexpressing transfected cell model. Interestingly, neither mAb8B9 or pAb188 antibodies showed cross-reactivity with hCTR, despite considerable sequence identity (Figure S1). The recognition of the hCTR by the antibodies must be disrupted by the six amino acids differing between the antigenic sequence and the corresponding region of human CTR. The mAb4614 antibody appeared to be less able to detect hCTR and rCTR when transfected with RAMP1, compared to hCTR or rCTR transfected with vector. It is possible that this is the result of epitope masking, where interacting proteins obscure the binding epitope for an antibody. Epitope masking has been observed previously for an anti-CTR antibody (19). This means that this antibody could underestimate the amount of CTR in a particular situation, should it be complexed with RAMP.

Table 1.

CTR antibody validation summary table. OVLT: vascular organ of the lamina terminalis; ACB: nucleus accumbens.

Antibody	ICC	WB	Calcr mouse model	Rat
pAb188	Stained rCTR, mCTR	Strong band, some non-specific	Stained OVLT and ACB	Staining in multiple regions, cell bodies and fibres
mAb8B9	Stained rCTR, mCTR	Weaker band, less non-specific than pAb188	Stained OVLT	Weak staining in multiple regions
mAb4614	Stained rCTR, mCTR, hCTR	Not compatible with WB	Stained OVLT	Weak staining in ACB, not used further

Western blots contributed to the characterisation profiles of these anti-CTR antibodies. There is some debate about the usefulness of western blots for characterising antibodies that are intended for other applications, as the denatured epitopes may show little resemblance to fixed epitopes in different sample types (33). However, in this study, the western blots using lysates from cells transfected with human, rat, or mouse CTR were informative. They suggested that the anti-CTR pAb188 and mAb8B9 antibodies could detect proteins at molecular weights consistent with CTR monomers and dimers. These bands were absent in lysates prepared from cells transfected with empty vector. The band patterns are broadly consistent with previous western blots using rat kidney and cell lysates (21,31,34). Interestingly, some low molecular weight bands were also present. It is possible that these are the products either of biosynthesis or degradation as the samples were prepared from whole cells and so all aspects of the receptor life-cycle are represented. mAb4614 was unsuitable for western blotting and may require alternative methods, such as native PAGE for further analysis. This could be because the antibody is raised against whole CTR protein and the epitope may rely on tertiary protein structure, which is denatured in western blotting.

The western blots using lysates of different tissue origin were difficult to interpret. While there were similarities between tissue types for rats and mice, overall, there was a variety of bands. In some cases, it is not clear whether these bands correspond to CTR or are non-specific. Our intentional use of relatively high protein loading amounts increased our ability to observe less prevalent proteins and therefore the potential limitations of these antibodies. Given the variations between tissues and species, these data could suggest tissue- and species-specific CTR isoforms or post-translational modifications. For example, the presence of CTR homodimers has previously been reported, but not in tissues (35). The use of mass spectrometry techniques or knockout tissue would be useful to investigate these differences further. More bands were present in the rodent tissue blots probed with anti-CTR pAb188. Due to the polyclonal nature of this antibody, it is not surprising that it may have a larger range of non-specific binding proteins (36). A limitation of this study was that comparisons were only made with transfected cell samples containing a single splice variant from each species. CTR is known to have species-specific isoforms in humans and rodents (31). In future, experiments could be performed using different rat and mouse splice variants to enable a more informative comparison to tissue samples. This work could also be done with and without deglycosylating enzymes to examine the contribution that glycosylation may be making to the observed molecular weights.

The use of genetically modified knockout animals is the gold standard control for validation of antibodies for IHC, as it convincingly links the target gene with patterns of staining. The anti-CTR pAb188 was able to stain the OVLT in wildtype but not Nestin-Calcr mice, suggesting that the Calcr gene product is responsible for the immunoreactivity of the anti-CTR pAb188 antibody. These results are supported by previous reports that tested the anti-CTR pAb188 antibody using Calcr knockout mice, looking at either the AP or the suprachiasmatic nucleus (22,37). The use of knockout animals is a superior control to the blocking peptide used for the rat tissue. Blocking peptide controls can only confirm that the antibody binding to the tissue is epitope-driven, rather than non-specific binding. However, a blocking peptide control cannot confirm that the epitope belongs to the target of interest (38).

Overall, the antibody validation provides a comprehensive profile of three antibodies against the CTR (Table 1). The differences between antibody applications demonstrate that antibodies need to be tested for their application, and comprehensive antibody work-up needs to be performed for all antibodies.

Expression of CTR in rodent brain

The results of the antibody validation supported the use of the anti-CTR pAb188 to investigate CTR expression in the adult rat brain (9). Although anti-CTR mAb8B9 generated a weaker signal, it did produce some immunoreactive patterns that mirrored the results of anti-CTR pAb188 for some regions. This may be related to the lower sensitivity of mAb8B9 and differences in CTR expression levels (27,36). Overall, these staining results could help understand whether there is a possible interaction between CTR (as part of the AMY₁ or other AMY receptors), and CGRP or amylin in the central nervous system. Staining results can also be compared with radioligand binding studies, as part of determining the molecular correlates of the binding sites.

The immunohistochemical results confirm and expand reports that CTR expression occurs in the AP, ACB and OVLT of adult rat brain (9). Multiple lines of evidence suggest that CTR is present in these structures, including mRNA studies, immunoreactivity, and the radioactive ligand binding profiles for these regions (4,5,9,12,13,39,40). As circumventricular organs, the AP and OVLT sample the peripheral circulation, and therefore could be exposed to circulating ligands. The AP is clearly relevant for the actions of amylin in inducing satiation, along with the NTS (10,22,41). The relationship between the CTR and CGRP in the AP is not clear but could potentially be relevant for migraine, particularly as the AP is known as the nausea centre of the brain, a common migraine symptom (32). Cell bodies and fibres have been reported to express CGRP-like immunoreactivity in the NTS and AP (42 –45).

Our results also highlight some regions of the brain where CTR expression is less well-established, including those that are involved in CGRP and/or migraine biology. Studies show strong binding of both CGRP and amylin to the LC, a region of dense CTR-like immunoreactivity in this study (4,46). High levels of CGRP-like immunoreactivity have been detected in the LC in rodents and human in cell bodies and fibres, and dysfunction of the LC has been proposed in migraine (43,47,48,49). In contrast, there is no such data for robust amylin expression in the LC. It is possible that the AMY₁ receptor could be involved in CGRP function in the LC. These observations are also true for the NRM, where both CTR-like and CGRP-like immunoreactivity have been observed (9,45). Similarly, the PB contains CGRP-expressing neurons involved in fear and aversion (50). A potential avenue of study is whether the expression of CTR in this region is involved in neurological circuits where CGRP is involved.

The observation of CTR-like immunoreactivity as distinctive fibres in the Sp5 has not previously been reported. CTR-like immunoreactivity has been reported in the human Sp5, but not in rats (51,52). The Sp5 is an important region transmitting signals to and from the peripheral trigeminal neurons, which are involved in migraine (32). CGRP-containing fibres are reported in the Sp5, as would be expected. In this study, fibres were observed in female rats but not formally compared between sexes. It would be interesting to know whether the presence of CTR fibres relates to potential sex differences in CGRP expression and migraine prevalence (53). The spatial relationship between CTR and CGRP should be explored in future studies.

A key remaining question is the expression of RAMP1 and other RAMPs in regions of the rodent brain that express CTR or CLR, to form functional CGRP-responsive receptors. Few studies have colocalised CTR or CLR expression with individual RAMPs to determine whether they are expressed within the same cells (54 –56). Unfortunately, many antibodies used to investigate RAMP expression are poorly characterised and lack sufficient validation, such as knockout mouse tissue, making interpretation and conclusions difficult (6). Studies investigating the distribution of RAMP2 and RAMP3 have mostly relied on mRNA (6). While this does give some indication of where they may be expressed, mRNA studies are not definitive. Ultimately, which receptors underlie the CGRP and amylin binding sites in the brain is still unknown and future studies should consider examining the relative expression of CTR with RAMP1, RAMP2 and RAMP3 in migraine-relevant structures with thoroughly validated tools.

Overall, CTR-like immunoreactivity and mRNA have been localised to many pain- and migraine-relevant brain structures (9,13). This has been expanded upon in this study, noting CTR-like immunoreactivity in both cell bodies and fibres in rat brain. There are hints that the distribution pattern of CGRP and CTR could have similarities. Binding sites for CGRP and amylin are widely distributed throughout the brain. The relative distribution of CTR and CGRP is yet to be investigated; however, this does indicate that they are likely present in the some of the same structures and that a CTR-based receptor could mediate CGRP’s activity in these structures. The involvement of many of these regions in migraine pain pathways suggests that a CTR-based receptor or receptors could contribute to CGRP’s role in migraine pathogenesis and provides another potentially druggable target. Additionally, the distribution and expression of CLR is poorly defined in migraine-relevant brain regions, with minimal or no available evidence of its expression (6). Further work investigating the expression of the CGRP family receptors is warranted, in both rodent and human brain.

Conclusions

In summary, this study has validated the use of anti-CTR pAb188 antibody for rat and mouse brain tissue. We have confirmed and profiled expression of CTR in the rat brain, where we identified discrete expression in migraine-relevant structures. Given the potential role of CTR in the CGRP-migraine pathway, further research is warranted. Characterising the relative expression profiles of CGRP and CTR would be an important step in understanding this complex peptide and receptor family with high therapeutic relevance for headache.

Article highlights

The CTR may be involved in CGRP processes in migraine.

The expression of the CTR is not well defined, even in rodents.

This study characterises anti-CTR antibodies as tools for exploring CTR expression in rodent tissues.

CTR is expressed in regions of the brainstem and midbrain of rats.

Supplemental Material

sj-pdf-1-cep-10.1177_03331024221084029 - Supplemental material for Calcitonin receptor antibody validation and expression in the rodent brain

Supplemental material, sj-pdf-1-cep-10.1177_03331024221084029 for Calcitonin receptor antibody validation and expression in the rodent brain by Erica R Hendrikse, Tayla A Rees, Zoe Tasma, Christelle Le Foll, Thomas A Lutz, Andrew Siow, Peter J Wookey, Christopher S Walker and Debbie L Hay: on behalf of the Hand Wrist Study Group in Cephalalgia

Footnotes

Acknowledgements

ERH would like to acknowledge Chia-Lin Chuang and Jennifer Chen for their expert help with tissue preparation and sectioning.

Author contributions

^#ERH and TAR contributed equally to this work. CLF and TAL generated, perfused, and provided transgenic mouse tissues. ERH and TAR performed all immunocytochemical and immunohistochemical experiments. ERH, TAR and ZT performed western blotting. AS performed peptide synthesis. PJW provided pAb188 and mAb8B9 antibodies. ERH, CLF, TAR, ZT, PJW, CSW and DLH drafted the manuscript. ERH, TAR, ZT, PJW, CLF, TAL, CSW and DLH edited and revised the manuscript. All authors approved the manuscript.

Declaration of conflicting interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: DLH is or has been a consultant or speaker for Lilly, Amgen, Teva, Intarcia, Merck Sharp & Dohme and has received research funding from Living Cell Technologies in the past 3 years. CSW has received research support from Living Cell Technologies. PJW is the director of Welcome Receptor Antibodies Pty Ltd, Melbourne, Australia.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was kindly supported by a Barbara Basham Doctoral Scholarship from the Auckland Medical Research Foundation, and funding from the Maurice Wilkins Centre to ERH. TAR acknowledges receipt of a University of Auckland Doctoral Scholarship. CSW is the recipient of a Sir Charles Hercus Health Research Fellowship from the Health Research Council, New Zealand. The authors acknowledge receipt of research funding from the National Institutes of Health, USA (Grant NS113839); the contents of this article do not represent the views of the United States government. TAL acknowledges funding from the Swiss National Science Foundation (Grant 31003A_175458).

ORCID iDs

Zoe Tasma

Andrew Siow

Christopher S Walker

Debbie L Hay

Supplemental material

The supplemental material for this article is available online.

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