Calcitonin gene-related peptide (CGRP) and its receptor components in human and rat spinal trigeminal nucleus and spinal cord at C1-level
© Eftekhari and Edvinsson; licensee BioMed Central Ltd. 2011
Received: 7 September 2011
Accepted: 10 November 2011
Published: 10 November 2011
Calcitonin gene-related peptide (CGRP) has a key role in migraine pathophysiology and is associated with activation of the trigeminovascular system. The trigeminal ganglion, storing CGRP and its receptor components, projects peripheral to the intracranial vasculature and central to regions in the brainstem with Aδ- and C-fibers; this constitutes an essential part of the pain pathways activated in migraine attacks. Therefore it is of importance to identify the regions within the brainstem that processes nociceptive information from the trigeminovascular system, such as the spinal trigeminal nucleus (STN) and the C1-level of the spinal cord. Immunohistochemistry was used to study the distribution and relation between CGRP and its receptor components - calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) - in human and rat STN and at the C1-level, using a set of newly well characterized antibodies. In addition, double-stainings with CGRP and myelin basic protein (MBP, myelin), synaptophysin (synaptic vesicles) or IB4 (C-fibers in general) were performed.
In the STN, the highest density of CGRP immunoreactive fibers were found in a network around fiber bundles in the superficial laminae. CLR and RAMP1 expression were predominately found in fibers in the spinal trigeminal tract region, with some fibers spanning into the superficial laminae. Co-localization between CGRP and its receptor components was not noted. In C1, CGRP was expressed in fibers of laminae I and II. The CGRP staining was similar in rat, except for CGRP positive neurons that were found close to the central canal. In C1, the receptor components were detected in laminae I and II, however these fibers were distinct from fibers expressing CGRP as verified by confocal microscopy.
This study demonstrates the detailed expression of CGRP and its receptor components within STN in the brainstem and in the spinal cord at C1-level, and shows the possibility of CGRP acting postjunctionally in these areas putatively involved in primary headaches.
Migraine is considered as a neurovascular disorder affecting more than 10% of the general population. Calcitonin gene-related peptide (CGRP) has a key role in migraine, where levels of CGRP are increased during acute migraine attacks . CGRP is expressed throughout the central and peripheral nervous systems, consistent with control of vasodilatation and transmission of nociceptive information. In migraine, CGRP is released from the trigeminal vascular system. At peripheral synapses, CGRP results in vasodilatation via receptors on the smooth muscle cells. At central synapses, CGRP has been suggested to act postjunctionally on second-order neurons to transmit pain centrally via the brainstem and midbrain to higher cortical pain regions .
There are two forms of this peptide; (i) αCGRP, which is predominantly expressed in the nervous system, and (ii) βCGRP, which is primarily expressed in the enteric sensory system. In the central nervous system (CNS), CGRP is expressed in several regions such as the striatum, amygdalae, hypothalamus, colliculi, brainstem, cerebellum and the trigeminal complex [3–7]. Moreover, CGRP is found in primary spinal afferent C- and Aδ-fibers, which project to the brainstem. However, CGRP and its receptor components have not fully been studied in man due to the fact that the receptor components only fairly recently were characterized.
The receptor for CGRP consists of a complex of a seven transmembrane-spanning protein, calcitonin receptor-like receptor (CLR), a single transmembrane-spanning protein designated receptor activity modifying protein 1 (RAMP1)  and an intracellular protein, receptor component protein (RCP) . Recently, CGRP antagonists have been developed with clinical efficacy for the treatment of acute migraine attacks [10–12]. Consequently, it is of considerable importance to clarify where the CGRP receptor is expressed which would indicate possible sites for the therapeutic effect of these antagonists. Hence, studies have focused on mapping CGRP and its receptor components in the trigeminovascular system and in the brainstem as recently reviewed .
A migraine active region has been demonstrated in the brainstem with positron emission tomography (PET) [14–16]. It has been hypothesized that brainstem stimulation can cause activation of the trigeminovascular system, resulting in CGRP-dependent vasodilatation .
We have investigated in detail the distribution and relationship of CGRP and its receptor components within human and rat spinal trigeminal nucleus (STN) in the brainstem and in the spinal cord at C1-level, using a set of newly characterized antibodies for immunohistochemistry . Our main findings in the present work were that CGRP and the receptor components appear in different structures/regions of STN, and in lamina I and II at C1-level, but do not co-localize. This suggests that C-fiber released CGRP acts postjunctionally on fibers expressing CLR/RAMP1 in these regions.
Postmortem human tissue samples
Samples of STN and C1 were obtained at autopsy from adult subjects in accordance with the Faculty of Medicine University of Szeged guidelines for ethics in human tissue experiments and were approved by the local Hungarian Ethics Committee. The tissue was bilaterally removed from 6 subjects (3 female; 3 male) with an age span of 65 to 86 years. None of the subjects suffered from any central nervous system disease and the cause of death was related to heart failure, septicemia or cancer. The tissues were collected within 24 to 36 hrs after death.
The samples were immersed overnight in fixative consisting of 4% paraformaldehyde (PFA) and in 0.1 mol/l phosphate buffer, pH 7.2. After fixation, the specimens were rinsed in sucrose-enriched (10%) Tyrode solution overnight, frozen and stored at -80°C. The samples were embedded in a gelatin medium (30% egg albumin and 3% gelatin in distilled water) and cryosectioned at 12 μm. The sections were stored at - 20°C until use.
Rat tissue samples
Brainstems were quickly removed from 5 male Sprague-Dawley rats weighing 300-350 g (Scanbur, Stockholm, Sweden). The STN samples were dissected at bregma -14.08, corresponding the caudal part of STN, Sp5C (using brain atlas Paxinos and Watson, second edition, 1986) and C1 were dissected at C1 vertebra. The tissues were immediately placed in 4% PFA and fixed for 2-4 hrs. After fixation the tissues were rinsed in raising concentrations of sucrose in Sörensen's phosphate buffer, embedded, sectioned and stored as the human samples.
Brainstems from 2 additional rats were after removal kept in the refrigerator at +4°C for 24 hrs before they were treated as above (to mimic the autopsy situation in man).
Also, comparison was made to tissue obtained from 2 additional rats that were perfusion fixed with 4% PFA (data not shown). We found no difference in the immunostaining patterns between the two procedures. The experiments were approved by the University Animal Ethics Committee (M8-09), Lund University, Sweden.
Human and rat sections were stained with Hematoxylin-Eosin (Htx-Eosin) using a standard protocol (Htx 3 min, water rinse, Eosin 1 min) for orientation and examination of the tissue condition. The areas within the rat and human brainstem were identified by the use of a brain atlas (Paxinos and Watson, second edition, 1986, and Koutcherov et al. chapter 10 in The Human Nervous System). Sections with STN (the caudal subdivision) or C1, were used and adjacent sections were employed for immunohistochemistry.
Details on primary antibodies used for immunohistochemistry
Name and product code
Calcitonin receptor-like receptor (CLR) 3152
C-terminal of human CLR
Merck & Co., Inc, USA
C-terminal of rat CLR
Merck & Co., Inc, USA
C-terminal of rat CLR
Merck & Co., Inc, USA
Receptor activity-modifying protein 1 (RAMP1) 844
C-terminal of human RAMP1
Merck & Co., Inc, USA
C-terminal of rat RAMP1
Merck & Co., Inc, USA
Calcitonin gene-related peptide (CGRP), polyclonal, B47-1
Europroxima; Arnhem, The Netherlands
CGRP, monoclonal, ab81887
Myelin basic protein (MBP) polyclonal, A0623
Myelin, Schwann cells
Dako; Cophenhagen, Denmark
Synaptophysin, polyclonal, A0010
Synaptic vesicle protein
Dako; Copenhagen, Denmark
Isolectin IB4 Alexa flour 594 conjugate, I21413
C-fibers (in general)
Invitrogen; CA, USA
The primary antibodies were diluted in PBST containing 1% BSA and 3% normal serum. After incubation with primary antibodies, sections were equilibrated to room temperature, rinsed in PBST for 3 × 15 min and exposed to secondary antibodies (for details, see Table 2) in PBST and 1% BSA for 1 hr at room temperature. The sections were subsequently washed with PBST for 3 × 15 min. Vectashield, an anti-fading medium, containing DAPI (Vectashield, Vector Laboratories., Burlingame CA) or glycerol in PBS were used as mounting media.
Secondary antibodies used for immunohistochemistry
Conjugate and host
Cayman Chemical, Ann Arbor, MI
Jackson Immuoresearch, West Grove, PA
Jackson Immuoresearch, West Grove, PA
Alexa 488 (donkey)
Jackson Immuoresearch, West Grove, PA
Jackson Immuoresearch, West Grove, PA
Jackson Immuoresearch, West Grove, PA
Jackson Immuoresearch, West Grove, PA
Controls and DAB staining
Omission of the primary antibody served as negative controls for all antibodies.
To evaluate secondary antibody staining, three different secondary antibodies (Table 2) were tested together with CLR or RAMP1, respectively.
Preabsorption controls with blocking peptides (details on these have been described before in ) were performed with all CLR and RAMP1 primary antibodies. Concentrations of the antibodies were the same as described in Table 1, peptide concentrations were 100:1. The blocking peptides were resuspended in PBS and then incubated at +4°C overnight in PBST containing 1% BSA and 3% normal serum, with or without primary antibodies. The immunostaining protocol was the same as described above. Sections incubated with antibodies alone versus blocked antibodies were compared.
In order to evaluate the fluorescence technique staining, 3,3'-diaminobenzidine (DAB) substrate together with Vectastain ABC kit standard PK-6100 (Vector Laboratories) was performed. In brief, sections were rinsed in PBST followed by incubation with methanolic hydrogen peroxidase (3% H2O2 and 10% MetOH in PBS) to remove endogenous activity. After incubation for 1 h with blocking solution of PBS and 5% normal swine or rabbit serum, the sections were incubated with primary antibodies against CGRP, CLR and RAMP1, at +4°C overnight. At the second day, sections were incubated with biotin-conjugated secondary antibodies, anti-rabbit or anti-goat for 1,5 h (1:400, Dako, Glostrup, Denmark). Visualization was achieved through the ABC kit using DAB/H2O2. Omission of primary antibody served as negative controls.
Sections were examined and images were obtained using a light- and epifluorescence microscope (Nikon 80i, Tokyo, Japan) coupled to a Nikon DS-2MV camera. Adobe Photoshop CS3 (v.8.0, Adobe Systems, Mountain View, CA) was used to visualise co-labelling by superimposing the digital images and to adjust brightness and contrast. In addition, confocal microscopy was performed using Nikon confocal microscope (EZ-cl, Germany), where detailed localization and/or co-localization of immunoreactivity were recorded using the confocal microscope. All pertinent questions were addressed with examination with confocal microscope analysis (see figures). The confocal microscopy was carried out using 20× or 60× oil immersion lenses. Z (frame) stacks were acquired using different laser channels one by one before next z position was acquired. Laser channels used were 488 nm excitation (filter 515/30) and 543 nm excitation (filter 605/75). Image analyses were conducted using NIS basic research software (Nikon, Japan). Briefly, z stacks were 3-dimensionally examined for detailed localization and distribution of immunoreactivity, and for possible co-localization of the antibodies used. The opportunity to move around 3-dimensionally within the 12 μm thick section allows for scrutinizing the detailed localization of the immunoreactivity, but also for thorough evaluation of the immunoreactive structures.
CLR and RAMP1 immunoreactivity
CLR and RAMP1 immunoreactivity
Controls and DAB staining
There was no immunoreactivity observed with preabsorbed CLR or RAMP1 antibodies, using their respective blocking peptides (additional file 3).
DAB stained sections showed similar staining pattern for CGRP, CLR and RAMP1 as seen with the immunofluorescence technique. DAB staining revealed CLR and RAMP1 expression in the walls of capillaries in rat (Figure 12E) and in laminae X, close to the central canal. Furthermore, three different secondary antibodies were tested to ensure that the capillary staining was not due to unspecific binding of the secondary antibody.
Two additional rat brainstems were kept at +4°C for 24 hrs before dissection, fixation and immunostaining to mimic the autopsy situation in man. As in our previous paper on the trigeminal ganglion ; this procedure did not differ in immunoreactivity for CGRP, CLR and RAMP1 as compared to fixation directly of fresh brainstems (data not shown).
We have previously reported in detail the distribution of CGRP and its receptor components in the human trigeminal ganglion . Small to medium sized neurons of the trigeminal ganglion store CGRP and have further connections with STN in the brainstem, and in related extensions down to the C1-2 level via un-myelinated C-fibers . CLR and RAMP1 are expressed in large neurons and thick fibers, which are co-expressed with a marker for Aδ-fibers. These fibers from the trigeminal ganglion extend further to the brainstem. Several studies point to the involvement of the brainstem in migraine pathophysiology [1, 19] and a region within the brainstem has been demonstrated to be active during attacks in migraine patients [14, 20].
The present study has revealed the detailed distribution and relations between CGRP and its receptor components in human and rat projection regions from the trigeminal ganglion. The study demonstrates for the first time the expression of CLR and RAMP1 within human STN in the brainstem and in the spinal cord at C1-level, using a series of novel well characterized antibodies. Thus, this indicates CGRP signaling in areas of the brainstem/spinal cord putatively involved in the migraine pathophysiology. The reason for choosing these regions in the present study is based on a series of detailed neuroanatomical mappings in rat showing pain pathways that may be involved in primary headaches .
Distribution of CGRP, CLR and RAMP1 in STN
We observed CGRP expression around nerve fiber bundles in the superficial laminae of human and rat STN. We found no CGRP expressing neurons in the STN neither in man nor in rat. However, in rat we observed a small group of CGRP positive neurons located close to the central canal, in the inferior olive and in the hypoglossal nucleus of the brainstem. Notably these neurons were absent in man. The results were confirmed using 2 different antibodies against CGRP. The functional role of CGRP in these regions remains to be disclosed. The presence of CGRP in the brainstem is supported by early autoradiography studies [22, 23]. In addition, CGRP expression in STN and spinal cord of different species has been studied [6, 24–26]
The expression of CGRP in the brainstem differs between species . It has been shown that the distribution of CGRP fibers is similar in rat and alpaca brainstem. However, CGRP containing neurons are more widespread in rat than in alpaca. In addition, the localization of CGRP positive neurons in the cat and alpaca brainstem differs . Hence, the distribution pattern observed between rat and man in our study is likely due to species differences.
CGRP and MBP double-staining showed no co-localization, indicating that CGRP is as expected expressed in un-myelinated fibers in STN and this is in agreement with a previous study on the trigeminal ganglion . Synaptophysin, used as a marker for synaptic vesicles, and CGRP were expressed in the same structures within the STN. This supports that CGRP is stored and released from nerve terminals as supported by a study on rat dorsal horn  and acts post-synaptic at CLR/RAMP1 expressing fibers.
In human STN, we found CLR and RAMP1 positive fibers mostly in the spinal trigeminal tract, spanning towards the superficial laminae. In rat STN, expression of CLR and RAMP1 were also found mostly in the spinal trigeminal tract, around fiber bundles and fibers in the spinal trigeminal tract spanning towards the superficial laminae. These results are in contrast to an earlier study by Lennerz et al., 2008, where CLR and RAMP1 expression was detected in the superficial laminae, partially co-localizing with CGRP. The reason for this difference could reside in that different antibodies were used.
There were no neuronal cell bodies immunopositive for CLR and RAMP1 in the human or rat STN. CLR and RAMP1 were observed to be co-expressed in the fibers, suggesting the presence of functional CGRP receptor in both types of species. Interestingly, we found that the receptor components were co-expressed on the walls of capillaries in rat STN
In rat STN, it was suggested that CGRP and its receptor components are localized in terminals from primary afferents . In contrast, CLR did not co-localize with neuropeptides of primary spinal afferents in the dorsal horn of rat . In the present work, we observed no co-localization between the receptor components and CGRP positive fibers, neither in man nor in rat. These results suggest that CGRP and the receptor components appear in nerve terminals, where C-fiber released CGRP may act post-synaptic at CGRP receptors on second-order neurons or modify the responses of trigeminal Aδ-fibers.
Distribution of CGRP, CLR and RAMP1 in C1
We examined the localization and expression of CGRP and its receptor components in the spinal cord at the C1-level, since the main part of the trigeminovascular projection occurs at this level . CGRP positive fibers were found in laminae I and II. Similar staining pattern has been demonstrated previously in the chick, quail dorsal horn of the spinal cord  and in the cat . In addition, we observed some CGRP positive neurons and fibers close to the central canal (laminae X). This finding is in agreement with a previous study in rat spinal cord with neurons being positive for CGRP .
To determine which fibers express CGRP, co-localization experiments were performed with CGRP and IB4. IB4 has previously been used as a marker for C-fibers . In the present study we found that IB4 and CGRP could be expressed in the same laminae, but most often in different types of fibers. This observation was confirmed by confocal microscopy. The IB4 staining was more prominent in the deeper laminae of the spinal cord, which is in agreement with a study of rat spinal cord . It has been shown that the degree of co-expression of CGRP and IB4 in neurons vary in the rat. More neurons expressing both markers are found in dorsal root ganglia compared to the trigeminal ganglion of rat . Electron microscopy showed that IB4 and CGRP expressing axons were distinct, but both could be present in the same bundle of un-myelinated fibers .
The MBP marker showed that in some areas within laminae I and II myelinated fibers are absent. Co-staining experiments with CGRP showed that CGRP is expressed in areas that are absent of MBP, suggesting that CGRP is indeed expressed in un-myelinated fibers.
To further scrutinize the CGRP positive fibers, double-staining of CGRP and synaptophysin was performed. In laminae I and II, where CGRP staining was found, co-expression of CGRP and synaptophysin was observed. With confocal microscopy, we obtained a detailed three-dimensional view of the staining pattern. This clearly showed that CGRP and synaptophysin were detected in the same fibers.
Reportedly, CLR and RAMP1 are expressed in fibers within laminae I and II in the dorsal horn of rat spinal cord . Similar observation but in different tissue was seen in our study; CLR and RAMP1 expressions were found within laminae I and II of human and rat C1. No CLR or RAMP1 positive neuronal cell bodies were observed at the C1-level. In rat, CGRP was expressed in the same laminae, but it did not co-localize with CLR or RAMP1. CLR and RAMP1 were co-expressed, suggesting expression of functional CGRP receptor in fibers within laminae I and II. The presence of receptor components in the spinal cord is supported by the mRNA expression of RAMP1 and RCP, detected with specific oligonucleotides for in situ hybridization .
CLR and RAMP1 were in addition detected in fibers close to the central canal. As described above, CGRP positive fibers were also detected in this area. Interestingly, tracing experiments in cat have revealed projections from the periaqueductal gray (PAG) region to the spinal cord. Horseradish peroxidase (HRP) injections into the PAG region resulted in labeled fibers close to the central canal, terminating in laminae X of C1 and C2-levels . The same authors found labeled fibers in segments C4 to T8 adjoining the ependymal layer of the central canal and next to the basal membrane of the nearby capillaries. Histochemical studies in different species have revealed neurons, axons and terminals within laminae X containing neuropeptides such as substance P . Thus, our results demonstrate fibers containing CGRP and its receptor components close to the central canal. The function of this is not known; one may speculate that these fibers can release CGRP directly into the cerebrospinal fluid or stimulate the ependymal cells of the central canal.
Methodology and technical considerations
The hematoxylin-eosin stained material revealed well-preserved human tissue adequate for immunofluorescence technique, even though the tissue samples were collected 24 to 36 hrs after death. Due to the relatively high age of the subjects, lipofuscin is accumulated in the tissue, causing auto-fluorescence. We have earlier examined, in rat trigeminal ganglion, if storage of the animals for 24 hrs at +4°C prior fixation would affect the immunohistochemistry; which was not the case . Similar results were obtained in the present study on the rat brainstem (data not shown). In rat we also performed a direct comparison of perfusion-fixed and immersion-fixed brainstems, and found no observable difference in antibody expression pattern.
The C1-level compared well in the Paxinos atlas for rat and man. The STN is a structure that is distributed for a considerable length in the brainstem and could therefore not be examined for its entire distribution. It is a limitation of the present study that we only studied a portion of the STN; the exact part is given in the method part.
In our previous study, antibodies against human and rat CLR and RAMP1 were generated, and the specificity of the antibodies was confirmed in HEK293 cells stably expressing the human CGRP receptor. The specificity of the raised antibodies was also confirmed by Western blotting . The same antibodies were used in this study.
The staining of the receptor components was weaker and a bit more diffuse in the human tissue compared to rat. This could be due of several factors: differences in antibodies recognizing the epitopes, tissue condition, or low level of CLR and RAMP1 in human tissue. In rat STN and C1, the RAMP1 antibody displayed a stronger staining pattern compared to the CLR antibody. If this was due to differences in antibody recognizing the epitopes or more expression of RAMP1 in these areas could not be verified.
Within the rat brainstem, we found expression of RAMP1 and CLR in the capillary walls. This was blocked with the specific blocking peptides (epitopes used in the production of the respective antibodies). The staining of the capillaries was similar in the endogenously activity blocked DAB-stained sections.
The neuropeptide CGRP is implicated in the pathophysiology of migraine and the CGRP receptor has long been regarded as a useful target for the development of novel antimigraine therapies. We have described in detail CGRP and its receptor components in the STN and C1 of man and rat using immunohistochemistry. Fibers expressing CGRP and its receptor components occur in STN and C1, however they were not co-expressed in the different areas and laminae. This suggests that CGRP released from C-fibers in the brainstem may act postjunctionally to modulate the activity in fibers that store the CGRP receptor in these regions. Differences in the CGRP expression between the species were observed in other parts of the brainstem. We have also demonstrated fibers and neurons expressing CGRP close to the central canal which suggests that CGRP may have a function within this area. Further efforts are essential to understand CGRP signaling and its function within the brainstem.
Bovine serum albumin
Calcitonin receptor-like receptor
Calcitonin gene-related peptide
Myelin basic protein
Phosphate buffered-saline (PBS) containing 0.25% Triton X-100
Receptor activity-modifying protein 1
Receptor component protein
Spinal trigeminal nucleus
Thanks are due to:
Grant support from the Swedish Research Council.
Christopher A. Salvatore for providing us with the antibodies against RAMP1 and CLR.
Dr. Janos Tajti for the human tissue.
Professor Karin Warfvinge, Warfvinge Science Support (http://www.sciencesupport.se) for assistance and valuable comments on the manuscript.
LRI Instrument AB, Lund, Sweden, especially Joakim Lindelöf, for help and guidance with the microscopic analysis.
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