Molecular studies of CGRP and the CGRP family of peptides in the central nervous system

Abstract

Background

Calcitonin gene-related peptide is an important target for migraine and other painful neurovascular conditions. Understanding the normal biological functions of calcitonin gene-related peptide is critical to understand the mechanisms of calcitonin gene-related peptide-blocking therapies as well as engineering improvements to these medications. Calcitonin gene-related peptide is closely related to other peptides in the calcitonin gene-related peptide family of peptides, including amylin. Relatedness in peptide sequence and in receptor biology makes it difficult to tease apart the contributions that each peptide and receptor makes to physiological processes and to disorders.

Summary

The focus of this review is the expression of calcitonin gene-related peptide, related peptides and their receptors in the central nervous system. Calcitonin gene-related peptide is expressed throughout the nervous system, whereas amylin and adrenomedullin have only limited expression at discrete sites in the brain. The components of two receptors that respond to calcitonin gene-related peptide, the calcitonin gene-related peptide receptor (calcitonin receptor-like receptor with receptor activity-modifying protein 1) and the AMY₁ receptor (calcitonin receptor with receptor activity-modifying protein 1), are expressed throughout the nervous system. Understanding expression of the peptides and their receptors lays the foundation for more deeply understanding their physiology, pathophysiology and therapeutic use.

Keywords

Amylin adrenomedullin calcitonin CGRP central nervous system migraine

Introduction

Calcitonin gene-related peptide (CGRP) is an important sensory neuropeptide that is involved in pain modulation (1,2). CGRP has attracted particular interest in regard to its role in migraine and likely plays a role in other primary headache disorders and painful conditions (2,3). Presently, three investigational classes of drug aim to reduce CGRP activity to prevent and treat migraine (3). These are small molecule antagonists against a CGRP receptor and antibodies that either block receptor activity or bind directly to the CGRP peptide. This ‘first generation’ of CGRP-based treatments will likely lead to further drugs over time, which will result from a deeper knowledge of the CGRP system at a cellular and molecular level and a greater understanding of the molecular neuroanatomy of CGRP and its receptors. This information guides understanding of the relative role of central and peripheral processes in which CGRP participates in the pathophysiology of migraine, and other disorders.

Understanding CGRP biology is complex due to the existence of similar peptides in the same family, and shared receptor complexes. The aim of this review is to summarize information about the expression of CGRP and to compare this to its binding sites and molecularly defined receptors. We will detail what is known about the expression of CGRP and related peptides, where binding sites for labelled peptides are found, and what information is available for the molecular correlate(s) to that binding. We focus our attention on the central nervous system (CNS) and peripheral ganglia, with emphasis on brain regions that are of particular relevance to migraine. The sensory circumventricular organs (CVOs) will also be considered, as circulating factors can act directly on these sites within the brain.

Migraine and the nervous system

Historically, there has been considerable debate around the importance of central and peripheral mechanisms of migraine. This initially took the form of the competing vascular and neuronal hypotheses for migraine. It is now clear, based on recent clinical trials, that migraine can be treated through peripheral blockade of CGRP action (3,4). This does not preclude a central origin of migraine. Although the peripheral aspects of the trigeminovascular system are important in migraine, it is still worthwhile to consider central contributions (5). Numerous regions of the CNS are activated during a migraine attack and central phenomena are associated with migraine symptoms, such as the link between cortical spreading depression and migraine aura (3). It is unclear whether blocking central CGRP action may be beneficial or perhaps a hindrance in treating migraine. However, central CGRP receptors should be an important consideration for developing a ‘second generation’ of CGRP system-based treatments with potentially greater effectiveness or fewer side effects.

This review will focus on the craniofacial pain pathway and other brain regions associated with migraine. The craniofacial pain pathway begins with Aδ and C fibres of the trigeminal nerve, whose cell bodies are located peripherally in the trigeminal ganglia and project centrally primarily into the spinal trigeminal nucleus (STN) of the brainstem and into the C1/C2 levels of the spinal cord. The STN is proposed as a possible site of migraine initiation and displays increased activity immediately prior to a migraine attack (6). Higher order processing of painful signals primarily involves the thalamus, insular cortex and somatosensory cortex. Interconnected with these regions are other sites, including the amygdala, raphe nuclei, periaqueductal gray (PAG), parabrachial area, gracile nucleus and locus coeruleus, which play roles in pain processing, proprioception, stress or aversion. Interestingly, in migraine patients, altered connectivity between these regions is reported, such as from the thalamus to the insular cortex and somatosensory cortex or from the PAG to the cortex and amygdala (7,8). Other brain regions involved in migraine may include the hypothalamus, implicated in attack initiation; the hippocampus, which displays greater pain-induced activity in migraine patients; and the cortex, cerebellum and visual network, which may be involved in cortical spreading depression and symptoms of migraine aura (9 –11).

The calcitonin gene-related peptide family of peptides

CGRP belongs to a small family of structurally related peptides (Figure 1). There are two forms of CGRP, α and β. The broad term “CGRP” is used for either α or β, unless specifically noted. The other major members of this family are amylin, adrenomedullin (AM) and adrenomedullin 2 (AM2). Another member of this family, the calcitonin receptor-stimulating peptide (CRSP) was reported in some mammals (12); however, CRSP is not expressed in primates or rodents. Each of these peptides contains a conserved pair of cysteine residues that form a disulfide bond, creating a loop. They also all contain a C-terminal amide. Amylin and CGRP are the most closely related peptides in terms of amino acid sequence, resulting in overlapping actions in pain modulation and nutrient balance (13,14). The similarities between the peptides within the CGRP family causes significant overlap in their ability to activate each other’s receptors. This “blurred” receptor pharmacology is particularly evident at rodent receptors, where there is noticeable cross-reactivity. This makes working with this peptide family challenging because it is often very difficult to determine which of the many possible molecularly defined receptors actually mediates an effect for a given peptide (15).

Figure 1.

Amino acid sequences of calcitonin gene-related peptide family members. Alignment of the amino acid sequences for human αCGRP, βCGRP, calcitonin (CT), AM, AM2 and amylin using the single letter code. AM and AM2 have been truncated at the N-terminal. All the peptides have a C-terminal amide (-NH₂). A conserved disulfide bond between the two N-terminal cysteine residues is indicated by a solid line.

Receptor composition and pharmacology

The molecular composition of the CGRP family of receptors is illustrated in Figure 2, which highlights the overlapping activity of the peptides at the different receptors. Overlap occurs, not only because the peptides have shared features but also because the receptors themselves have shared and closely related components. All of the receptors within this family are G protein-coupled receptors (GPCRs). Two specific GPCRs form high affinity receptors for the different peptides due to their association with accessory proteins. There are three of these accessory proteins, receptor activity-modifying protein (RAMP) 1, 2 and 3. The CGRP, AM₁ and AM₂ receptors consist of the calcitonin receptor-like receptor (CLR) and RAMP1, RAMP2 or RAMP3, respectively (16,17). The AMY₁, AMY₂ and AMY₃ receptors consist of the calcitonin receptor (CTR) with each RAMP (17,18). CLR alone does not appear to act as a functional receptor, whereas CTR is the receptor for calcitonin (16,17). Figure 2 shows that CGRP can activate both CLR/RAMP1 and CTR/RAMP1 equally. CGRP is weaker at other human receptor complexes, but there are clear deviations in other species (15). At rat CLR and CTR-based receptors, CGRP is potent at both RAMP3-based receptors (19). Amylin can potently activate CTR/RAMP1, CTR/RAMP2 and CTR/RAMP3 but not the CLR-based receptors. AM activates all three CLR/RAMP complexes but has a preference for CLR/RAMP2 and 3 (17).

Figure 2.

The calcitonin gene-related peptide (CGRP) receptor family. The relative potency of CGRP, calcitonin (CT), AM, AM2 or amylin is shown below a schematic representation of the appropriate receptor complex (17). The potential overlap between the CGRP and amylin receptors is highlighted in purple. The receptor components are described in the legend.

One pharmacological method for teasing apart closely-related receptors with overlapping peptide binding parameters is to use specific antagonists. Although several antagonists have been reported that are capable of antagonizing members of the CGRP receptor family, they generally lack the required specificity. For example, although CGRP_8-37, an N-terminally truncated form of CGRP, is often cited as a specific CGRP receptor antagonist, it does not display sufficient specificity to be practically useful. CGRP_8-37 is less than 10-fold selective for CGRP receptors over AMY₁ and is capable of antagonizing AM₂ and AMY₃ receptors (19 –22). Similarly, CTR and amylin receptor antagonists including truncated salmon calcitonin (sCT_8-32) and AC187 are potent antagonists, but do not distinguish between amylin receptor subtypes (19,21). The development of the small molecule “gepant” class of drugs has yielded some useful tools for separating the CGRP receptor from AM receptors and, if used carefully, can tell apart the CGRP and AMY₁ receptors (Table 1). For instance, olcegepant displays greater than 10,000-fold selectivity for the CGRP over AM receptors and 100–200 fold selectivity for the CGRP receptor over the AMY₁ receptor (23 –25). However, other gepants including telcagepant and MK-3207 are less selective and the degree of selectivity measured may depend on other variables, including the signaling pathway measured (23,26). With the careful selection of several different agonist and antagonist concentrations, it may be possible to pharmacologically characterize members of the CGRP receptor family present in a particular cell line or tissue. However, for practical use in in vivo systems more specific pharmacological tools are required.

Table 1.

Summary of small molecule CGRP receptor antagonists (gepants) for migraine treatment.

Small molecule	Olcegepant	Telcagepant	MK-3207	BHV-5000	Atogepant	Ubrogepant	Rimegepant
Blocks CGRP receptor	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Blocks AMY₁ receptor¹	Yes (∼100–200 fold lower)	Yes (∼50–100 fold lower)	Yes (∼50 fold lower)	No data	No data	No data	No data
Clinical development	Development stopped	Development stopped	Development stopped	Preclinical	Phase 2	Phase 3	Phase 3

No data, no published data is available.

The relative affinity or potency for the AMY₁ receptor compared to the CGRP receptor is reported in parenthesis (20,23,26,154).

Challenges associated with determining the molecular target for a given peptide

To understand the biology of the CGRP peptide family, it is crucial to define the molecular identity of the peptide and receptor responsible for a biological effect in physiological systems. The overlapping pharmacology described above represents a major challenge associated with studying this important family of receptors. Therefore, pharmacological approaches should be complemented with other methods. The measurement of mRNA can be a useful guide. However, the detection of mRNA does not necessarily mean that protein is present, especially in neurons where proteins can be transported to projections distant from the cell body (27). Stable membrane proteins including CLR, CTR and RAMPs may not need high levels of mRNA expression to maintain protein expression in steady-state cells. This has been illustrated where moderate to high levels of immunoreactivity were observed in regions where the corresponding mRNA was low or not detected (28,29).

The development of techniques including high-resolution confocal imaging has allowed researchers to detect the precise cellular localization of a receptor using antibodies. However, when used to directly detect protein expression, antibodies also have limitations. They require comprehensive validation to confirm specificity and selectivity for their targets (30). In many cases, antibodies are used without sufficient evidence of validation and it may not be possible to draw the conclusions that the authors suggest. For example, studies may simply lack the required information regarding the antibodies used to allow evaluation of the data (31) or the antibodies used have been shown to recognize multiple proteins. This prevents observed immunoreactivity from being conclusively associated with a single protein (30). The latter situation has been a significant issue for antibodies designed to detect RAMPs (32).

Even if a receptor subunit has been identified with a well-validated antibody, this alone is not meaningful for this receptor family, except when considering calcitonin activity at CTR alone. Expression of CLR or CTR by themselves says little about the receptor phenotype without further examining potential co-localization with a RAMP. RAMPs are also known to modify the signaling of other receptors, forming alternative complexes that complicate interpretation (33). However, valuable information has been obtained from studies showing co-localization between CLR and receptor-component protein or between CLR and RAMP1, indicating the likely presence of the CGRP receptor (34 –36). The ideal solution maybe to develop antibodies that specifically target the heteromeric complex. This strategy was employed to detect the CLR/RAMP1 complex in the trigeminal ganglia, dura mater and the spinal trigeminal nucleus (37). However, antibodies that recognize heteromeric complexes are difficult to generate and characterize. Thus, protein expression data should be used alongside other experimental observations. For example, mRNA and peptide binding correlations were informative in early studies validating CLR/RAMP complexes as receptors for CGRP and AM (38). Another consideration relates to the level of expression reported. For many receptors, there is considerable amplification in signal following ligand-receptor binding. Hence, relatively low expression does not automatically translate into little function.

Regardless of method, much research relies on qualitative description of the biological target within anatomical locations. This is a highly subjective process, and is open to individual interpretation of intensity and location. Collating data, improving its accessibility, and standardisation all help to address these issues. For example, the Human Protein Atlas, the Allen Brain Atlas and other web-based tools provide excellent resources for expression data, assisting identification of patterns (39 –41).

Peptide accessibility to the CNS and relevance of receptors in different locations

The blood-brain barrier (BBB) is an important consideration when looking at the origin, expression and activity of neuropeptides in the CNS. Peptides present in the blood may cross the BBB either through active transport or passive diffusion (42,43). Alternatively, circulating peptides can interact directly with CVOs. CVOs are highly vascularized, with fenestrated capillaries, allowing peptides access to these discrete parts of the CNS. The CGRP family of peptides do not freely cross the BBB (44). The proportion of peripheral amylin that can cross the BBB is low and is unlikely to be sufficient to activate receptors inside the BBB at physiological concentrations (45,46). Interestingly, the highest amount of labelled amylin that was reported to penetrate the brain was observed in the hypothalamus and medulla (46), which are associated with sensory CVOs and thus are permeable to circulating hormones. Hence, relatively high levels of amylin experimentally reaching these regions is not surprising. The reported actions of exogenous amylin at sites inside the BBB, such as the ventral tegmental area, are unlikely to be explained by amylin crossing the BBB (47 –49). Alternative explanations could include local production of amylin or another member of the CGRP peptide family triggering receptor activation in these regions. The BBB penetrance of CGRP and AM have been examined primarily in vascular models, which suggest that neither peptide can cross the BBB when the vascular endothelium is intact (50 –52). A more definitive study has been performed for AM, which, under normal conditions, does not appear to cross the BBB and penetrate the brain (53). Curiously, definitive experiments have not been performed for CGRP. However, given the lack of brain penetrance displayed by calcitonin, amylin and AM and data from vascular models, significant crossing of the BBB by CGRP seems unlikely. Overall, the low BBB permeability for CGRP family peptides suggests that CNS expression of these peptides is required for them to have activity inside the BBB.

AM and AM2

AM and AM2 expression in the CNS

AM is best known as a regulator of the cardiovascular and lymphatic systems. It is a potent vasodilator and is highly expressed in the vascular endothelium (54,55). Vascular AM may be involved in maintaining the BBB, cerebral circulation and the volume of cerebrovascular fluid (56 –58). Expression in the vasculature may complicate expression analysis that relies on homogenized tissue, including mRNA analysis and membrane binding. mRNA and immunoreactivity for AM has been reported in the human and rat brain, localized to the vasculature, the choroid plexus and in neurons and glia of the hypothalamus, cerebellum and medulla (59 –63). However, the cerebellum, which has been implicated in AM-regulated blood pressure control, was the only brain region where mature AM peptide was detected (59,64). Interestingly, in a genetic mouse model that was modified to lack AM expression in the CNS, altered responses to pain, anxiety and stress were observed (65,66). Similarly, AM has been reported to induce pain, and AM-like immunoreactivity was detected in dorsal root ganglia neurons and axon terminals in the dorsal horn from rats (67,68). These studies suggest that AM may play a physiological role in the CNS despite limited expression.

The physiology of AM2 is poorly understood; however, central or peripheral administration can modulate blood pressure (69). AM2 mRNA and immunoreactivity has been found in the pituitary and hypothalamus of humans and rats as well as the spinal cord and dorsal root ganglia of rats (70 –74). AM2 may be involved in the hypothalamo-pituitary axis and the central control of blood pressure in the hypothalamus (69,75 –77).

AM and AM2 binding sites in the CNS

AM binding sites have been measured in rat and human brain homogenates, with high levels of ¹²⁵I-AM binding in the hypothalamus, thalamus and spinal cord (72,78 –80). AM2 binding sites in the CNS have not been specifically studied. More work is required to determine the prevalence and location of AM and AM2 binding sites within the brain.

Molecular composition of AM and AM2 binding sites in the CNS

CLR, RAMP2 and RAMP3 are the most relevant to the AM peptides (Figure 2; Hay et al., 2017). The expression of CLR mRNA and protein has been studied extensively within the nervous system, where it is widespread (Table 2 (28,70,81 –83)). Thus, the presence of functional AM₁ and AM₂ receptors is dependent upon the expression of RAMP2 or RAMP3, respectively. The expression of mRNA for these receptor components has been well documented in the vasculature (50,84,85). However, there is only limited data describing RAMP2 or RAMP3 in discrete rat nervous system regions (Table 2). In rats, abundant mRNA for RAMP2 was detected in the hypothalamus, and RAMP3 was detected in nuclei of the thalamus (86,87). Relatively high RAMP2 expression was also detected in the hippocampus and spinal cord (70,86). RAMP2 and RAMP3 have not been examined in a human brain (Table 3). Unfortunately, there are currently no antibodies for the RAMP2 or RAMP3 proteins that have been sufficiently characterized for use in immunohistochemical mapping (33). Characterized antibodies that have been used for the detection and mapping of CLR, CTR and RAMP1 are discussed later.

Table 2.

Expression of CGRP family receptor components and peptides and their binding sites in rodents.

	Receptor subunit					Binding sites			Peptide
Region	CLR	CTR	RAMP1	RAMP2	RAMP3	¹²⁵I-CGRP	¹²⁵I-Amy	¹²⁵I-sCT	CGRP	Amylin	References
Trigeminal ganglia	MP	P	MP						MP	P	(20, 29, 35, 92, 93, 132, 133, 138, 139, 155 -158)
Dorsal root ganglia	MP	P	MP¹	M	M				MP	MP	(29, 45, 70, 107, 128, 131, 158 -161)
CVOs
Area postrema		MP	M	M	M	Y	Y	Y	P¹		(99, 105, 115, 139, 145, 146, 150, 151, 162, 163)
Subfornical organ		M	M	M	M	Y	Y	Y	P¹		(87, 99, 115, 145, 146, 150, 151)
OVLT		M				Y	Y	Y	P		(99, 115, 139, 145, 146, 162)
Pituitary		M		M			N	Y	P		(87, 99, 115, 139, 164)
Median eminence							N				(115)
Subcommisural organ			M				N				(115, 151)
Pineal gland
Spinal cord	MP	MP	MP	M	M	Y		Y	MP	P	(29, 67, 70, 82, 86, 87, 92, 97, 101, 131, 139, 140, 146, 158, 161, 165)
Medulla	MP	MP	MP	M	M	Y	Y	Y	MP		(29, 81, 82, 86, 87, 92, 97 -99, 105, 115, 132, 139, 145 -148, 150, 151, 158, 162 -164)
Cerebellum	MP	P	MP	M	M	Y	N	N	P		(81, 86, 87, 97 -99, 105, 115, 143, 145 -148, 151)
Pons	P	MP	P	M		Y	Y	Y	MP		(29, 81, 87, 92, 97, 105, 115, 145 -147, 162, 163, 166)
Midbrain		MP	MP	M	M	Y	Y	Y	MP		(29, 47, 81, 92, 97 -99, 105, 115, 139, 145 -148, 162 -164, 167)
Periaqueductal grey	P	MP	M		M	Y	N	Y	P		(29, 86, 92, 97, 98, 105, 115, 139, 145, 153, 162)
Hypothalamus	MP	MP	M¹	M	M	Y	Y	Y	MP	MP	(29, 81, 86, 87, 92, 97, 98, 105, 114, 115, 139, 145 -148, 150, 158, 162 -164, 168)
Thalamus	P	M	M¹P	M	M	Y	Y¹	Y	MP		(81, 86, 87, 92, 99, 115, 145, 146, 150, 151, 162, 169)
Amygdala	M	MP¹	M	M	M	Y	Y	Y	P		(86, 87, 92, 97 -99, 105, 115, 139, 145, 146, 148, 151, 152, 162 -164, 170)
Basal ganglia	M	MP	M	M		Y	Y	Y	MP		(29, 86, 87, 92, 97 -99, 105, 115, 139, 145, 146, 148, 151, 152, 162 -164, 170, 171)
Hippocampus	P	MP¹	MP	M		Y	N		MP		(29, 81, 86, 87, 115, 139, 145, 147, 151, 158, 170)
Septal area		MP	P	M		Y	N	Y	P		(29, 81, 86, 87, 97, 99, 105, 115, 139, 145, 162, 170)
Cerebral cortex	P	M	MP	M	M	Y	N	N	P		(81, 86, 87, 97, 98, 115, 145, 146, 150, 151, 158, 170)

M: mRNA detected by polymerase chain reaction or in situ hybridisation; P: protein/immunoreactivity in tissues; Y: specific radioligand binding detected; N: no binding detected; Empty space: no expression or data not available; CVOs: circumventricular organs; OVLT: organum vasculosum of lamina terminalis.

Very low expression.

Table 3.

Expression of CGRP family receptor components and peptides and their binding sites in primates including humans.

	Receptor subunit					Binding sites			Peptide
Region	CLR	CTR	RAMP1	RAMP2	RAMP3	¹²⁵I-CGRP	¹²⁵I-Amy	¹²⁵I-sCT	CGRP	Amylin	References
Trigeminal ganglia	MP	P	P						MP		(20, 35, 37, 83, 133, 172)
Dorsal root ganglia	MP								MP		(140)
CVOs
Area postrema	MP	P	MP				Y	Y			(28, 95, 96, 106, 163)
Subfornical organ							Y	Y			(95)
OVLT							Y	Y			(95)
Pituitary	MP		MP			N					(28, 101)
Median eminence	MP		MP								(28)
Subcommisural organ
Pineal gland	MP		MP								(28)
Spinal cord	MP		MP					Y	P		(28, 82, 95, 101, 140)
Medulla	MP	P	MP			Y	Y	Y	P		(20, 28, 37, 82, 95, 96, 106, 147, 163, 173)
Cerebellum	MP		MP			Y	NS	N	P		(34, 95, 101, 147)
Pons	MP		MP			Y	Y	Y	P		(28, 95, 147, 163, 173)
Midbrain	MP		MP			Y	Y	Y			(28, 95, 101, 147, 163)
Periaqueductal grey	MP		MP			Y	N	Y	P		(28, 95, 147, 173)
Hypothalamus	MP		MP			Y	Y	Y			(28, 95, 101, 147, 163)
Thalamus	P					Y	Y	Y			(95, 147)
Amygdala						Y	Y	Y			(95, 147, 163)
Basal ganglia						Y	Y	Y			(95, 147, 163)
Hippocampus							NS	Y			(95)
Septal area							Y	Y			(95)
Cerebral cortex								Y			(95)

M: mRNA detected by polymerase chain reaction or in situ hybridisation; P: protein/immunoreactivity in tissues; Y: specific radioligand binding detected; N: no binding detected; Blank space: no expression or data not available; NS: non-specific binding; CVOs: circumventricular organs; OVLT: organum vasculosum of lamina terminalis.

Very low expression or binding.

Calcitonin

Calcitonin expression in the CNS

Canonically, calcitonin is expressed by thyroid C cells and regulates calcium homeostasis (88 –90). The CALCA gene encodes calcitonin and CGRP mRNA (91). Tissue-specific alternative splicing of CALCA mRNA results in the preferential expression of CGRP mRNA in the nervous system; calcitonin mRNA is not detectable in these regions (1,88,92,93).

Calcitonin binding sites in the CNS

Although calcitonin does not appear to be expressed in the nervous system, binding sites for calcitonin are found in many brain structures (94 –96). Salmon calcitonin has frequently been used as a tool in calcitonin studies because it has higher affinity than human or rat calcitonin and can bind CTR alone, AMY₁, AMY₂ and AMY₃ receptors with high affinity (18). Thus, depending upon the presence or lack of a RAMP, salmon calcitonin binding may indicate the presence of a receptor for amylin, CGRP or calcitonin. Salmon calcitonin binding sites have been examined in rat and primate nervous systems using autoradiography (Tables 2 and 3). The data are generally consistent between these studies with only minor differences. Overall, autoradiography shows high levels of salmon calcitonin binding in the hypothalamus, sensory CVOs and the nucleus accumbens, along with moderate binding in the bed nucleus of the stria terminalis (BNST), PAG and locus coeruelus (95,97 –100). The spinal cord has minimal salmon calcitonin binding in the dorsal horn (95,97). There also seems to be very little binding in the cerebral cortex and in the cerebellum. Data from the human brain is more limited. In a single study, salmon calcitonin binding was examined by autoradiography in the human medulla (Table 3). Discrete binding sites were observed in several nuclei, including high densities of binding in the NTS and raphe nuclei. Very little binding was observed in the fibre tracts (96). In another study, human hypothalamic membranes showed high levels of salmon calcitonin binding (101). The variability in displacement of salmon calcitonin by human calcitonin between brain regions suggests that AMY receptors account for a significant proportion of salmon calcitonin binding sites (102,103). However, more work is required to determine the prevalence and location of calcitonin binding sites within the human brain, and their direct relevance to calcitonin, amylin and/or CGRP activity.

Molecular composition of calcitonin binding sites in the CNS

Consistent with widespread calcitonin binding sites, CTR mRNA and protein expression has been observed throughout the nervous system of rodents (Table 2). Several splice variants of the CTR have been reported. These variants are not necessarily conserved across species and can display altered functions (104). This review does not distinguish between these, because there are no tools to determine the expression profile of specific splice variants at the protein level. We discuss CTR only in general due to limited data, even at the mRNA level. In the peripheral nervous system, CTR staining was observed in neuronal cell bodies in the trigeminal and dorsal root ganglia (20,29). Centrally, dense protein expression was reported in fibres and cell bodies of brain regions including the nucleus accumbens, the substantia nigra, the NTS and the area postrema (105). Moderate staining of cell bodies and fibres was observed in the spinal cord, amygdala, PAG and dorsal raphe nucleus (105). Limited but discrete staining has also been observed in the thalamus, BNST and regions of the somatosensory cortex (29,105). Interestingly, CTR protein has also been reported in several regions involved in the perception and higher-order processing of craniofacial pain (Table 2). The pattern of CTR expression in human neural tissues is similar to that of rodents, although data are limited to the trigeminal ganglia and the medulla (Table 3; (20,106). Widespread expression of CTR was observed in the human medulla, including the NTS, STN, cuneate, gracile and hypoglossal nuclei (106). Considering the lack of calcitonin expression in central and peripheral nervous tissues and its limited ability to cross the BBB, it is surprising that CTR is so widely expressed within the nervous system. This suggests that the CTR probably functions in concert with one or more RAMPs inside the nervous system to bind amylin, CGRP, or another peptide.

Amylin

Amylin expression in the CNS

The pancreatic β-cell is a major site of amylin expression and releases amylin into the circulation to induce meal-ending satiation (13). Early reports show a lack of amylin expression in the nervous system (107,108). However, later work reported limited amylin expression within the brain and in sensory neurons (Table 2; (109 –111). It is likely that some amylin antibodies can also detect CGRP and some CGRP antibodies can detect amylin. Potential cross-reactivity between amylin and CGRP antibodies may account for some discrepancies between studies (112). Hence, care should be taken to confirm that antibodies detect only the desired target. More recently, amylin mRNA expression was reported in the preoptic area and BNST neurons of lactating dams (113). Amylin mRNA and immunoreactivity has also been reported in hypothalamic neurons in mice (114). Although amylin expression has not been examined in the human brain (Table 3), the limited amylin expression in the CNS suggests that its central actions likely occur predominantly outside the BBB through the CVOs, although discrete expression in a limited number of CNS regions is possible.

Amylin binding sites in the CNS

Despite the reportedly low expression of amylin in the CNS, ¹²⁵I-amylin binds with widespread distribution in rat brain when measured using autoradiography (Table 2; (115)). This included moderate to high levels of binding in the nucleus accumbens, BNST, amygdala, hypothalamus, locus coeruleus, the NTS and the sensory CVOs – the area postrema and subfornical organ. These CVOs can access circulating amylin, consistent with the physiology of amylin following its release from the pancreas (13,116). A similar pattern of binding was observed in primates, with the hypothalamus described as having the densest binding of ¹²⁵I-rat amylin (Table 3; (95)). If amylin is produced in the hypothalamus, then hypothalamic binding sites for amylin imply a paracrine action of the peptide. However, the physiological relevance of other amylin binding sites inside the BBB is not clear.

Molecular composition of amylin binding sites in the CNS

Amylin potently activates CTR in complex with RAMP1, RAMP2 or RAMP3. As described above, CTR is present in many regions of the brain. In a similar manner to the AM receptors, the presence of CTR alone is not sufficient evidence for the existence of a high affinity amylin receptor. RAMP must be co-localized. For RAMP2 and RAMP3 there are only mRNA studies in rodents (Table 2; (86)). Interestingly, CTR, RAMP1, RAMP2 and RAMP3 mRNA were all identified in single area postrema neurons (117). For RAMP1, there is protein data in the context of the CGRP receptor, which is discussed below (Tables 2 and 3). There has been very little investigation of CTR alongside RAMP1. To date, only a single study has examined the co-expression of CTR and RAMP1 proteins using validated antibodies. This study suggested that these proteins are co-expressed in neuron cell bodies from human or rat trigeminal ganglia and in fibres from the human spinal trigeminal tract (20). As noted above, given the lack of amylin in the brain, it is unclear why CTR and RAMP proteins are co-expressed or why is there so much CTR in the brain. One possibility is that the CTR acts as a receptor component for another endogenous ligand. It has been suggested that the AMY₃ receptor could be activated by amyloid β42 and may contribute to Alzheimer’s disease (118). However, this is difficult to rationalize based on the known structure-function relationships between the receptors and their ligands for the CGRP peptide family (119 –121), and contradicts the generally positive benefits amylin is reported to have in Alzheimer’s disease models (122). Furthermore, amyloid β42 was unable to activate amylin receptors in a separate study (123). A more likely explanation is that CTR complexes with RAMP1 to form receptors for CGRP in the CNS. Perhaps both the CLR/RAMP1 CGRP receptor and AMY₁ are needed to exert the full repertoire of effects of CGRP.

Calcitonin gene-related peptide (CGRP)

CGRP expression in the CNS

CGRP is implicated in a wide range of physiological effects including vasodilation, nociception and energy metabolism (2,124 –126). In most studies, αCGRP and βCGRP cannot be distinguished, partly due to a lack of specific antibodies. However, αCGRP and βCGRP mRNA are both expressed in the central and peripheral nervous systems and can coexist in a single neuron (127 –130). CGRP immunoreactivity was reported throughout the nervous system of αCGRP knockout mice, suggesting that βCGRP is present throughout the nervous system (131).

CGRP expression has been primarily studied in rats; despite its value, primate and human data are more limited (Tables 2 and 3). Unsurprisingly, CGRP has widespread expression. Peripherally, CGRP is expressed in Aδ and C fibres of the trigeminal nerve and neuronal cell bodies in the trigeminal ganglia and in dorsal root ganglia neurons (1,2,70,82,93,132 –138).

In the spinal cord, CGRP is expressed in fibres in both the ventral and dorsal horns of rats, predominantly in the outer laminae (70,82,92,139,140). Some cell bodies containing CGRP were also detected in the ventral horn (110). In human tissue, CGRP immunoreactivity was present in laminae I and II, consistent with rat studies (141).

In the brain, CGRP expression is abundant and there are discrete patterns of expression in regions studied in the rat (2,81). Areas positive for CGRP mRNA or protein in rats include the brainstem, cerebellum, midbrain, pituitary, hypothalamus, hippocampus, thalamus, amygdala and the BNST (Table 2). In most brain regions, CGRP immunoreactivity was present in the soma of neurons (81). CGRP-expressing fibres are less common, with the exception of the septal nucleus (81,142). The brainstem appears to have particularly high expression of CGRP, with staining in neuronal cell bodies of all brainstem nuclei, including the locus coeruleus, raphe nuclei, gracile nucleus and STN, consistent with a central role in migraine (81,92,139,141). In contrast, the hippocampus has generally weak CGRP staining in neurons (81,92). The Purkinje cells of the cerebellum stain strongly for CGRP in rat (143). Data describing the expression of CGRP in peripheral and central glial cells is somewhat conflicting, with both positive and negative reports (35,132,144). Of all the CGRP family peptides, CGRP appears to be the most abundant and most widespread in the CNS.

CGRP binding sites in the CNS

Consistent with its widespread expression, autoradiography has detected CGRP binding sites throughout the brain and spinal cord (Tables 2 and 3; (145 –147)), with some discrepancies between studies. For example, contrary results were reported for ¹²⁵I-CGRP binding in the nucleus accumbens (147,148). Species differences may also exist – for example, in rat brain, ¹²⁵I-CGRP binding sites in the nucleus accumbens were more sensitive to competition with salmon calcitonin than corresponding sites in monkey brain. Experimental differences make these results difficult to compare, but this may suggest differences in the underlying molecular nature of the CGRP receptors present (148,149). The reported CGRP binding sites could be a mixture of CGRP and AMY₁, with contributions from other receptors such as AM₂ and AMY₃ receptors, depending on the species.

Molecular composition of CGRP binding sites in the CNS

CGRP and AMY₁ receptors comprise either CLR or CTR and RAMP1 proteins (17). The majority of research has focused on examining the canonical CGRP receptor: CLR and RAMP1 (Table 2; (70,86,150 –152). The availability of specific CGRP receptor antibodies has been relatively recent and their usage limited; however, several studies have mapped the expression of these receptor proteins in the nervous system (Tables 2 and 3; (28,35,70,81).

In rat and human trigeminal ganglia, CLR and CTR mRNA and protein have been detected (Tables 2 and 3). CLR and CTR appeared to have limited co-localization, whereas both co-localize with RAMP1 in neuron cell bodies (20,35,132). This suggests that both AMY₁ and CGRP receptors are present. Interestingly, CGRP and CLR/RAMP1 are rarely localized within the same neurons, indicating that CGRP released from neuron cell bodies may act locally upon a distinct population of neurons (35,132,133). It is not known whether CTR/RAMP1 and CGRP are co-localized in the trigeminal ganglia, but it is possible that CGRP acts in an autocrine manner via this receptor, and a paracrine manner via CLR/RAMP1.

In the rat, CNS, CLR and RAMP1 appear to be widely expressed (Table 2; (81,82)). CLR and RAMP1 immunoreactivity was reported primarily in neuronal processes or fibres. In the brainstem, CLR and RAMP1 staining was observed in fibres from several nuclei including the STN, gracile and hypoglossal nuclei. Staining was also observed in cell bodies and some fibres in the locus coeruleus and raphe nuclei (81). Fibres that span the cerebral cortex of rats were reported to express CLR and RAMP1 (81). Staining was also reported in the Purkinje cells and neuronal processes in the molecular layer of the cerebellum (81,143). Some staining for CLR and RAMP1 was observed in fibres from thalamic nuclei and the cell bodies of neurons in the PAG (81,153). Data from human and primate CNS are more limited (Table 3). In the human brain, CLR/RAMP1 protein has been co-localized in the STN and cerebellum (34,82). The pattern of expression is similar to that reported in rats. It has been suggested that there may be more RAMP1 present than CLR in some regions, and that CGRP binding does not always coincide with CLR mRNA expression (28,86,152). Attempts have been made to describe the ratio of CLR to RAMP1, but this has not been conclusively quantified (81,86). The presence of AMY₁ receptors, which could act as a receptor for CGRP, could account for any discrepancies.

The widespread expression of CTR mRNA and immunoreactivity throughout the nervous system is described above. CTR is reported at many sites that express RAMP1 throughout the brain (29,105,106). Additionally, CTR and RAMP1 protein were co-localized in the human spinal trigeminal tract (20). Further studies are required to determine the extent of AMY₁ receptor expression within the nervous system and the importance of this receptor for the physiological actions of CGRP. Tables 2 and 3 clearly identify the knowledge gaps that need to be filled in order to understand the contribution of this receptor to CGRP (or amylin) biology.

Concluding remarks

The expression of CGRP and related peptides has been extensively studied in the CNS. Coupled with receptor binding and expression studies, this research provides some insight into how these peptides may contribute to various biological functions. However, the overlapping expression and receptor pharmacology of these peptides, coupled with the lack of specific antibodies and effective pharmacological tools, has made determining the specific receptors involved in pharmacological and physiological responses difficult. There is still more that needs to be learned. The physiological contributions of the two receptors for CGRP remains poorly described. The co-expression of the subunits for the AMY₁ receptor (subtype) has only been detailed in the trigeminal ganglia and spinal trigeminal tract. Although the molecular composition of receptors for CGRP in the CNS is not always clear, CGRP and its receptor components are widespread throughout the CNS. In particular, CGRP and components for both CGRP and AMY₁ receptors are present at several central sites involved in processing of pain and other migraine symptoms. The potential role of the CVOs in responding to circulating signals, such as increased peripheral CGRP during a migraine attack, should also be considered. In the future, both peripheral and central sites could be targeted to more effectively treat migraine.

Article highlights

CGRP is expressed throughout the nervous system. Amylin and adrenomedullin display limited expression at discrete sites in the brain.

CGRP peptide family members do not cross the blood-brain barrier in a physiologically relevant manner.

The components of two receptors that respond to CGRP, the CGRP receptor (CLR and RAMP1) and the AMY₁ receptor (CTR and RAMP1), are expressed throughout the nervous system.

Footnotes

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: Christopher S Walker and Debbie L Hay have received research support from Alder Biopharmaceuticals.

Funding

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Christopher S Walker is supported by a Sir Charles Hercus Health Research Fellowship from the Health Research Council of New Zealand. Debbie L Hay is supported by a James Cook Research Fellowship of the Royal Society of New Zealand. Erica R Hendrikse is supported by the Barbara Basham Doctoral Scholarship administered by the Auckland Medical Research Foundation and Perpetual Guardian.

References

Russell

King

Smillie

et al.

Calcitonin gene-related peptide: Physiology and pathophysiology. Physiol Rev 2014; 94: 1099–1124.

Iyengar

Ossipov

Johnson

. The role of calcitonin gene–related peptide in peripheral and central pain mechanisms including migraine. Pain 2017; 158: 543–559.

Goadsby

Holland

Martins-Oliveira

et al.

Pathophysiology of migraine: A disorder of sensory processing. Physiol Rev 2017; 97: 553–622.

Edvinsson

Warfvinge

. Recognizing the role of CGRP and CGRP receptors in migraine and its treatment. Cephalalgia. 2019; 39: 366–373.

Edvinsson

. The trigeminovascular pathway: Role of CGRP and CGRP receptors in migraine. Headache 2017; 57: S47–S55.

Stankewitz

Aderjan

Eippert

et al.

Trigeminal nociceptive transmission in migraineurs predicts migraine attacks. J Neurosci 2011; 31: 1937–1943.

Mainero

Boshyan

Hadjikhani

. Altered functional magnetic resonance imaging resting-state connectivity in periaqueductal gray networks in migraine. Ann Neurol 2011; 70: 838–845.

Amin

Hougaard

Cramer

et al.

Intact blood−brain barrier during spontaneous attacks of migraine without aura: A 3T DCE-MRI study. Eur J Neurol 2017; 24: 1116–1124.

Charles

. The evolution of a migraine attack – a review of recent evidence. Headache 2013; 53: 413–419.

10.

Schwedt

Chong

Chiang

et al.

Enhanced pain-induced activity of pain-processing regions in a case-control study of episodic migraine. Cephalalgia 2014; 34: 947–958.

11.

Schulte

Allers

May

. Hypothalamus as a mediator of chronic migraine: Evidence from high-resolution fMRI. Neurology 2017; 88: 2011–2016.

12.

Katafuchi

Minamino

. Structure and biological properties of three calcitonin receptor-stimulating peptides, novel members of the calcitonin gene-related peptide family. Peptides 2004; 25: 2039–2045.

13.

Hay

Chen

Lutz

et al.

Amylin: Pharmacology, physiology, and clinical potential. Pharmacol Rev 2015; 67: 564–600.

14.

Lima

Marques-Oliveira

da Silva

et al.

Role of calcitonin gene-related peptide in energy metabolism. Endocrine 2017; 58: 3–13.

15.

Hay

Garelja

Poyner

et al.

Update on the pharmacology of calcitonin/CGRP family of peptides: IUPHAR Review 25. Br J Pharmacol 2018; 175: 3–17.

16.

McLatchie

Fraser

Main

et al.

RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 1998; 393: 333–339.

17.

Hay

Garelja

Poyner

et al.

Update on the pharmacology of calcitonin/CGRP family of peptides: IUPHAR Review: “25”. Brit J Pharmacol 2018; 175: 3–17.

18.

Christopoulos

Perry

Morfis

et al.

Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product. Mol Pharmacol 1999; 56: 235–242.

19.

Bailey

Walker

Ferner

et al.

Pharmacological characterization of rat amylin receptors: Implications for the identification of amylin receptor subtypes. Brit J Pharmacol 2012; 166: 151–167.

20.

Walker

Eftekhari

Bower

et al.

A second trigeminal CGRP receptor: Function and expression of the AMY1 receptor. Ann Clin Trans Neurol 2015; 2: 595–608.

21.

Hay

Christopoulos

et al.

Pharmacological discrimination of calcitonin receptor: Receptor activity-modifying protein complexes. Mol Pharmacol 2005; 67: 1655–1665.

22.

Hay

Howitt

Conner

et al.

CL/RAMP2 and CL/RAMP3 produce pharmacologically distinct adrenomedullin receptors: A comparison of effects of adrenomedullin22–52, CGRP8–37 and BIBN4096BS. Brit J Pharmacol 2003; 140: 477–486.

23.

Moore

Salvatore

. Targeting a family B GPCR/RAMP receptor complex: CGRP receptor antagonists and migraine. Brit J Pharmacol 2012; 166: 66–78.

24.

Hay

Christopoulos

et al.

Determinants of 1-piperidinecarboxamide, N-[2-[[5-amino-l-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-d ibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazoliny l) (BIBN4096BS) affinity for calcitonin gene-related peptide and amylin receptors – the role of receptor activity modifying protein 1. Mol Pharmacol 2006; 70: 1984–1991.

25.

Hay

Howitt

Conner

et al.

CL/RAMP2 and CL/RAMP3 produce pharmacologically distinct adrenomedullin receptors: A comparison of effects of adrenomedullin22-52, CGRP8-37 and BIBN4096BS. Brit J Pharmacol 2003; 140: 477–486.

26.

Walker

Raddant

Woolley

et al.

CGRP receptor antagonist activity of olcegepant depends on the signalling pathway measured. Cephalalgia. 2018; 38: 437–451.

27.

Liu

Beyer

Aebersold

. On the dependency of cellular protein levels on mRNA abundance. Cell 2016; 165: 535–550.

28.

Eftekhari

Gaspar

Roberts

et al.

Localization of CGRP receptor components and receptor binding sites in rhesus monkey brainstem: A detailed study using in situ hybridization, immunofluorescence, and autoradiography. J Comp Neurol 2016; 524: 90–118.

29.

Tolcos

Tikellis

Rees

et al.

Ontogeny of calcitonin receptor mRNA and protein in the developing central nervous system of the rat. J Comp Neurol 2003; 456: 29–38.

30.

Uhlen

Bandrowski

Carr

et al.

A proposal for validation of antibodies. Nature Meth 2016; 13: 823–827.

31.

Zhang

Yang

Wang

et al.

Multiple target of hAmylin on rat primary hippocampal neurons. Neuropharmacol 2017; 113: 241–251.

32.

Zhao

Bell

Smith

et al.

Differential expression of components of the cardiomyocyte adrenomedullin/intermedin receptor system following blood pressure reduction in nitric oxide-deficient hypertension. J Pharmacol Exp Ther 2006; 316: 1269–1281.

33.

Hay

Pioszak

. Receptor activity-modifying proteins (RAMPs): New insights and roles. Ann Review Pharmacol Toxicol 2016; 56: 469–487.

34.

Eftekhari

Salvatore

Gaspar

et al.

Localization of CGRP receptor components, CGRP, and receptor binding sites in human and rhesus cerebellar cortex. Cerebellum 2013; 12: 937–949.

35.

Eftekhari

Salvatore

Calamari

et al.

Differential distribution of calcitonin gene-related peptide and its receptor components in the human trigeminal ganglion. Neuroscience 2010; 169: 683–696.

36.

Bullock

Wookey

Bennett

et al.

Peripheral calcitonin gene-related peptide receptor activation and mechanical sensitization of the joint in rat models of osteoarthritis pain. Arthritis Rheumatol 2014; 66: 2188–2200.

37.

Miller

Liu

Warfvinge

et al.

Immunohistochemical localization of the calcitonin gene-related peptide binding site in the primate trigeminovascular system using functional antagonist antibodies. Neuroscience 2016; 328: 165–183.

38.

Chakravarty

Suthar

Coppock

et al.

CGRP and adrenomedullin binding correlates with transcript levels for Calcitonin Receptor-Like Receptor (CRLR) and Receptor Activity Modifying Proteins (RAMPs) in rat tissues. Brit J Pharmacol 2000; 130: 189–195.

39.

Uhlén

Fagerberg

Hallström

et al.

Tissue-based map of the human proteome. Science 2015; 347: 394–394.

40.

Lein

Hawrylycz

et al.

Genome-wide atlas of gene expression in the adult mouse brain. Nature 2007; 445: 168–176.

41.

Hawrylycz

Lein

Guillozet-Bongaarts

et al.

An anatomically comprehensive atlas of the adult human brain transcriptome. Nature 2012; 489: 391–399.

42.

Kastin

Pan

. Blood-brain barrier and feeding: Regulatory roles of saturable transport systems for ingestive peptides. Curr Pharm Des 2008; 14: 1615–1619.

43.

Keaney

Campbell

. The dynamic blood-brain barrier. The FEBS journal 2015; 282: 4067–4079.

44.

Morimoto

Nishimura

Miyauchi

et al.

Calcitonin in plasma and cerebrospinal fluid from normal subjects and patients with medullary thyroid carcinoma: Possible restriction of calcitonin by the blood-brain barrier. J Clin Endocrinol Metab 1982; 55: 594–596.

45.

Banks

Kastin

Maness

et al.

Permeability of the blood-brain barrier to amylin. Life Sci 1995; 57: 1993–2001.

46.

Banks

Kastin

. Differential permeability of the blood–brain barrier to two pancreatic peptides: Insulin and amylin. Peptides 1998; 19: 883–889.

47.

Reiner

Mietlicki-Baase

Olivos

et al.

Amylin acts in the lateral dorsal tegmental nucleus to regulate energy balance through gamma-aminobutyric acid signaling. Biol Psych 2017; 82: 828–838.

48.

Mietlicki-Baase

Rupprecht

Olivos

et al.

Amylin receptor signaling in the ventral tegmental area is physiologically relevant for the control of food intake. Neuropsychopharmacol 2013; 38: 1685–1697.

49.

Mietlicki-Baase

McGrath

Koch-Laskowski

et al.

Amylin receptor activation in the ventral tegmental area reduces motivated ingestive behavior. Neuropharmacol 2017; 123: 67–79.

50.

Jansen-Olesen

Jorgensen

Engel

et al.

In-depth characterization of CGRP receptors in human intracranial arteries. Eur J Pharmacol 2003; 481: 207–216.

51.

Edvinsson

Nilsson

Jansen-Olesen

. Inhibitory effect of BIBN4096BS, CGRP(8-37), a CGRP antibody and an RNA-Spiegelmer on CGRP induced vasodilatation in the perfused and non-perfused rat middle cerebral artery. Brit J Pharmacol 2007; 150: 633–640.

52.

Passaglia

Gonzaga

Tirapelli

et al.

Pharmacological characterisation of the mechanisms underlying the relaxant effect of adrenomedullin in the rat carotid artery. J Pharm Pharmacol 2014; 66: 1734–1746.

53.

Kastin

Akerstrom

Hackler

et al.

Adrenomedullin and the blood-brain barrier. Hormone Metab Res 2001; 33: 19–25.

54.

Hinson

Kapas

Smith

. Adrenomedullin, a multifunctional regulatory peptide. Endocrine Rev 2000; 21: 138–167.

55.

Kato

Kitamura

. Bench-to-bedside pharmacology of adrenomedullin. Eur J Pharmacol 2015; 764: 140–148.

56.

Kis

Kaiya

Nishi

et al.

Cerebral endothelial cells are a major source of adrenomedullin. J Neuroendocrinol 2002; 14: 283–293.

57.

Kis

Deli

Kobayashi

et al.

Adrenomedullin regulates blood–brain barrier functions in vitro. Neuroreport 2001; 12: 4139–4142.

58.

Honda

Nakagawa

Hayashi

et al.

Adrenomedullin improves the blood–brain barrier function through the expression of claudin-5. Cell Mol Neurobiol 2006; 26: 109–118.

59.

Serrano

Uttenthal

Martínez

et al.

Distribution of adrenomedullin-like immunoreactivity in the rat central nervous system by light and electron microscopy. Brain Res 2000; 853: 245–268.

60.

Ueta

Kitamura

Isse

et al.

Adrenomedullin-immunoreactive neurons in the paraventricular and supraoptic nuclei of the rat. Neurosci Lett 1995; 202: 37–40.

61.

Washimine

Asada

Kitamura

et al.

Immunohistochemical identification of adrenomedullin in human, rat, and porcine tissue. Histochem Cell Biol 1995; 103: 251–254.

62.

Takahashi

Satoh

Sone

et al.

Expression of adrenomedullin mRNA in the human brain and pituitary. Peptides 1997; 18: 1051–1053.

63.

Macchi

Porzionato

Belloni

et al.

Immunohistochemical mapping of adrenomedullin in the human medulla oblongata. Peptides 2006; 27: 1397–1404.

64.

Figueira

Israel

. Role of cerebellar adrenomedullin in blood pressure regulation. Neuropeptides 2015; 54: 59–66.

65.

Fernández

Serrano

Tessarollo

et al.

Lack of adrenomedullin in the mouse brain results in behavioral changes, anxiety, and lower survival under stress conditions. Proc Nat Acad Sci 2008; 105: 12581–12586.

66.

Fernández

Serrano

Martínez-Murillo

et al.

Lack of adrenomedullin in the central nervous system results in apparently paradoxical alterations on pain sensitivity. Endocrinology 2010; 151: 4908–4915.

67.

Chabot

J-G

Quirion

. A role for adrenomedullin as a pain-related peptide in the rat. Proc Nat Acad Sci 2006; 103: 16027–16032.

68.

Hong

Liu

Chabot

J-G

et al.

Upregulation of adrenomedullin in the spinal cord and dorsal root ganglia in the early phase of CFA-induced inflammation in rats. Pain 2009; 146: 105–113.

69.

Hong

Hay

Quirion

et al.

The pharmacology of adrenomedullin 2/intermedin. Brit J Pharmacol 2012; 166: 110–120.

70.

Cottrell

Roosterman

Marvizon

et al.

Localization of calcitonin receptor-like receptor and receptor activity modifying protein 1 in enteric neurons, dorsal root ganglia, and the spinal cord of the rat. J Compar Neurol 2005; 490: 239–255.

71.

Roh

Chang

Bhalla

et al.

Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complexes. J Biol Chem 2004; 279: 7264–7274.

72.

Taylor

Meeran

O’Shea

et al.

Adrenomedullin inhibits feeding in the rat by a mechanism involving calcitonin gene-related peptide receptors. Endocrinology 1996; 137: 3260–3264.

73.

Hashimoto

Kitamura

Kawasaki

et al.

Adrenomedullin 2/intermedin-like immunoreactivity in the hypothalamus and brainstem of rats. Autonom Neurosci 2008; 139: 46–54.

74.

Takei

Inoue

Ogoshi

et al.

Identification of novel adrenomedullin in mammals: A potent cardiovascular and renal regulator. FEBS Lett 2004; 556: 53–58.

75.

Krukoff

. Decrease in arterial pressure induced by adrenomedullin in the hypothalamic paraventricular nucleus is mediated by nitric oxide and GABA. Regul Peptides 2004; 119: 21–30.

76.

Takahashi

Morimoto

Hirose

et al.

Adrenomedullin 2/intermedin in the hypothalamo–pituitary–adrenal axis. J Mol Neurosci 2011; 43: 182–192.

77.

Zhou Y-B, Sun H-J, Chen D, et al. Intermedin in paraventricular nucleus attenuates sympathetic activity and blood pressure via nitric oxide in hypertensive rats. Hypertension 2013; 63: 330–337.

78.

Owji

Gardiner

Upton

et al.

Characterisation and molecular identification of adrenomedullin binding sites in the rat spinal cord: A comparison with calcitonin gene-related peptide receptors. J Neurochem 1996; 67: 2172–2179.

79.

Juaneda

Dumont

Chabot

J-G

et al.

Adrenomedullin receptor binding sites in rat brain and peripheral tissues. Eur J Pharmacol 2003; 474: 165–174.

80.

Sone

Takahashi

Satoh

et al.

Specific adrenomedullin binding sites in the human brain. Peptides 1997; 18: 1125–1129.

81.

Warfvinge

Edvinsson

. Distribution of CGRP and CGRP receptor components in the rat brain. Cephalalgia. 2019; 39: 342–353.

82.

Eftekhari

Edvinsson

. Calcitonin gene-related peptide (CGRP) and its receptor components in human and rat spinal trigeminal nucleus and spinal cord at C1-level. BMC Neurosci 2011; 12.

83.

Tajti

Uddman

Möller

et al.

Messenger molecules and receptor mRNA in the human trigeminal ganglion. J Autonom Nerv Sys 1999; 76: 176–183.

84.

Sams

Jansen-Olesen

. Expression of calcitonin receptor-like receptor and receptor-activity-modifying proteins in human cranial arteries. Neurosci Lett 1998; 258: 41–44.

85.

Oliver

Wainwright

Edvinsson

et al.

Immunohistochemical localization of calcitonin receptor-like receptor and receptor activity-modifying proteins in the human cerebral vasculature. J Cereb Blood Flow Metab 2002; 22: 620–629.

86.

Oliver

Kane

Salvatore

et al.

Cloning, characterization and central nervous system distribution of receptor activity modifying proteins in the rat. Eur J Neurosci 2001; 14: 618–628.

87.

Stachniak

Krukoff

. Receptor activity modifying protein 2 distribution in the rat central nervous system and regulation by changes in blood pressure. J Neuroendocrinol 2003; 15: 840–850.

88.

Zaidi

Breimer

MacIntyre

. Biology of peptides from the calcitonin genes. Exper Physiol 1987; 72: 371–408.

89.

Sabate

Stolarsky

Polak

et al.

Regulation of neuroendocrine gene expression by alternative RNA processing. Colocalization of calcitonin and calcitonin gene-related peptide in thyroid C-cells. J Biol Chem 1985; 260: 2589–2592.

90.

Findlay

Sexton

. Calcitonin. Growth Factors 2004; 22: 217–224.

91.

Amara

Jonas

Rosenfeld

et al.

Alternative RNA processing in calcitonin gene expression generates mRNAs encoding different polypeptide products. Nature 1982; 298: 240–244.

92.

Kresse

Jacobowitz

Skofitsch

. Detailed mapping of CGRP mRNA expression in the rat central nervous system: Comparison with previous immunocytochemical findings. Brain Res Bull 1995; 36: 261–274.

93.

Rosenfeld

Mermod

Amara

et al.

Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 1983; 304: 129–135.

94.

Sexton

. Central nervous system binding sites for calcitonin and calcitonin gene-related peptide. Mol Neurobiol 1991; 5: 251–273.

95.

Paxinos

Chai

Christopoulos

et al.

In vitro autoradiographic localization of calcitonin and amylin binding sites in monkey brain. J Chem Neuroanat 2004; 27: 217–236.

96.

Sexton

Paxinos

Huang

et al.

In vitro autoradiographic localization of calcitonin binding sites in human medulla oblongata. J Compar Neurol 1994; 341: 449–463.

97.

Olgiati

Guidobono

Netti

et al.

Localization of calcitonin binding sites in rat central nervous system: Evidence of its neuroactivity. Brain Res 1983; 265: 209–215.

98.

Henke

Tobler

Fischer

. Localization of salmon calcitonin binding sites in rat brain by autoradiography. Brain Res 1983; 272: 373–377.

99.

Hilton

Chai

Sexton

. In vitro autoradiographic localization of the calcitonin receptor isoforms, C1a and C1b, in rat brain. Neuroscience 1995; 69: 1223–1237.

100.

Sexton

McKenzie

Mendelsohn

. Evidence for a new subclass of calcitonin/calcitonin gene-related peptide binding site in rat brain. Neurochem Int 1988; 12: 323–335.

101.

Tschopp

Henke

Petermann

et al.

Calcitonin gene-related peptide and its binding sites in the human central nervous system and pituitary. Proc Nat Acad Sci 1985; 82: 248–252.

102.

Nakamuta

Itokazu

Koida

et al.

Autoradiographic localization of human calcitonin sensitive binding sites in rat brain. Jpn J Pharmacol 1991; 56: 551–555.

103.

Sexton

Paxinos

Huang

et al.

In vitro autoradiographic localization of calcitonin binding sites in human medulla oblongata. J Comp Neurol 1994; 341: 449–463.

104.

Furness

Wootten

Christopoulos

et al.

Consequences of splice variation on Secretin family G protein-coupled receptor function. Brit J Pharmacol 2012; 166: 98–109.

105.

Becskei

Riediger

Zünd

Wookey

Lutz

. Immunohistochemical mapping of calcitonin receptors in the adult rat brain. Brain Res 2004; 1030: 221–233.

106.

Bower

Eftekhari

Waldvogel

et al.

Mapping the calcitonin receptor in human brain stem. Am J Physiol – Reg Int Comp Physiol 2016; 310: R788–R793.

107.

Ferrier

Pierson

Jones

et al.

Expression of the rat amylin (IAPP/DAP) gene. J Mol Endocrinol 1989; 3: R1–R4.

108.

Asai

Nakazato

Miyazato

et al.

Regional distribution and molecular forms of rat islet amyloid polypeptide. Biochem Biophys Res Comm 1990; 169: 788–795.

109.

D’Este

Casini

Wimalawansa

et al.

Immunohistochemical localization of amylin in rat brainstem. Peptides 2000; 21: 1743–1749.

110.

Skofitsch

Gubisch

Wimalawansa

et al.

Comparative immunohistochemical distribution of amylin-like and calcitonin gene related peptide like immunoreactivity in the rat central nervous system. Can J Physiol Pharmacol 1995; 73: 945–956.

111.

Chance

Balasubramaniam

Zhang

et al.

Anorexia following the intrahypothalamic administration of amylin. Brain Res 1991; 539: 352–354.

112.

Tingstedt

J-E

Edlund

Madsen

et al.

Gastric amylin expression: Cellular identity and lack of requirement for the homeobox protein PDX-1. A study in normal and PDX-1-deficient animals with a cautionary note on antiserum evaluation. J Histochem Cytochem 1999; 47: 973–980.

113.

Dobolyi

. Central amylin expression and its induction in rat dams. J Neurochem 2009; 111: 1490–1500.

114.

Kelly

Gergi

et al.

Hypothalamic amylin acts in concert with leptin to regulate food intake. Cell Metab 2015; 22: 1059–1067.

115.

Sexton

Paxinos

Kenney

et al.

In vitro autoradiographic localization of amylin binding sites in rat brain. Neuroscience 1994; 62: 553–567.

116.

Barth

Riediger

Lutz

et al.

Peripheral amylin activates circumventricular organs expressing calcitonin receptor a/b subtypes and receptor-activity modifying proteins in the rat. Brain Res 2004; 997: 97–102.

117.

Liberini

Boyle

Cifani

et al.

Amylin receptor components and the leptin receptor are co-expressed in single rat area postrema neurons. Eur J Neurosci 2016; 43: 653–661.

118.

Ruangkittisakul

MacTavish

et al.

Amyloid β (Aβ) peptide directly activates amylin-3 receptor subtype by triggering multiple intracellular signaling pathways. J Biol Chem 2012; 287: 18820–18830.

119.

Booe

Walker

Barwell

et al.

Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor. Mol Cell 2015; 58: 1040–1052.

120.

Watkins

Hay

. The structure of secretin family GPCR peptide ligands: Implications for receptor pharmacology and drug development. Drug Disc Today 2012; 17: 1006–1014.

121.

Liang

Khoshouei

Radjainia

et al.

Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature 2017; 546: 118–123.

122.

Zhu

Xue

Wang

et al.

Amylin receptor ligands reduce the pathological cascade of Alzheimer's disease. Neuropharmacol 2017; 119: 170–181.

123.

Gingell

Burns

Hay

. Activity of pramlintide, rat and human amylin but not Aβ1–42 at human amylin receptors. Endocrinology 2014; 155: 21–26.

124.

Walker

Whiting

et al.

Mice lacking the neuropeptide α-calcitonin gene-related peptide are protected against diet-induced obesity. Endocrinology 2010; 151: 4257–4269.

125.

Russell

King

Smillie

et al.

Calcitonin gene-related peptide. Physiol Pathophysiol Physiol Rev 2014; 94: 1099–1142.

126.

Lima

Marques-Oliveira

da Silva

et al.

Role of calcitonin gene-related peptide in energy metabolism. Endocrine 2017; 58: 3–13.

127.

Mulderry

Ghatki

Spokks

et al.

Differential expression of α-CGRP and β-CGRP by primary sensory neurons and enteric autonomic neurons of the rat. Neuroscience 1988; 25: 195–205.

128.

Noguchi

Senba

Morita

et al.

Co-expression of α-CGRP and β-CGRP mRNAs in the rat dorsal root ganglion cells. Neurosci Lett 1990; 108: 1–5.

129.

Amara

Arriza

Leff

et al.

Expression in brain of a messenger RNA encoding a novel neuropeptide homologous to calcitonin gene-related peptide. Science 1985; 229: 1094–1097.

130.

Gibson

Polak

Giaid

et al.

Calcitonin gene-related peptide messenger RNA is expressed in sensory neurones of the dorsal root ganglia and also in spinal motoneurones in man and rat. Neurosci Lett 1988; 91: 283–288.

131.

Schütz

Mauer

Salmon

et al.

Analysis of the cellular expression pattern of β-CGRP in α-CGRP-deficient mice. J Compar Neurol 2004; 476: 32–43.

132.

Lennerz

Rühle

Ceppa

et al.

Calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and calcitonin gene-related peptide (CGRP) immunoreactivity in the rat trigeminovascular system: Differences between peripheral and central CGRP receptor distribution. J Compar Neurol 2008; 507: 1277–1299.

133.

Eftekhari

Salvatore

Johansson

et al.

Localization of CGRP, CGRP receptor, PACAP and glutamate in trigeminal ganglion. Relation to the blood-brain barrier. Brain Res 2015; 1600: 93–109.

134.

Mason

Peterfreund

Sawchenko

et al.

Release of the predicted calcitonin gene-related peptide from cultured rat trigeminal ganglion cells. Nature 1984; 308: 653–655.

135.

Uddman

Edvinsson

Ekman

et al.

Innervation of the feline cerebral vasculature by nerve fibers containing calcitonin gene-related peptide: Trigeminal origin and co-existence with substance P. Neurosci Lett 1985; 62: 131–136.

136.

Lundberg JM, Franco-Cereceda A, Alving K, et al. Release of calcitonin gene-related peptide from sensory neurons. Ann N Y Acad Sci 1992; 657: 187–193.

137.

Eftekhari

Edvinsson

. Possible sites of action of the new calcitonin gene-related peptide receptor antagonists. Ther Adv Neurol Dis 2010; 3: 369–378.

138.

Eftekhari

Warfvinge

Blixt

et al.

Differentiation of nerve fibers storing CGRP and CGRP receptors in the peripheral trigeminovascular system. J Pain 2013; 14: 1289–1303.

139.

Skofitsch

Jacobowitz

. Calcitonin gene-related peptide: Detailed immunohistochemical distribution in the central nervous system. Peptides 1985; 6: 721–745.

140.

Gibson

Polak

Bloom

et al.

Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species. J Neurosci 1984; 4: 3101–3111.

141.

Uddman

Tajti

Hou

et al.

Neuropeptide expression in the human trigeminal nucleus caudalis and in the cervical spinal cord C1 and C2. Cephalalgia 2002; 22: 112–116.

142.

Warfvinge

Edvinsson

. Pearls and pitfalls in neural CGRP immunohistochemistry. Cephalalgia 2013; 33: 593–603.

143.

Edvinsson

Eftekhari

Salvatore

et al.

Cerebellar distribution of calcitonin gene-related peptide (CGRP) and its receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) in rat. Mol Cell Neurosci 2011; 46: 333–339.

144.

Morara

Rosina

Provini

et al.

Calcitonin gene-related peptide receptor expression in the neurons and glia of developing rat cerebellum: An autoradiographic and immunohistochemical analysis. Neuroscience 2000; 100: 381–391.

145.

Sexton

McKenzie

Mason

et al.

Localization of binding sites for calcitonin gene-related peptide in rat brain by in vitro autoradiography. Neuroscience 1986; 19: 1235–1245.

146.

Skofitsch

Jacobowitz

. Autoradiographic distribution of 125 I calcitonin gene-related peptide binding sites in the rat central nervous system. Peptides 1985; 6: 975–986.

147.

Inagaki

Kito

Kubota

et al.

Autoradiographic localization of calcitonin gene-related peptide binding sites in human and rat brains. Brain Res 1986; 374: 287–298.

148.

Dennis

Fournier

Guard

et al.

Calcitonin gene-related peptide (hCGRPα) binding sites in the nucleus accumbens. Atypical structural requirements and marked phylogenic differences. Brain Res 1991; 539: 59–66.

149.

Dennis

Fournier

St Pierre

et al.

Structure-activity profile of calcitonin gene-related peptide in peripheral and brain tissues. Evidence for receptor multiplicity. J Pharmacol Exp Ther 1989; 251: 718–725.

150.

Barth

Riediger

Lutz

et al.

Peripheral amylin activates circumventricular organs expressing calcitonin receptor a/b subtypes and receptor-activity modifying proteins in the rat. Brain Res 2004; 997: 97–102.

151.

Ueda

Ugawa

Saishin

et al.

Expression of receptor-activity modifying protein (RAMP) mRNAs in the mouse brain. Mol Brain Res 2001; 93: 36–45.

152.

Oliver

Wainwright

Heavens

et al.

Distribution of novel CGRP 1 receptor and adrenomedullin receptor mRNAs in the rat central nervous system. Mol Brain Res 1998; 57: 149–154.

153.

Pozo-Rosich

Storer

Charbit

et al.

Periaqueductal gray calcitonin gene-related peptide modulates trigeminovascular neurons. Cephalalgia 2015; 35: 1298–1307.

154.

Salvatore

Moore

Calamari

et al.

Pharmacological properties of MK-3207, a potent and orally active calcitonin gene-related peptide receptor antagonist. J Pharmacol Exp Ther 2010; 333: 152–160.

155.

Zhang

Winborn

De Prado

et al.

Sensitization of calcitonin gene-related peptide receptors by receptor activity-modifying protein-1 in the trigeminal ganglion. J Neurosci 2007; 27: 2693–2703.

156.

Tajti

Kuris

Vécsei

et al.

Organ culture of the trigeminal ganglion induces enhanced expression of calcitonin gene-related peptide via activation of extracellular signal-regulated protein kinase 1/2. Cephalalgia 2011; 31: 95–105.

157.

Vause

Durham

. Calcitonin gene-related peptide stimulation of nitric oxide synthesis and release from trigeminal ganglion glial cells. Brain Res 2008; 1196: 22–32.

158.

Huang

Yang

Chang

et al.

Amylin suppresses acetic acid-induced visceral pain and spinal c-fos expression in the mouse. Neuroscience 2010; 165: 1429–1438.

159.

Gebre-Medhin

Mulder

Zhang

et al.

Reduced nociceptive behavior in islet amyloid polypeptide (amylin) knockout mice. Mol Brain Res 1998; 63: 180–183.

160.

Nicholl

Bhatavdekar

Mak

et al.

Extra-pancreatic expression of the rat islet amyloid polypeptide (amylin) gene. J Mol Endocrinol 1992; 9: 157–163.

161.

Mulder

Leckstrom

Uddman

et al.

Islet amyloid polypeptide (amylin) is expressed in sensory neurons. J Neurosci 1995; 15: 7625–7632.

162.

Nakamoto

Soeda

Takami

et al.

Localization of calcitonin receptor mRNA in the mouse brain: Coexistence with serotonin transporter mRNA. Mol Brain Res 2000; 76: 93–102.

163.

Christopoulos

Sexton

Paxinos

et al.

Comparative distribution off receptors for amylin and the related peptides calcitonin gene related peptide and calcitonin in rat and monkey brain. Can J Physiol Pharmacol 1995; 73: 1037–1041.

164.

Sheward

Lutz

Harmar

. The expression of the calcitonin receptor gene in the brain and pituitary gland of the rat. Neurosci Lett 1994; 181: 31–34.

165.

Marvizón

JCG

Pérez

Song

et al.

Calcitonin receptor-like receptor and receptor activity modifying protein 1 in the rat dorsal horn: Localization in glutamatergic presynaptic terminals containing opioids and adrenergic α 2C receptors. Neuroscience 2007; 148: 250–265.

166.

Carter

Soden

Zweifel

et al.

Genetic identification of a neural circuit that suppresses appetite. Nature 2013; 503: 111–114.

167.

Van Rossum

Menard

Fournier

et al.

Autoradiographic distribution and receptor binding profile of [125I] Bolton Hunter-rat amylin binding sites in the rat brain. J Pharmacol Exp Ther 1994; 270: 779–787.

168.

Dunn-Meynell

Le Foll

Johnson

et al.

Endogenous VMH amylin signaling is required for full leptin signaling and protection from diet-induced obesity. Am J Physiol-Regul, Integ Compar Physiol 2016; 310: R355–R365.

169.

Summ

Charbit

Andreou

et al.

Modulation of nocioceptive transmission with calcitonin gene-related peptide receptor antagonists in the thalamus. Brain 2010; 133: 2540–2548.

170.

Filiz

Tepe

Eftekhari

et al.

CGRP receptor antagonist MK-8825 attenuates cortical spreading depression induced pain behavior. Cephalalgia. 2019; 39: 354–365.

171.

Van Rossum

Menard

Chang

et al.

Comparative affinities of human adrenomedullin for 125I-labelled human alpha calcitonin gene related peptide ([125I] hCGRP alpha) and 125I-labelled Bolton-Hunter rat amylin ([125I] BHrAMY) specific binding sites in the rat brain. Can J Physiol Pharmacol 1995; 73: 1084–1088.

172.

Edvinsson

Cantera

Jansen-Olesen

et al.

Expression of calcitonin gene-related peptide 1 receptor mRNA in human trigeminal ganglia and cerebral arteries. Neurosci Lett 1997; 229: 209–211.

173.

Tajti

Uddman

Edvinsson

. Neuropeptide localization in the ‘migraine generator’ region of the human brainstem. Cephalalgia 2001; 21: 96–101.