Clinical studies have suggested a link between the sensory trigeminal system and the parasympathetic ganglia. Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide which plays an important role in vasodilatation and pain transmission in craniocervical structures. The present study was designed to examine if CGRP and CGRP receptor components are present in the human sphenopalatine ganglion (SPG) in order to reveal an interaction between the sensory and parasympathetic systems. Indirect immunofluorescence technique was used for immunohistochemical demonstration of CGRP, the calcitonin receptor-like receptor (CLR) and the receptor activity modifying protein 1 (RAMP1) in human and rat SPG. Cryostat sections were examined and images were obtained using a light- and epifluorescence microscope coupled to a camera to visualize co-labeling by superimposing the digital images. In addition, Western blot technique was used to demonstrate the existence of CGRP receptor components in rat SPG. CGRP immunoreactive fibers were frequently found intraganglionic in the SPG in the vicinity of neurons. CLR immunoreactivity was observed in satellite glial cells (SGCs) as well as in nerve fibers, but not in neurons. RAMP1 immunoreactivity was localized in many neurons and SGCs. Thus, the two CGRP receptor components together were found in the SGCs. In addition, Western blot revealed the presence of RAMP1 and CLR in rat SPG. Our results suggest a possible sensory influence in the parasympathetic cranial ganglia. The sensory CGRP-containing fibers probably originate in the trigeminal ganglion, project to the SPG and act on CGRP receptors on SGCs.

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.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator and a neuromodulator implicated in the pathophysiology of migraine. It binds to the extracellular domains of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein (RAMP) 1 that together form the CGRP receptor. Antagonist antibodies against CGRP and its binding site at the receptor are clinically effective in preventing migraine attacks. The blood-brain barrier penetration of these antagonist antibodies is limited, suggesting that a potential peripheral site of action is sufficient to prevent migraine attacks. To further understand the sites of CGRP-mediated signaling in migraine, we used immunohistochemical staining with recently developed antagonist antibodies specifically recognizing a fusion protein of the extracellular domains of RAMP1 and CLR that comprise the CGRP binding pocket at the CGRP receptor in monkey and man. We confirmed binding of the antagonist antibodies to human vascular smooth muscle cells (VSMCs) of dural meningeal arteries and neurons in the trigeminal ganglion, both of which are likely sites of action for therapeutic antibodies in migraine patients. We further used one of these antibodies for detailed mapping on cynomolgus monkey tissue and found antagonist antibody binding sites at multiple levels in the trigeminovascular system: in the dura mater VSMCs, in neurons and satellite glial cells in the trigeminal ganglion, and in neurons in the spinal trigeminal nucleus caudalis. These data reinforce and clarify our understanding of CGRP receptor localization in a pattern consistent with a role for CGRP receptors in trigeminal sensitization and migraine pathology.

Functional imaging studies have revealed that certain brainstem areas are activated during migraine attacks. The neuropeptide calcitonin gene-related peptide (CGRP) is associated with activation of the trigeminovascular system and transmission of nociceptive information and plays a key role in migraine pathophysiology. Therefore, to elucidate the role of CGRP, it is critical to identify the regions within the brainstem that process CGRP signaling. In situ hybridization and immunofluorescence were performed to detect mRNA expression and define cellular localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), respectively. To define CGRP receptor binding sites, in vitro autoradiography was performed with [(3)H]MK-3207 (a CGRP receptor antagonist). CLR and RAMP1 mRNA and protein expression were detected in the pineal gland, medial mammillary nucleus, median eminence, infundibular stem, periaqueductal gray, area postrema, pontine raphe nucleus, gracile nucleus, spinal trigeminal nucleus, and spinal cord. RAMP1 mRNA expression was also detected in the posterior hypothalamic area, trochlear nucleus, dorsal raphe nucleus, medial lemniscus, pontine nuclei, vagus nerve, inferior olive, abducens nucleus, and motor trigeminal nucleus; protein coexpression of CLR and RAMP1 was observed in these areas via immunofluorescence. [(3)H]MK-3207 showed high binding densities concordant with mRNA and protein expression. The present study suggests that several regions in the brainstem may be involved in CGRP signaling. Interestingly, we found receptor expression and antagonist binding in some areas that are not protected by the blood-brain barrier, which suggests that drugs inhibiting CGRP signaling may not be able to penetrate the central nervous system to antagonize receptors in these brain regions.

Calcitonin gene-related peptide (CGRP) receptor antagonists have demonstrated anti-migraine efficacy. One remaining question is where do these blockers act? We hypothesized that the trigeminal ganglion could be one possible site. We examined the binding sites of a CGRP receptor antagonist (MK-3207) and related this to the expression of CGRP and its receptor in rhesus trigeminal ganglion. Pituitary adenylate cyclase-activating polypeptide (PACAP) and glutamate were examined and related to the CGRP system. Furthermore, we examined if the trigeminal ganglion is protected by the blood-brain barrier (BBB). Autoradiography was performed with [(3)H]MK-3207 to demonstrate receptor binding sites in rhesus trigeminal ganglion (TG). Immunofluorescence was used to correlate binding and the presence of CGRP and its receptor components, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), and the distribution of PACAP and glutamate in rhesus and rat TG. Evans blue was used to examine large molecule penetration into the rat TG. High receptor binding densities were found in rhesus TG. Immunofluorescence revealed expression of CGRP, CLR and RAMP1 in trigeminal cells. CGRP positive neurons expressed PACAP but not glutamate. Some neurons expressing CLR and RAMP1 co-localized with glutamate. Evans blue revealed that the TG is not protected by BBB. This study demonstrates CGRP receptor binding sites and expression of the CGRP receptor in rhesus and rat TG. The expression pattern of PACAP and glutamate suggests a possible interaction between the glutamatergic and CGRP system. In rat the TG is outside the BBB, suggesting that molecules do not need to be CNS-penetrant to block these receptors.

The calcitonin gene-related peptide (CGRP) family of neuropeptides, consists of CGRP, adrenomedullin, amylin, and calcitonin. The receptors consist of either calcitonin receptor-like receptor (CLR) or calcitonin receptor (CTR) which for function needs an accessory protein, receptor activity-modifying proteins (RAMPs). CGRP has a pivotal role in primary headaches but the role of the other members of the CGRP family of peptides in headaches is not known. Here, we describe the expression of these molecules in the trigeminal ganglion (TG) to understand more on their possible role(s). Single or double immunohistochemistry were applied on frozen sections of rat TG using primary antibodies against CGRP, procalcitonin, calcitonin, adrenomedullin, amylin, RAMP1/2/3, CLR, and CTR. In addition, mRNA expression was measured by quantitative qPCR on TGs. CGRP and calcitonin showed rich expression in the cytoplasm of small to medium-sized neurons, and co-localized sometimes. Procalcitonin was observed in the glial cells. Immunoreactive fibers storing both CGRP and calcitonin were also observed. Adrenomedullin immunoreactivity was found in the satellite glial cells and in fibers, probably the myelinating Schwann cells. Amylin was found in the cytoplasm in many TG neurons. Levels of mRNA expression for adrenomedullin, amylin, CLR, RAMP1, RAMP2, RAMP3, and CTR were measured using qPCR. The experiments verified the expression of mRNA in the TG with the exception of CTR, which was above the limit of detection indicating little or no mRNA expression. In addition to the well-known CGRP receptor (CLR/RAMP1) and the receptor for calcitonin-CTR, we propose that other receptors exist in the rat TG: adrenomedullin receptor AM2 (CLR/RAMP3) in mainly the satellite glial cells, amylin receptors AMY1 (CTR/RAMP1) in mainly neurons, and AMY3 (CTR/RAMP3) in the satellite glial cells. It is important to compare peptides and receptors side-by-side in studies to help address questions of actions resulting from cross-reactivity between receptors. Several of the diverse biological actions of the CGRP family of peptides are clinically relevant. Our findings demonstrate the specific ligand and receptor sites in the rat trigeminal ganglion, highlighting recognition mechanisms to facilitate drug development.