Localization of CGRP receptor components and receptor binding sites in rhesus monkey brainstem: A detailed study using in situ hybridization, immunofluorescence, and autoradiography

Abstract

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.

Localization of CGRP, CGRP receptor, PACAP and glutamate in trigeminal ganglion. Relation to the blood–brain barrier

“…It will be of great interest to assess if CNS-penetrant CGRP receptor antagonists demonstrate better clinical efficacy. Interestingly, we have shown binding of a CGRP receptor antagonist via in vitro autoradiography in the pineal gland and area postrema of rhesus monkey (Eftekhari et al, 2013a). These CNS areas are not protected by the BBB suggesting that these areas can also be reached by drugs such as CGRP receptor antagonists.…”

Wiping Out CGRP: Potential Cardiovascular Risks

“…Thus, structures that are located outside the BBB appear to be more likely therapeutic targets for the CGRP antibodies. Such structures include not only cranial blood vessels [28], but also neuronal structures that are not fully protected by the BBB, such as the trigeminal ganglion and the paraventricular structures within the brain stem [29][30][31].…”

“…Thus, structures that are located outside the BBB appear to be more likely therapeutic targets for the CGRP antibodies. Such structures include not only cranial blood vessels [28], but also neuronal structures that are not fully protected by the BBB, such as the trigeminal ganglion and the paraventricular structures within the brain stem [29][30][31].Little is known about the pharmacokinetic and pharmacodynamic properties of the CGRP (receptor) antibodies. Given that CGRP plasma concentrations and half-life are known approximately and by assuming (i) linear efficacy during the whole dosing period and (ii) that a given dose of CGRP antibody will completely enter the circulation and will only disappear from the circulation after having bound to a molecule of CGRP, we may estimate that CGRP antibodies might scavenge CGRP for up to 1.5 months (Box 1).…”

The role of calcitonin gene–related peptide in peripheral and central pain mechanisms including migraine

“…65 High densities of the CGRP receptor were found in the pineal gland, medial mammillary nucleus, median eminence, infundibular stem, periaqueductal gray (PAG), dorsal raphe, posterior hypothalamic area, trochlear nucleus, and the medial lemniscus. 65 Binding to [ 3 H]MK-3207, indicative of CGRP receptors, was also detected in the spinal TNC, nucleus gracilis, dorsal and ventral horns of the spinal cord, pontine raphe nucleus, pontine nuclei, area postrema, inferior olive, the centrointermediate part of the dorsal motor nucleus of vagus, and the hypoglossal nucleus. 65 Many of these regions, such as the PAG, the raphe areas, and the nucleus gracilis, receive somatosensory inputs, are implicated in the transmission or modulation of nociceptive signaling, and have also been implicated in migraine headache.…”

“…3,65,67,177 In a 2001 study, it was estimated that up to 80% of cell bodies in the locus coeruleus, which is implicated in cardiovascular, autonomic, and nociceptive functions, expressed CGRP. 177 In that study, no CGRP was detected in cell bodies of the PAG or NRM.…”