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

Highlights

• High in vitro binding of CGRP receptor antagonist in rhesus trigeminal ganglion.

• Expression of CGRP and its receptor components in the rhesus trigeminal ganglion.

• 23% of the immunoreactive trigeminal neurons co-express CGRP and PACAP.

• Some neurons expressing the CGRP receptor components store glutamate.

• The trigeminal ganglion is not protected by the blood–brain barrier.

Abstract

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 [³H]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.

Introduction

Migraine is today recognized as a neurovascular disorder which originates in the brain, involving the hypothalamus and thalamus, as well as certain brainstem regions (Goadsby, 2012). The acute migraine attack is often preceded by prodromal symptoms, which suggest the central nervous system (CNS) as a likely point of origin (Charles, 2013). The pain during a migraine attack is associated with the release of the peptide calcitonin gene-related peptide (CGRP) which has a key role in migraine pathophysiology (Goadsby et al., 1988, Edvinsson and Goadsby, 1990, Ho et al., 2010). Clinical studies have demonstrated increased levels of CGRP putatively from the trigeminal system that can be found in serum, cerebrospinal fluid, and saliva (Goadsby et al., 1990, Goadsby and Edvinsson, 1993, Bellamy et al., 2006, Cernuda-Morollon et al., 2013). In addition, systemic infusion of CGRP can trigger a “migraine-like headache” in patients (Hansen et al., 2010). It is hypothesized that CGRP acts at second order neurons in the trigeminal nucleus caudalis (TNC) and at the C_1–2 level of the spinal cord, to transmit pain signals to thalamus and higher cortical pain regions (Goadsby, 2007, Levy et al., 2005). The trigeminovascular system is involved in the regulation of the cranial vasculature and is a key element in the transmission of pain. The trigeminal ganglion contains neurons that peripherally innervate the intracranial vasculature and dura mater. The trigeminal ganglion also centrally projects to the brainstem, in related extensions down to the spinal cord and to parts of the CNS where nociceptive information is processed to higher cortical regions (Liu et al., 2009; Goadsby, 2012). Stimulation of the trigeminal ganglion resulted in release of CGRP and elevation of CGRP in the external jugular vein (Goadsby et al., 1988, Limmroth et al., 2001). Neural activity in the trigeminal nociceptive system in migraine patients has been demonstrated using imaging techniques (Borsook et al., 2006). The wide distribution of CGRP receptors in the trigeminovascular system is consistent with a role in migraine pathophysiology (Lennerz et al., 2008, Eftekhari and Edvinsson, 2010, Eftekhari et al., 2010, Bhatt et al., 2014).

The most important evidence for the role of CGRP in migraine pain came from the development of CGRP receptor antagonists (Olesen et al., 2004, Ho et al., 2008a, Ho et al., 2008b) which act by blocking the action of CGRP on the CGRP-receptor complex. The CGRP receptor is a G protein-coupled receptor of the B-type consisting of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), both necessary to yield a functional CGRP receptor (Walker and Hay, 2013). It has been suggested that elevated neuronal RAMP1 could potentially sensitize the trigeminal ganglia of individuals to CGRP actions (Zhang et al., 2007). However, little is currently known about the regulation of RAMP1 levels in migraine.

Recent findings have identified the blockade of the CGRP receptor as a mechanism to reduce migraine pain (Salvatore and Kane, 2011, Moore and Salvatore, 2012). Clinical studies using CGRP receptor antagonists have demonstrated clinical efficacy comparable to that of triptans in the treatment of acute migraine attacks (Edvinsson and Linde, 2010, Ho et al., 2010, Salvatore and Kane, 2011). Therefore, it is of great interest to define where the CGRP receptor is expressed and on which possible sites drugs blocking CGRP signaling may have their therapeutic effect.

Two other neuronal messenger molecules that have been suggested to have an important role in migraine pathophysiology are pituitary adenylate cyclase-activating polypeptide (PACAP) and glutamate. Recent studies suggest that PACAP may have similar actions as CGRP and has been suggested to be involved in migraine pathogenesis (Kaiser and Russo, 2013, Tuka et al., 2013, Edvinsson, 2014). Also glutamate is implicated in migraine pathophysiology involving trigeminovascular activation, central sensitization and cortical spreading depression. Therefore, the use of glutamate receptor antagonists has been suggested for migraine treatment (Andreou and Goadsby, 2009, Marin and Goadsby, 2010). In relation to the trigeminal system both PACAP and glutamate are found in neurons in the trigeminal ganglion and in the trigeminocervical complex (Kai-Kai and Howe, 1991, Tajti et al., 1999, Uddman et al., 2002). However, their relation to the CGRP system (mainly the CGRP receptor) in the trigeminal ganglion has not been fully evaluated.

In order to better understand if the trigeminal ganglion may be one possible site of action for drugs such as the CGRP receptor antagonists, we have studied CGRP receptor binding sites with a CGRP receptor antagonist (MK-3207) in rhesus monkey. To confirm expression and cellular distribution of CGRP and its receptor components in rhesus monkey trigeminal ganglion immunofluorescence was used. In addition, the distribution of PACAP and glutamate were analyzed for possible co-expression with CGRP and the CGRP receptor in the rhesus monkey and rat trigeminal ganglion. Additionally, we used Evans blue in rats to study the relation of trigeminal ganglion to the blood brain-barrier (BBB).