Background: Monoclonal antibodies (mAbs) towards CGRP or the CGRP receptor show good prophylactic antimigraine efficacy. However, their site of action is still elusive. Due to lack of passage of mAbs across the bloodbrain barrier the trigeminal system has been suggested a possible site of action because it lacks blood-brain barrier and hence is available to circulating molecules. The trigeminal ganglion (TG) harbors two types of neurons; half of which store CGRP and the rest that express CGRP receptor elements (CLR/RAMP1). Methods: With specific immunohistochemistry methods, we demonstrated the localization of CGRP, CLR, RAMP1, and their locations related to expression of the paranodal marker contactin-associated protein 1 (CASPR). Furthermore, we studied functional CGRP release separately from the neuron soma and the part with only nerve fibers of the trigeminal ganglion, using an enzyme-linked immunosorbent assay. Results: Antibodies towards CGRP and CLR/RAMP1 bind to two different populations of neurons in the TG and are found in the C-and the myelinated Aδ-fibers, respectively, within the dura mater and in trigeminal ganglion (TG). CASPR staining revealed paranodal areas of the different myelinated fibers inhabiting the TG and dura mater. Double immunostaining with CASPR and RAMP1 or the functional CGRP receptor antibody (AA58) revealed co-localization of the two peptides in the paranodal region which suggests the presence of the CGRP-receptor. Double immunostaining with CGRP and CASPR revealed that thin C-fibers have CGRP-positive boutons which often localize in close proximity to the nodal areas of the CGRP-receptor positive Aδ-fibers. These boutons are pearl-like synaptic structures, and we show CGRP release from fibers dissociated from their neuronal bodies. In addition, we found that adjacent to the CGRP receptor localization in the node of Ranvier there was PKA immunoreactivity (kinase stimulated by cAMP), providing structural possibility to modify conduction activity within the Aδ-fibers. Conclusion: We observed a close relationship between the CGRP containing C-fibers and the Aδ-fibers containing the CGRP-receptor elements, suggesting a point of axon-axon interaction for the released CGRP and a site of action for gepants and the novel mAbs to alleviate migraine.

The fifth cranial nerve is the common denominator for many headaches and facial pain pathologies currently known. Projecting from the trigeminal ganglion, in a bipolar manner, it connects to the brainstem and supplies various parts of the head and face with sensory innervation. In this review, we describe the neuroanatomical structures and pathways implicated in the sensation of the trigeminal system. Furthermore, we present the current understanding of several primary headaches, painful neuropathies and their pharmacological treatments. We hope that this overview can elucidate the complex field of headache pathologies, and their link to the trigeminal nerve, to a broader field of young scientists.

Background Migraine occurs 2–3 times more often in females than in males and is in many females associated with the onset of menstruation. The steroid hormone, 17β-estradiol (estrogen, E2), exerts its effects by binding and activating several estrogen receptors (ERs). Calcitonin gene-related peptide (CGRP) has a strong position in migraine pathophysiology, and interaction with CGRP has resulted in several successful drugs for acute and prophylactic treatment of migraine, effective in all age groups and in both sexes. Methods Immunohistochemistry was used for detection and localization of proteins, release of CGRP and PACAP investigated by ELISA and myography/perfusion arteriography was performed on rat and human arterial segments. Results ERα was found throughout the whole brain, and in several migraine related structures. ERβ was mainly found in the hippocampus and the cerebellum. In trigeminal ganglion (TG), ERα was found in the nuclei of neurons; these neurons expressed CGRP or the CGRP receptor in the cytoplasm. G-protein ER (GPER) was observed in the cell membrane and cytoplasm in most TG neurons. We compared TG from males and females, and females expressed more ER receptors. For neuropeptide release, the only observable difference was a baseline CGRP release being higher in the pro-estrous state as compared to estrous state. In the middle cerebral artery (MCA), we observed similar dilatory ER-responses between males and females, except for vasodilatory ERβ which we observed only in female arteries. Conclusion These data reveal significant differences in ER receptor expression between male and female rats. This contrasts to CGRP and PACAP release where we did not observe discernable difference between the sexes. Together, this points to a hypothesis where estrogen could have a modulatory role on the trigeminal neuron function in general rather than on the acute CGRP release mechanisms and vasomotor responses.

Background Several neurotransmitters are expressed in the neurons of the trigeminal ganglion. One such signalling molecule is the pituitary adenylate cyclase-activating peptide (PACAP). PACAP signalling has been suggested to have a possible role in the pathophysiology of primary headaches. Objective The present study was designed to investigate the relationship between PACAP and calcitonin gene-related peptide, currently the two most relevant migraine peptides. Methods In the current study, we used ELISA to investigate PACAP and calcitonin gene-related peptide release in response to 60 mM K+ or capsaicin using a rat hemi-skull model. We combined this analysis with qPCR and immunohistochemistry to study the expression of PACAP and calcitonin gene-related peptide receptors and ligands. Results Calcitonin gene-related peptide (CGRP) is released from the trigeminal ganglion and dura mater. In contrast, PACAP is only released from the trigeminal ganglion. We observed a weak correlation between the stimulated release of the two neuropeptides. PACAP-38 immunoreactivity was expressed alone and in a subpopulation of neurons in the trigeminal ganglion that also store calcitonin gene-related peptide. The receptor subtype PAC1 was mainly expressed in the satellite glial cells (SGCs), which envelop the neurons in the trigeminal ganglion, in some neuronal processes, inside the Aδ-fibres and in the outermost layer of the myelin sheath that envelopes the Aδ-fibres. Conclusion Unlike CGRP, PACAP is only released within the trigeminal ganglion. This raises the question of whether a migraine therapy aimed at preventing peripheral PACAP signalling would be as successful as the CGRP signalling targeted treatments.

Background Recent clinical findings suggest that oxytocin could be a novel treatment for migraine. However, little is known about the role of this neuropeptide/hormone and its receptor in the trigeminovascular pathway. Here we determine expression, localization, and function of oxytocin and oxytocin receptors in rat trigeminal ganglia and targets of peripheral (dura mater and cranial arteries) and central (trigeminal nucleus caudalis) afferents. Methods The methods include immunohistochemistry, messenger RNA measurements, quantitative PCR, release of calcitonin gene-related peptide and myography of arterial segments. Results Oxytocin receptor mRNA was expressed in rat trigeminal ganglia and the receptor protein was localized in numerous small to medium-sized neurons and thick axons characteristic of A∂ sensory fibers. Double immunohistochemistry revealed only a small number of neurons expressing both oxytocin receptors and calcitonin gene-related peptide. In contrast, double immunostaining showed expression of the calcitonin gene-related peptide receptor component receptor activity-modifying protein 1 and oxytocin receptors in 23% of the small cells and in 47% of the medium-sized cells. Oxytocin immunofluorescence was observed only in trigeminal ganglia satellite glial cells. Oxytocin mRNA was below detection limit in the trigeminal ganglia. The trigeminal nucleus caudalis expressed mRNA for both oxytocin and its receptor. K+-evoked calcitonin gene-related peptide release from either isolated trigeminal ganglia or dura mater and it was not significantly affected by oxytocin (10 µM). Oxytocin directly constricted cranial arteries ex vivo (pEC50 ∼ 7); however, these effects were inhibited by the vasopressin V1A antagonist SR49059. Conclusion Oxytocin receptors are extensively expressed throughout the rat trigeminovascular system and in particular in trigeminal ganglia A∂ neurons and fibers, but no functional oxytocin receptors were demonstrated in the dura and cranial arteries. Thus, circulating oxytocin may act on oxytocin receptors in the trigeminal ganglia to affect nociception transmission. These effects may help explain hormonal influences in migraine and offer a novel way for treatment.

BackgroundOnabotulinumtoxin type A (BoNT-A) has been found to reduce pain in chronic migraine. The aim of the present study was to ask if BoNT-A can interact directly on sensory mechanisms in the trigeminal ganglion (TG) using an organ culture method.MethodsTo induce inflammation, rat TGs were incubated for 24 hrs with either the mitogen MEK1/2 inhibitor U0126, BoNT-A or NaCl. After this the TGs were prepared for immunohistochemistry. Sections of the TG were then incubated with primary antibodies against CGRP (neuronal transmitter), iNOS (inflammatory marker), IL-1β (Interleukin 1β), SNAP-25 (synaptic vesicle docking protein) or SV2-A (Botulinum toxin receptor element).ResultsWe report that CGRP, iNOS, IL-1β, SNAP-25 and SV2-A were observed in fresh TG with a differential distribution. Interestingly, NaCl organ culture of the TG resulted in enhanced expression of CGRP and SNAP-25 in neurons and iNOS in SGCs. Co-incubation with U0126 or BoNT-A retained the increased expression of SNAP-25, while it decreased the IL-1β immunoreactivity in neurons. The iNOS expression in SGCs returned to levels observed in fresh specimens. Moreover, we observed no alteration SV2-A expression in SGCs. Thus, the overall picture is that both U0126 and BoNT-A have the ability to modify the expression of certain molecules in the TG.ConclusionWe hypothesize that chronic migraine might be associated with some degree of inflammation in the TG that could involve both neurons and SGCs. It is clinically well recognized that treatment with corticosteroids will reduce the symptoms of chronic migraine; however this remedy is associated with long-term side effects. Understanding the mechanisms involved in the expressional alterations may suggest novel ways to modify the changes and indicate novel therapeutics. The results of the present work illustrate one way by which BoNT-A may modify these expressional alterations.

Migraine is one of the most common of neurological disorders with a global prevalence of up to 15%. One in five migraineurs have frequent episodic or chronic migraine requiring prophylactic treatment. In recent years, specific pharmacological treatments targeting calcitonin gene‐related peptide (CGRP) signalling molecules have provided safe and effective treatments, monoclonal antibodies for prophylaxis and gepants for acute therapy. Albeit beneficial, it is important to understand the molecular mechanisms of these new drugs to better understand migraine pathophysiology and improve therapy. Here, we describe current views on the role of the CGRP family of peptides ‐ CGRP, calcitonin, adrenomedullin, amylin ‐ and their receptors in the trigeminovascular system. All these molecules are present within the trigeminovascular system but differ in expression and localization. It is likely that they have different roles, which can be utilized in providing additional drug targets. LINKED ARTICLES This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc

Substance P (SP) and calcitonin gene-related peptide (CGRP) have both been considered potential drug candidates in migraine therapy. In recent years, CGRP receptor inhibition has been established as an effective treatment, in particular as a prophylactic for chronic migraine. Curiously, inhibition of neurokinin receptor 1 (NK1R) failed to alleviate acute migraine attacks in clinical trials, and the neurokinins were consequently abandoned as potential antimigraine candidates. The reason behind this has remained enigmatic. Utilizing immunohistochemistry and semi-quantitative cell counts the expression of neurokinins and their associated receptors was examined in the rat trigeminal ganglion. Immunohistochemistry results revealed SP co-localization in CGRP positive neurons and C-fibres, where it mainly concentrated at boutons. Neurokinin A (NKA) was observed in a population of C-fibres and small neurons where it could co-localize with SP. In contrast, neurokinin B (NKB) did not co-localize with SP and was observed in large/medium sized neurons and Aδ-fibres. All neurokinin receptors (NK1-3R) were found to be expressed in a majority of trigeminal ganglion neurons and A-fibres. The functional release of SP and CGRP in the trigeminovascular system was stimulated with either 60 mM K+ or 100 nM capsaicin and measured with an enzyme-linked immunosorbent assay (ELISA). ELISA results established that SP can be released locally from trigeminovascular system. The released SP was comparatively minor compared to the CGRP release from stimulated dura mater, trigeminal ganglion neurons and fibres. We hypothesize that SP and CGRP signalling pathways may work in tandem to exacerbate painful stimuli in the TGV system.