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Cutaneous afferent nerves convey sensory information from the external, visceral nerves from the internal environment. The saphenous nerve arising from lumbar dorsal root ganglia and the vagus nerve originating in the nodosum ganglia are prototypic examples of such cutaneous and visceral nerves. Despite a common sensory role, these two nerves have distinct embryonic origin and vary in neuropeptide expression. Because of their distinct physiological roles, it is plausible that they differ also in conductive properties. We have tested calcitonin gene-related peptide (CGRP) release in these nerves in response to electrical and chemical stimulation. Electrical stimulation at 3, 6, and 9 Hz increased the release in saphenous but not vagus nerves, with 6 Hz being the most potent stimulus. Similarly, both capsaicin and a depolarizing solution of 60 mM KCl evoked CGRP release in saphenous but not vagus nerves. Simultaneous recording of the superimposed (compound) action potentials of these nerves revealed that only saphenous nerves exhibit a progressive and marked activity-dependent slowing of conduction velocity in response to electrical stimulation at 3, 6, and 9 Hz (30%, 44%, and 50%, respectively). Capsaicin caused an unexpected decrease in conduction latency (i.e., speeding) in contrast to the slowing seen in other nerves. Exposure of axons to 1 µM TTX rapidly blocked conduction in all nerves. Together our results demonstrate that vagus and saphenous primary afferents reveal different activation and conductive properties, presumably correlating their particular physiological roles in transmitting sensory signals. © 2018 Wiley Periodicals, Inc.

Background and purpose Meningeal blood flow is controlled by CGRP released from trigeminal afferents and NO mainly produced in arterial endothelium. The vasodilator effect of NO may be due to the NO-derived compound, nitroxyl (HNO), generated through reaction with endogenous H2 S. We investigated the involvement of HNO in CGRP release and meningeal blood flow. Experimental approach Blood flow in exposed dura mater of rats was recorded by laser Doppler flowmetry. CGRP release from the dura mater in the hemisected rat head was quantified using an elisa. NO and H2 S were localized histochemically with specific sensors. Key results Topical administration of the NO donor diethylamine-NONOate increased meningeal blood flow by 30%. Pretreatment with oxamic acid, an inhibitor of H2 S synthesis, reduced this effect. Administration of Na2 S increased blood flow by 20%, an effect abolished by the CGRP receptor antagonist CGRP8-37 or the TRPA1 channel antagonist HC030031 and reduced when endogenous NO synthesis was blocked. Na2 S dose-dependently increased CGRP release two- to threefold. Co-administration of diethylamine-NONOate facilitated CGRP release, while inhibition of endogenous NO or H2 S synthesis lowered basal CGRP release. NO and H2 S were mainly localized in arterial vessels, HNO additionally in nerve fibre bundles. HNO staining was lost after treatment with L-NMMA and oxamic acid. Conclusions and implications NO and H2 S cooperatively increased meningeal blood flow by forming HNO, which activated TRPA1 cation channels in trigeminal fibres, inducing CGRP release. This HNO-TRPA1-CGRP signalling pathway may be relevant to the pathophysiology of headaches.

Objective To reinvestigate the innervation pattern of the dura mater of rat and human middle cranial fossa, the morpho-functional substrate of headache generation, and adjacent extracranial tissues with neuronal in vitro tracing. Background This study was initiated by recent structural and functional findings of meningeal afferent fibers which innervate the cranial dura mater and may project to extracranial tissues. Methods Anterograde and retrograde neuronal in vitro tracing was made in formaldehyde fixed hemisected rat and human skulls. The fluorescent tracer DiI was applied to proximally cut meningeal nerves in rat and to distal branches of the spinosus nerve in human calvaria lined by dura mater. After several weeks, the dura mater and deep extracranial tissues were examined with fluorescence microscopy. Results In addition to a network of meningeal nerve fibers, several fiber bundles were observed, leaving the skull through emissary canals and fissures to innervate the pericranial temporal, parietal, and occipital periosteum. Traced fibers were seen spreading into deep layers of the temporal and upper neck muscles. Retrograde neuronal tracing revealed labeled cell bodies exclusively in the mandibular and maxillary division of the rat trigeminal ganglion, and centrally projecting fibers were identified in the spinal trigeminal tract. Electron microscopy of the cross-sected spinosus nerve showed myelinated and unmyelinated axons with similar numbers in human and rat. Conclusions We conclude that a proportion of meningeal afferents innervates extracranial tissues like periosteum and pericranial muscles via collaterals projecting through the skull. These afferents may be nociceptive, some may subserve proprioceptive functions. The finding of extracranial projections of meningeal afferents may be important for our understanding of extracranial impacts on headache generation and therapy.

Calcitonin gene-related peptide (CGRP) and metabolic products of nitric oxide (NO) are increased in jugular venous plasma during migraine attacks and other primary headaches. Patients suffering from primary headaches are particularly sensitive to CGRP and NO donors responding with delayed headaches to an infusion of either of these substances. Accordingly, both CGRP and NO are considered as key mediators in migraine, and clinical trials have shown that inhibitors of CGRP receptors and NO synthase are effective in treating migraine. There is an implicit understanding that CGRP and NO systems interact, and here, we review the body of preclinical work on these systems focusing on the trigeminovascular system in migraine. NO derives from various cell types via 3 isoforms of NO synthase, whereas CGRP is produced from a subset of trigeminal afferents. In rodents, NO donors cause activity alterations on different levels of the trigeminal system including enhancement of CGRP release, which in turn results in arterial vasodilatation and possibly mast cell degranulation in the meninges. The activity of spinal trigeminal neurons, which is a sensitive integrative measure for trigeminal activity, is partly under the control of CGRP and NO. Both mediators facilitate nociceptive transmission, possibly via presynaptic mechanisms. These functions are supported by immunolocalization of CGRP receptor components on 3 trigeminovascular levels: cranial dura mater, trigeminal ganglion, and spinal trigeminal nucleus. Current data support a relationship of CGRP and NO actions on all levels of the trigeminovascular system and emphasize central CGRP receptors as possible therapeutic targets.

BACKGROUND AND PURPOSE Calcitonin gene-related peptide (CGRP) is widely distributed in the trigeminovascular system and released from sensory fibres of the cranial dura mater upon noxious stimulation. Such release may be a mechanism underlying migraine headache. Based on data from guinea pig basilar artery preparations, we have here studied CGRP release and uptake in an organ preparation of the hemisected rat skull. EXPERIMENTAL APPROACH CGRP release from the cranial dura was quantified by a commercial enzyme-linked immunoassay. CGRP was depleted using repetitive challenges of capsaicin. After incubating the tissue with CGRP for 20 min and extensive washing, another capsaicin challenge was performed. Immunohistochemistry was used to visualize CGRP immunofluorescence in dural nerve fibres. KEY RESULTS Capsaicin-induced CGRP release was attenuated by the transient receptor potential vanilloid receptor type I antagonist capsazepine or by Ca2+-free solutions. After the CGRP-depleted preparation had been exposed to exogenous CGRP, capsaicin-induced CGRP release was increased compared to the challenge just prior to incubation. CGRP uptake was not influenced by Ca2+-free solutions. Olcegepant and CGRP8–37 (CGRP receptor antagonists) did not affect uptake of CGRP. However, a monoclonal CGRP-binding antibody decreased CGRP uptake significantly. Release of CGRP after incubation was attenuated by Ca2+-free solutions and by capsazepine. Immunohistochemical assays indicated a weak trend towards CGRP uptake in rat dura mater. CONCLUSION AND IMPLICATIONS We have presented evidence for CGRP uptake in nerves and its re-release in rat dura mater. This may have implications for the pathophysiology and treatment of migraine.

Axonal conduction velocity varies according to the level of preceding impulse activity. In unmyelinated axons this typically results in a slowing of conduction velocity and a parallel increase in threshold. It is currently held that Na+–K+-ATPase-dependent axonal hyperpolarization is responsible for this slowing but this has long been equivocal. We therefore examined conduction velocity changes during repetitive activation of single unmyelinated axons innervating the rat cranial meninges. In direct contradiction to the currently accepted postulate, Na+–K+-ATPase blockade actually enhanced activity-induced conduction velocity slowing, while the degree of velocity slowing was curtailed in the presence of lidocaine (10–300 μm) and carbamazepine (30–500 μm) but not tetrodotoxin (TTX, 10–80 nm). This suggests that a change in the number of available sodium channels is the most prominent factor responsible for activity-induced changes in conduction velocity in unmyelinated axons. At moderate stimulus frequencies, axonal conduction velocity is determined by an interaction between residual sodium channel inactivation following each impulse and the retrieval of channels from inactivation by a concomitant Na+–K+-ATPase-mediated hyperpolarization. Since the process is primarily dependent upon sodium channel availability, tracking conduction velocity provides a means of accessing relative changes in the excitability of nociceptive neurons.

Several endogenous factors such as calcitonin gene-related peptide (CGRP), nitric oxide, and histamine have been found to affect meningeal blood flow. A possible regulation of meningeal blood flow by thermosensitive neurons has not been examined so far. We monitored meningeal arterial blood flow with laser Doppler flowmetry in anesthetized rats. A vortex thermode was used to control the temperature of the exposed dura mater. After fast heating from 36 degrees C to 45 degrees C, meningeal blood flow increased slowly within 5 min. Immediately after fast cooling to 36 degrees C, the flow transiently increased before it returned to the initial basal level. After the application of lidocaine onto the dura mater, the heating-induced flow increase was greater and the transient cooling-induced flow increase was reduced, indicating the involvement of neuronal mechanisms. Furthermore, after topical application of phenylephrine, the cooling-induced response was increased, and following topical application of phentolamine the heating-induced flow increase was higher. The flow changes evoked by thermal stimulation were unaffected by high concentrations of 1) cimetidine, 2) a vasoactive intestinal polypeptide antagonist and the CGRP receptor antagonists, 3) CGRP(8-37), and 4) BIBN4096BS. We conclude that activation of temperature-sensitive meningeal afferents can regulate the dura mater blood flow by a sympathetic reflex.

1 Calcitonin gene-related peptide (CGRP) released from trigeminal afferents is known to play an important role in the control of intracranial blood flow. In a rat preparation with exposed cranial dura mater, periods of electrical stimulation induce increases in meningeal blood flow. These responses are due to arterial vasodilatation mediated in part by the release of CGRP. In this preparation, the effect of a CGRP-binding mirror-image oligonucleotide (Spiegelmer NOX-C89) was examined. 2 Spiegelmer NOX-C89 applied topically at concentrations between 10−7and 10−5 M to the exposed dura mater led to a dose-dependent inhibition of the electrically evoked blood flow increases. The highest dose reduced the mean increases in flow to 56% of the respective control levels. A nonfunctional control Spiegelmer (not binding to CGRP) was ineffective in changing blood flow increases. Intravenous injection of NOX-C89 (5 mg kg−1) reduced the evoked blood flow increases to an average of 65.5% of the control. The basal blood flow was not changed by any of the applied substances. 3 In addition, an ex vivo preparation of the hemisected rat skull was used to determine CGRP release from the cranial dura mater caused by antidromic activation of meningeal afferents. In this model, 10−6 M of NOX-C89 reduced the evoked CGRP release by about 50%. 4 We conclude that increases in meningeal blood flow due to afferent activation can be reduced by sequestering the released CGRP and thus preventing it from activating vascular CGRP receptors. Moreover, the Spiegelmer NOX-C89 may inhibit CGRP release from meningeal afferents. Therefore, the approach to interfere with the CGRP/CGRP receptor system by binding the CGRP may open a new opportunity for the therapy of diseases that are linked to excessive CGRP release such as some forms of primary headaches. British Journal of Pharmacology (2006) 148, 536–543. doi:10.1038/sj.bjp.0706742

1. Vasodilatation in the dura mater has been suggested to play an important role in the pathophysiology of vascular headaches. Histamine may contribute to these vascular changes. The aim of the present study was to examine the role of different histamine receptors in histamine-induced meningeal hyperperfusion using laser Doppler flowmetry. 2. The blood flow in the medial meningeal artery was monitored in the exposed parietal dura mater encephali of barbiturate anaesthetized rats. Local application of histamine (10(-5) and 10(-4) M) onto the dura caused increases in flow to 114.2+/-9.6 and 135.1+/-19.1%, respectively, of the basal flow. 3. Flow increases induced by topical application of histamine (10(-4) M) were reduced by local pretreatment with the H(2)-receptor antagonist cimetidine (0.4 and 4 mM) to 63.4+/-17 and 37.8+/-18.8%, respectively. Systemic pre-administration of cimetidine (5 mg kg(-1) i.v.) did not change histamine-induced flow increases. 4. Local pretreatment with the H(1)-receptor antagonist cetirizine (2 micro M) further increased the flow evoked by topical histamine administration (10(-4) M) to 123.5+/-14.7% of the histamine control. 5. Increases in blood flow induced by i.v. administration of histamine (10 micro g kg(-1)) were reduced by i.v. pre-injection of cetirizine (50 micro g kg(-1)) to 31.9+/-9% but not by i.v. cimetidine (5 mg kg(-1)). 6. We conclude that histamine-induced relaxation of dural arterial vessels is mediated by H(2)-receptors, most likely located on vascular smooth muscle cells, and by endothelial H(1)-receptors. In addition, H(1)-receptors on smooth muscle cells may mediate vasoconstriction.

1. Nitric oxide (NO) has been proposed to be a key molecule in the pathogenesis of migraine pain and other headaches that are linked to vascular disorders. Several lines of evidence indicate that the meningeal vascularization is crucially involved in the generation of these headaches. In an experimental model in the rat a dominating role of calcitonin gene-related peptide (CGRP) in causing neurogenic vasodilatation and increased blood flow has been shown. The aim of the present study was to clarify the role of NO in this model with regard to the meningeal blood flow. 2. The blood flow in and around the medial meningeal artery (dural arterial flow) was recorded in the exposed parietal dura mater encephali of barbiturate anaesthetized rats using laser Doppler flowmetry. Local electrical stimulation of the dura mater (pulses of 0.5 ms delivered at 7.5 - 17.5 V and 5 or 10 Hz for 30 s) caused temporary increases in dural arterial flow for about 1 min that reached peaks of 1.6 - 2.6 times the basal flow. The effects of NO synthase (NOS) inhibitors on the basal flow and the electrically evoked increases in flow were examined. 3. Systemic (i. v.) administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) at cumulative doses of 10 and 50 mg kg(-1) lowered the basal flow to 87 and 72%, respectively, of the control and reduced the evoked increases in blood flow to 82 and 44% on an average. Both these effects could partly be reversed by 300 mg kg(-1) L-arginine. The systemic arterial pressure was increased by L-NAME at both doses. Injection of the stereoisomer D-NAME at same doses did not change basal flow and evoked increases in flow. 4. 4. Topical application of L-NAME (10(-4) - 10(-2) M) was effective only at the highest concentration, which caused lowering of the basal blood flow to 78% of the control; the evoked increases in flow were not changed. Topical application of 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a specific inhibitor of the inducible NOS, at concentrations of 10(-4) - 10(-2) M lowered the basal flow to 89, 87.5 and 85%, respectively, but did not significantly change the evoked flow increases. Same concentrations of 7-nitroindazole monosodium salt (7-NINA), a specific inhibitor of the neuronal NOS, had no significant effects on basal flow and evoked increases in flow. 5. It is concluded that NO is involved in the maintenance of the basal level of dural arterial blood flow as well as in the electrically evoked flow increases, which have been shown to be mainly mediated by CGRP released from dural afferent fibres. The most important source of NO is probably the endothelium of dural arterial vessels. The synergistic effect of NO and CGRP on the stimulated blood flow may be in part due to a NO mediated facilitation of the CGRP release.

1. The dura mater encephali of the rat was exposed and the blood flow around branches of the medial meningeal artery was monitored with a laser Doppler flowmeter. Changes in the meningeal blood flow (MBF) following electrical stimulation of the dura mater at a parasagittal site were registered. The effects of human calcitonin gene-related peptide (h-alpha CGRP) and the CGRP antagonist (h-alpha CGRP8-37) on the MBF were tested. 2. Electrical stimulation with rectangular pulses of 0.5 ms, 10-20 V, 5-10 Hz and a duration of 30 s caused an increase of the MBF in 14 out of 16 rats tested. The increases were dependent on stimulus strength and frequency. 3. The increase in MBF was inhibited in a dose-dependent manner by topical application of 0.1 ml of h-alpha CGRP8-37 at concentrations of 10(-7) - 10(-5) M. The highest dose abolished the increase in MBF. 4. Topical administration of 0.1 ml of h-alpha CGRP at a concentration of 10(-4) M increased the basal MBF by 15% on average. 5. It is suggested that the increase in MBF following electrical stimulation of the dura mater is mediated by the release of CGRP. The contribution of the dural afferent and sympathetic and parasympathetic efferent nerve fibres to this response are discussed.

Afferent group III and IV nerve fibers of the knee joint markedly differ in their responsiveness to mechanical stimulation, which may be reflected in the structure and location of their terminals. Therefore, in sympathectomized cats, the fine afferent innervation of the knee joint capsule was studied via ultrastructural three-dimensional reconstructions over distances of up to 300 microns. Small peripheral nerves and "free" (noncorpuscular) sensory nerve endings were found in a superficial layer of the outer fibrous part of the capsule, in the patellar retinaculum, and in the outer and inner surface layers of the medial collateral and patellar ligaments. Group III nerve fibers showed a proximal myelinated portion inside the nerve, an intermediate portion that lacks a myelin sheath and is only surrounded by perineurium, and a distal portion outside of the perineurium that forms the sensory ending proper. Group IV fibers showed only two distinct portions, an intraperineurial (proximal) and an extraperineurial (distal) portion without any further morphological differences. Outside of the perineurium, a network formed by Schwann cells ("Schwann cell reticulum") provides a pathway for the distal portion of the sensory axons. No distinct subgroups of the sensory terminal fibers could be defined according to the configuration of the Schwann cells and the nerve fiber terminals. Sensory terminals were located adjacent to different structures such as venous and lymphatic vessels, fat cells, and collagenous fibers. Distinct parts of the same terminal nerve fiber were found in close contact to a vessel wall; others were surrounded by dense collagenous tissue. Close to sensory endings, mast cells and mast cell-like cells were frequently found, indicating a functional relationship.

Neurogenic inflammation of the dura mater encephali has been suggested to play an important role in the pathophysiology of headaches. Although functional studies using extravasation techniques indicate an enhanced permeability of blood vessels after chemical or electrical stimulation of C-fibres supplying the dura mater, histological demonstration of leaky blood vessels is still a problem. We used the vascular labelling method combined with i.v. injection of colloidal silver solution to test the permeability increasing effect of intravenous administration of substance P, topical application of mustard oil or acidic phosphate buffer and local electrical stimulation of the exposed dura mater. Histological characteristics of increased vascular permeability were observed exclusively after mustard oil and acidic phosphate buffer. This observation may indicate different mechanisms of increased vascular permeability involving pinocytosis and formation of interendothelial gaps selectively visualized by the vascular labelling method.

SUMMARY Two different techniques for three-dimensional reconstructions of nerve fibres or other extended fine tissue structures were developed to study the afferent innervation of the cat's knee-joint capsule. The re-embedding technique starts with series of semithin sections for light microscopic reconstructions. In a second step, selected semithin sections can be re-embedded for ultramicrotomy to examine ultrastructural details. This method offers the possibility to investigate fine structures over a distance of several hundred micrometres without any loss of ultrastructural information. The serial section-ESI technique is based on the electron spectroscopic imaging of semithin sections. Small tissue blocks are cut into series of either semithin or alternate semi- and ultrathin sections which can be directly used for a complete ultrastructural investigation. Finally, true-to-scale three-dimensional reconstructions are performed by graphical techniques or computer-aided methods.