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Objective.— To determine whether controlled changes in barometric pressure activate rat spinal trigeminal neurons as a possible animal correlate of headaches. Background.— Changes in weather accompanied by changes in atmospheric pressure are suggested to trigger primary headaches. Mechanisms that increase neuronal activity in the rat spinal trigeminal nucleus may parallel those that contribute to the generation of headaches. Methods.— Urethane anesthetized rats were placed in a climatic chamber, in which the air pressure could be selectively manipulated. The parietal cranial dura mater and the spinal dura mater covering the medulla were exposed. Electrolyte-filled electrodes were introduced into the spinal trigeminal nucleus to record from neurons with receptive fields in facial areas and the cranial dura mater and/or the cornea and/or the temporal muscle. Arterial pressure and heart rate were monitored. The barometric pressure was lowered by 40 hPa during 8 minutes, kept at this level for 8 minutes and returned to the previous level. Results.— During lowering of the barometric pressure and the low pressure period a sample of neurons showed increased discharge rates. Group analysis revealed that it was the group of units with receptive fields in the cornea, but not in the dura mater or the temporal muscle, which was significantly activated when the animal was exposed to low atmospheric pressure. Exposure of the cranial dura and opening of the cisterna magna did not prevent an increase in activity. In another sample of units the activity recorded after infusion of the nitric oxide donor sodium nitroprusside did not change under low pressure exposure. Arterial pressure and heart rate changed slightly during barometric pressure changes. Conclusions.— We conclude that distinct neurons in the trigeminal nucleus caudalis, particularly with preferential afferent input from the eye, respond to lowering of atmospheric pressure. Similar mechanisms may contribute to the generation of headaches during changes in weather.

Changes in blood vessel diameter can be measured manually, but this is time-consuming and often impractical. For automatic measurement commercial solutions are available, but the proprietary algorithms and their potential shortcomings are not known to the user. We present an approach with a CCD camera for image acquisition combined with free and open source ImageJ software for offline analysis. A subtraction image allows for the evaluation of the diameter changes throughout the field of view. A full width at half-maximum algorithm plug-in was written to measure the vessel diameter. For a given line across a vessel, the results of five measurements with parallel shifts throughout an image stack are copied to the clipboard. For validation of this method an established in vivo model was used, namely vascular changes in the rat dura mater, reflecting the activity of the afferent neurons. Vasoconstriction of the meningeal arterioles induced by local electrical stimulation of the dura was inhibited by intravenous administration of the adrenoceptor antagonist phentolamine and amplified by the CGRP receptor antagonist olcegepant. The described methods allow the user to quickly evaluate vessel diameter changes in the whole acquired field at any selected position.

Several lines of evidence suggest a major role of calcitonin gene-related peptide (CGRP) in the pathogenesis of migraine and other primary headaches. Inhibition of CGRP receptors by olcegepant and telcagepant has been successfully used to treat acute migraine and to reduce the activity of spinal trigeminal neurons involved in meningeal nociception in rodents. The site of CGRP receptor inhibition is unclear, however. In adult Wistar rats anaesthetized with isofluorane systemic intravenous infusion (0.9 mg/kg) or unilateral facial injection (1 mM in 100 microl) of capsaicin was used to induce activity in the trigeminal nociceptive system. Animals were pre-treated either by saline or olcegepant. In comparison with vehicle infusion or the non-injected side of the face, capsaicin significantly increased the expression of the activation markers Fos in the spinal trigeminal nucleus and phosphorylated extracellular signal-regulated kinase in the trigeminal ganglion. Pre-treatment with olcegepant (900 microg/kg) inhibited the capsaicin-induced expression of Fos throughout the spinal trigeminal nucleus by 57%. In contrast, the expression of phosphorylated extracellular signal-regulated kinase in the trigeminal ganglion was not changed by olcegepant pre-treatment. CGRP receptor inhibition, which has been shown to decrease spinal trigeminal activity, is likely to occur in the central nervous system rather than in the periphery including the trigeminal ganglion. This may be important for future therapeutic interventions with CGRP receptor antagonists in migraine.

Background Morphine and its derivatives are key drugs in pain control. Despite its well-known analgesic properties morphine at high concentrations may be proalgesic. Particularly, short-lasting painful sensations have been reported upon dermal application of morphine. To study a possible involvement of TRP receptors in the pro-nociceptive effects of morphine (0.3 – 10 mM), two models of nociception were employed using C57BL/6 mice and genetically related TRPV1 and TRPA1 knockout animals, which were crossed and generated double knockouts. Hindpaw skin flaps were used to investigate the release of calcitonin gene-related peptide indicative of nociceptive activation. Results Morphine induced release of calcitonin gene-related peptide and sensitized the release evoked by heat or the TRPA1 agonist acrolein. Morphine activated HEK293t cells transfected with TRPV1 or TRPA1. Activation of C57BL/6 mouse dorsal root ganglion neurons in culture was investigated with calcium imaging. Morphine induced a dose-dependent rise in intracellular calcium in neurons from wild-type animals. In neurons from TRPV1 and TRPA1 knockout animals activation by morphine was markedly reduced, in the TRPV1/A1 double knockout animals this morphine effect was abrogated. Naloxone induced an increase in calcium levels similar to morphine. The responses to both morphine and naloxone were sensitized by bradykinin. Conclusion Nociceptor activation and sensitization by morphine is conveyed by TRPV1 and TRPA1.

Neurogenic inflammation of the dura mater encephali is considered to be involved in the generation of primary vascular headaches. Different components of neurogenic inflammation – increased blood flow and vascular permeability, activation of endothelial and mast cells – seem to have differential significance in the pathophysiology of primary headaches. While the release of vasoactive substances from primary sensory neurons, endothelium, or mast cells has clearly been shown to occur during headache attacks, vascular permeability changes seem not to play any role. Clinical and experimental observations indicate that the sensory neuropeptide calcitonin gene-related peptide (CGRP) has a key position in meningeal vasodilatation. Beside its direct vasorelaxant effect, CGRP releases the inflammatory mediator histamine from mast cells and interacts with another potent vasodilator, nitric oxide, originating mainly from dural endothelial cells. Parasympathetic nerve fibers of the dura mater are also activated in some types of primary headaches, possibly by a trigeminal-parasympathetic reflex involving brain stem mechanisms. Interactions between different vasoactive regulators of meningeal blood flow may contribute to the pathogenesis of primary headaches.

Infusion of nitric oxide (NO) donors is known to induce delayed attacks of migraine and cluster headache or aggravate tension-type headaches in patients suffering from these primary headaches. Previously we have reported that infusion of NO donors in the rat causes delayed neuronal activity in the spinal trigeminal nucleus, which parallels the above clinical observations. Suggesting that endogenous NO production is involved in the generation of primary headaches, we used this animal model of meningeal nociception to determine whether a prolonged increase in NO levels causes an increase in neuronal activity. In anaesthetized rats spinal trigeminal neurons with afferent input from the exposed dura were recorded. Continuous intravenous infusion of the NO donors sodium nitroprusside (25 μg/kg/h) or glycerol trinitrate (250 μg/ kg/h) for 2h induced a persisting increase in neuronal activity but no change in systemic blood pressure. In this activated trigeminal system the calcitonin gene-related peptide (CGRP) receptor antagonist BIBN4096BS (900 μg/ kg) was infused. Spinal trigeminal activity was significantly reduced within minutes and to a similar extent as previously reported in animals not treated with NO. Slow continuous NO infusion may be a model of the active headache phase, and inhibition of CGRP receptors can reverse the induced neuronal activity.

Migraine is a complex neurological disease with a genetic background. Headache is the most prominent and clinically important symptom of migraine but its origin is still enigmatic. Numerous clinical, histochemical, electrophysiological, molecular and genetical approaches form a puzzle of findings that slowly takes shape. The generation of primary headaches like migraine pain seems to be the consequence of multiple pathophysiological changes in meningeal tissues, the trigeminal ganglion, trigeminal brainstem nuclei and descending inhibitory systems, based on specific characteristics of the trigeminovascular system. This contribution reviews the current discussion of where and how the migraine pain may originate and outlines the experimental work to answer these questions.

Calcitonin gene-related peptide (CGRP) is a key mediator in primary headaches including migraine. Animal models of meningeal nociception demonstrate both peripheral and central CGRP effects; however, the target structures remain unclear. To study the distribution of CGRP receptors in the rat trigeminovascular system we used antibodies recognizing two components of the CGRP receptor, the calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1). In the cranial dura mater, CLR and RAMP1 immunoreactivity (-ir) was found within arterial blood vessels, mononuclear cells, and Schwann cells, but not sensory axons. In the trigeminal ganglion, besides Schwann and satellite cells, CLR- and RAMP1-ir was found in subpopulations of CGRP-ir neurons where colocalization of CGRP- and RAMP1-ir was very rare ( approximately 0.6%). CLR- and RAMP1-ir was present on central, but not peripheral, axons. In the spinal trigeminal nucleus, CLR- and RAMP1-ir was localized to "glomerular structures," partly colocalized with CGRP-ir. However, CLR- and RAMP1-ir was lacking in central glia and neuronal cell bodies. We conclude that CGRP receptors are associated with structural targets of known CGRP effects (vasodilation, mast cell degranulation) and targets of unknown function (Schwann cells). In the spinal trigeminal nucleus, CGRP receptors are probably located on neuronal processes, including primary afferent endings, suggesting involvement in presynaptic regulation of nociceptive transmission. Thus, in the trigeminovascular system CGRP receptor localization suggests multiple targets for CGRP in the pathogenesis of primary headaches.

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 microm) and carbamazepine (30-500 microm) 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.

Fischer MJM & Messlinger K. Cannabinoid and vanilloid effects of R(+)-methanandamide in the hemisected meningeal preparation. Cephalalgia 2007; 27:422–428. London. ISSN 0333-1024 The endogenous cannabinoid R(+)-methanandamide (mAEA) exerts differential anti- and pronociceptive effects by activating both cannabinoid (CB1) and vanilloid (TRPV1) receptors of nociceptive primary afferents. The significance of these effects in meningeal nociception was evaluated by modulation of calcitonin gene-related peptide (CGRP) release from meningeal afferents measured in an in vitro preparation of the hemisected rat skull. Temperature steps to 39°C and 45°C caused heat-dependent increases in CGRP release. One micromolar mAEA inhibited CGRP release at 32°C but facilitated it at 45°C. This effect was abolished in the presence of the TRPV1 receptor antagonist capsazepine. Lower doses of mAEA had no effect on basal or heat-evoked release. In the presence of the CB1 receptor antagonist SR141716 (0.2 µm) heat-stimulated increase in CGRP release was facilitated. CGRP release in the presence of SR141716 (0.2 µm) was further increased by adding mAEA at a concentration which had no effect on its own. These results confirm an opposing functional role for anandamide at CB1 and TRPV1 receptors on meningeal afferents.

Calcitonin gene-related peptide (CGRP), nitric oxide (NO) and histamine are implicated in primary headaches but their role in vascular and nociceptive events in the dura mater is not well described. In an in vitro preparation of the hemisected rat skull, CGRP and histamine release from the cranial dura was measured using enzyme-linked immunoassays. While the NO donator NONOate (10-4 M) was without effect, CGRP (10-5 M) induced considerable histamine release from the rat cranial dura, which was blocked by the CGRP receptor antagonist CGRP8-37 (10-5 M). Conversely, histamine (10-4 M) did not stimulate CGRP release. In vitro recordings from single rat meningeal afferents showed that only one of 12 mechanically identified units but several mechanically insensitive units responded to histamine (up to 10-5 M). Increases in meningeal blood flow after histamine application (10-4 M) to the rat cranial dura remained unchanged during CGRP receptor blockade with CGRP8-37, inhibition of NO synthesis with L-NAME (20 mg/kg i.v.) and H3 receptor blockade with thioperamide (10-4 M). We conclude that histamine produces direct vasodilatation and activates a subset of largely non-mechanically sensitive, non-CGRP containing afferents in the rat meninges. Histamine is released from meningeal mast cells which are stimulated by CGRP. Similar mechanisms may be involved in the pathogenesis of headaches.

Calcitonin gene-related peptide (CGRP), a potent vasodilator released during activation from a subset of sensory Adelta- and C-fiber afferents, has been suggested to play a beneficial role in myocardial ischemia. Variations in CGRP release can possibly be correlated with diseases that involve changes in activity or degeneration of cardiac afferent fibers. The aim of the present study was to examine basal and stimulated CGRP release from cardiac tissue in patients who underwent cardiopulmonary bypass surgery and to compare patients with and without known history of diabetes mellitus. Small pieces of the right atrium routinely excised during the bypass operations were passed through series of oxygenated solutions. The TRPV1 receptor agonist capsaicin and the nitric oxide donor NONOate were added for stimulation of cardiac afferent fibers. The eluates were processed using an enzyme immuno-assay (EIA) for measurement of CGRP concentrations. Both capsaicin and NONOate caused significant increases in CGRP release. No significant differences in CGRP release between patients with and without diabetes mellitus were examined. The present study evaluates a simple and reproducible model for measuring stimulated CGRP release from the human right atrium.

Heat sensitivity shows considerable functional variability in humans and laboratory animals, and is fundamental to inflammatory and possibly neuropathic pain. In the mouse, at least, much of this variability is genetic because inbred strains differ robustly in their behavioral sensitivity to noxious heat. These strain differences are shown here to reflect differential responsiveness of primary afferent thermal nociceptors to heat stimuli. We further present convergent behavioral and electrophysiological evidence that the variable responses to noxious heat are due to strain-dependence of CGRP expression and sensitivity. Strain differences in behavioral response to noxious heat could be abolished by peripheral injection of CGRP, blockade of cutaneous and spinal CGRP receptors, or long-term inactivation of CGRP with a CGRP-binding Spiegelmer. Linkage mapping supports the contention that the genetic variant determining variable heat pain sensitivity across mouse strains affects the expression of the Calca gene that codes for CGRPα.

Calcitonin gene-related peptide (CGRP) has been suggested to play a major role in the pathogenesis of migraines and other primary headaches. CGRP may be involved in the control of neuronal activity in the spinal trigeminal nucleus (STN), which integrates nociceptive afferent inputs from trigeminal tissues, including intracranial afferents. The activity of STN neurons is thought to reflect the activity of central trigeminal nociceptive pathways causing facial pain and headaches in humans.In a rat model of meningeal nociception, single neuronal activity in the STN was recorded. All units had receptive fields located in the exposed parietal dura mater. Heat and cold stimuli were repetitively applied to the dura in a fixed pattern of ramps and steps. The nonpeptide CGRP receptor antagonist BIBN4096BS was topically applied onto the exposed dura or infused intravenously.BIBN4096BS (300 μg/kg, i.v.) reduced spontaneous activity by ∼30%, the additional dose of 900 μg/kg intravenously by ∼50% of the initial activity, whereas saline had no effect. The activity evoked by heat ramps was also reduced after BIBN4096BS (900 μg/kg, i.v.) by ∼50%. Topical administration of BIBN4096BS (1 mm) did not significantly change the spontaneous neuronal activity within 15 min.We conclude that the endogenous release of CGRP significantly contributes to the maintenance of spontaneous activity in STN neurons. Blockade of CGRP receptors, possibly at central and peripheral sites, may therefore be an effective way to decrease nociceptive transmission. This may offer a new therapeutic strategy for the treatment of facial pain and primary headaches.

TRPV1 gene disruption results in a loss of capsaicin and proton responsiveness, but has minimal effects on heat-induced nocifensive behavior, suggesting that sensory transduction of heat is independent of TRPV1. TRPV3, another heat-activated ion channel but insensitive to capsaicin, was shown to be expressed in keratinocytes as well as in sensory neurons projecting to the skin. Recently, 2-aminoethoxydiphenyl borate was introduced as a TRPV3 agonist, but its selectivity was questioned by showing that it activated recombinant TRPV1 and TRPV2 as well. We used the isolated mouse skin-saphenous nerve preparation and whole-cell patch-clamping of cultured dorsal root ganglia neurons from TRPV1-/- and wildtype mice. We found no phenotypic differences between the heat responses of polymodal C-fibers, whereas cultured dorsal root ganglia neurons of TRPV1-/- hardly showed any heat-activated currents. Only C-fibers of wildtype but not TRPV1-/- mice were clearly sensitized to heat by 2-aminoethoxydiphenyl borate 10 and 100 microM; heat-activated current in wildtype neurons was only facilitated at 100 microM. Noxious heat-induced calcitonin gene-related peptide release showed clear deficits (50%) in TRPV1 deficient skin, but the stimulated calcitonin gene-related peptide release from the isolated skull dura was unaffected. In both models, 2-aminoethoxydiphenyl borate was able to potentiate the heat response (46 degrees C, 5 min) in a concentration-dependent manner, again, only in wildtype but not TRPV1-/- mice, suggesting that TRPV2/3 are not involved in this sensitization to heat. The results further suggest that TRPV1 is not responsible for the normal heat response of native nociceptors but plays the essential role in thermal sensitization and a prominent one in controlling dermal calcitonin gene-related peptide release, i.e. neurogenic inflammation.

Morphine exerts its analgesic effect via the endogenous pain control system consisting of the periaqueductal grey (PAG) and the rostral ventromedial medulla (RVM). Acetylsalicylic acid (ASA) may also act via this system, but so far this has only been demonstrated for the inhibitory effect on the tail-flick reflex with extremely high doses (200–300 mg/kg). Both drugs show synergistic effects on PAG neurons in vitro. It is unclear whether this mechanism accounts for the well-known analgesic synergism of these drugs in vivo. Thus, the effects of ASA (30 mg/kg) and morphine on off- and on-cells in the RVM and the jaw-opening reflex (JOR) were investigated in anesthetized rats. Under morphine, off-cell activity increased (+34%), on-cell activity decreased (−98%) and the reflex was suppressed (−53%). ASA increased off-cell activity (+20%) and decreased the activity of on-cells (−52%). After preceding ASA administration, the effects of morphine on off- and on-cells and on the reflex did not alter statistically. The experiments document the modulatory effect of a clinically relevant dose of ASA on RVM cells. This effect resembles that of morphine. The results do not support the hypothesis of a mediation of the analgesic synergism of morphine and ASA by the PAG–RVM-network in vivo.

Nitric oxide is thought to control transmitter release and neuronal activity in the spinal dorsal horn and the spinal trigeminal nucleus, where nociceptive information from extra- and intracranial tissues is processed. Extracellular impulse activity was recorded from neurons in the rat spinal trigeminal nucleus with afferent input from the cranial dura mater. In contrast to the inactive isomer D-NAME, infusion of the nitric oxide synthase inhibitor L-NAME (20 mg/kg) significantly reduced neuronal activity and increased systemic blood pressure. It is concluded that nitric oxide production contributes to the ongoing activity of sensitized neurons in the spinal trigeminal nucleus. The results suggest that nitric oxide may be involved in the generation and maintenance of primary headaches such as migraine.

Nitric oxide (NO) and calcitonin-gene-related peptide (CGRP) are implicated in the pathophysiology of vascular headaches. We studied the interaction of these two vasodilatory mediators in an animal model and suggest that NO may increase meningeal blood flow not only by its direct vasodilatory action but also by stimulating CGRP release. First, CGRP release from the rat cranial dura mater was measured in vitro using an enzyme immunoassay. Hemisected skulls with adhering dura mater were filled with synthetic interstitial fluid and stimulated with the NO donor diethylamine-NONOate (10–5–10–3M) or with NO gas (1,000 ppm), which caused concentration-dependent increases in CGRP release up to 166.8%. Second, meningeal blood flow was recorded in vivo in the exposed dura mater using laser Doppler flowmetry. Topical application of the NO donors NONOate, S-nitroso-N-acetylpenicillamine and N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethenamine (10–5–10–3M) caused concentration-dependent increases in blood flow. These increases were significantly reduced by local preliminary application of the CGRP receptor antagonist CGRP8–37 (10–4M). We conclude that NO stimulates the release of CGRP from dural afferents. The blood-flow-increasing effect of NO seems to be partly mediated by CGRP. The interaction of NO and CGRP may be relevant for the development of vascular headaches.

Headaches, which are usually referred to characteristic sites of the skull, are believed to involve meningeal nociceptors located in the dura mater encephali. Animal experiments show that these meningeal nociceptors are polymodal and usually highly sensitive to mechanical stimulation. These properties are also characteristic for the second order neurons in the spinal trigeminal nucleus, most of which receive convergent input from facial receptive sites. Sensitization of primary and secondary neurons by chemical irritants to mechanical stimuli may be an important mechanism in the generation of headaches. The convergent input from extracranial structures, which seems to be differentially organized in rodents and man, may explain the typical features of referred headache. Targets for analgesics used in the therapy of headaches (non-steroidal antiinflammatory drugs, 5-HT(1) receptor agonists) are probably meningeal nociceptors and different sites of the central trigeminal nociceptive and antinociceptive pathways.

The effect of somatostatin (SOM) on neurogenic increases in meningeal blood flow was examined in barbiturate anaesthetized rats. The parietal skull was trepanized and the blood flow in the medial meningeal artery was monitored using a laser Doppler flowmeter with needle probes. Electrical stimulation (pulses of 8–10 V at 5–10 Hz for 30 s) close to the superior sagittal sinus evoked reproducible increases in blood flow. These increases were reduced by topical applications of SOM at concentrations of 10 −5 –10 −3 M in a dose-dependent manner. The effect was most pronounced within 10 min after application of SOM followed by a recovery of the flow responses. We conclude that stimulus-evoked increases in dural arterial flow, which are most likely caused by afferent activation and can be regarded as an element of neurogenic inflammation, are reduced by anti-inflammatory peptides such as SOM.

Headache is often accompanied by referred pain in the face. This phenomenon is probably due to a convergence of afferent inputs from the meninges and the face onto central trigeminal neurons within the medullary dorsal horn (MDH). The possible existence and extent of this convergence was examined in rat and man. MDH neurons activated by stimulation of the parietal meninges were tested for convergent tactile and noxious mechanical input from all three facial branches of the trigeminal nerve. All 21 units with meningeal input could also be activated by facial stimuli. Brush stimuli applied to the supraorbital nerve area activated 86%, to the infraorbital nerve area 29%, and to the mental nerve area none of the units. Pinch stimuli applied to the supraorbital nerve area activated 95%, to the infraorbital nerve area 86%, and to the mental nerve area 52% of the units. The results suggest convergence of meningeal and facial inputs concentrated on the supraorbital nerve in rat. In man convergence was examined by probing neuronal excitability of MDH applying the blink reflex (BR) during Valsalva maneuver which probably increases intracranial pressure. The BR evoked by supraorbital nerve stimulation remained unchanged, while the BR evoked by mental nerve stimulation was significantly facilitated. This facilitation may be due to convergence of meningeal and facial inputs onto trigeminal neurons in man.

Although the pathogenesis of migraine headache is still a matter of speculation rather than of understanding, clinical and experimental evidence has long focused attention on pathological processes involving the perivascular innervation of mainly intracranial blood vessels (i.e. pial and durai vessels) as the likely source of the pain in this disease [1– 3]. The discovery of neuropeptides in perivascular nerve fibres during recent years has had a major influence here, and greatest interest has been directed to the possible actions of two vasoactive peptides found in trigeminal sensory neurons, calcitonin gene-related peptide (CGRP) and substance P (SP). Though meningeal as well as extracranial vessels are innervated by trigeminal nerve fibres containing these neuropeptides [4, 5] and extracranial vascular mechanisms may partly be involved [6], experimental and clinical work, initiated by the pioneering studies of Ray and Wolff [7] and Penfield and McNaughton in 1940 [8], implicated a dominant role of intracranial blood vessels, i.e. arteries of the dura mater encephali, the sagittal sinus, and to some extent basal intracerebral arteries, in the generation of headaches [3, 9]. Therefore the dura mater has become the preferential target for studying mechanisms of meningeal nociception and vascular headache [9– 12]. The neurogenic inflammation of the meninges in particular (an experimental inflammation characterized by oedema, hyperaemia, activation of platelets, endothelium and mast cells following antidromic stimulation of trigeminal afferents [13,14]), has attracted the attention of several investigators.

The blood flow in and around the medial meningeal artery (dural arterial flow) was recorded in the exposed parietal dura mater encephali of the anesthetized rat 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. The effects of the 5-HT1 receptor agonists sumatriptan and CP 93,129 on the basal flow and the electrically evoked increases in flow were examined. Topical administration of undiluted sumatriptan (12 mg/ml) lowered the basal and the evoked flow by 20% on average. Systemic (i.v.) administration of sumatriptan (0.24, 0.72 and 3.6 mumol/kg) caused a short-lasting reduction of the evoked flow increases only at the higher doses while the basal flow was not significantly altered. Systemic administration of CP 93, 129 (0.46 and 4.6 mumol/kg) caused no significant changes of the basal and the evoked flow. At a dose of 23 mumol/kg CP 93,129 lowered the basal flow by 20% and the evoked flow by 30% for 20 min. The systemic arterial pressure was not significantly altered by sumatriptan and CP 93,129 within the whole range of doses. It is suggested that sumatriptan and CP 93,129 at high doses exert inhibitory effects on those fine afferent nerve fibers which release the calcitonin gene-related peptide, since this neuropeptide mediates the evoked increases in dural arterial flow.

Background Nitric oxide (NO) is thought to play an important role in the pathophysiology of migraine. Infusion of the nitrovasodilator glyceroltrinitrate (nitroglycerin, GTN), which mobilizes NO in the organism, is an approved migraine model in humans. Calcitonin gene-related peptide (CGRP) is regarded as another key mediator in migraine. Increased plasma levels of CGRP have been found during spontaneous as well as nitrovasodilator-induced migraine attacks. The nociceptive processes and interactions underlying the NO and CGRP mediated headache are poorly known but can be examined in animal experiments. In the present study we examined changes in immunofluorescence of CGRP receptor components (CLR and RAMP1) and soluble guanylyl cyclase (sGC), the intracellular receptor for NO, in rat trigeminal ganglia after pretreatment with GTN. Methods Isoflurane anaesthetised rats were intravenously infused with GTN (1 mg/kg) or saline for four hours and two hours later the trigeminal ganglia were processed for immunohistochemistry. Different primary antibodies recognizing CLR, RAMP1, CGRP and sGC coupled to fluorescent secondary antibodies were used to examine immunoreactive cells in serial sections of trigeminal ganglia with epifluorescence and confocal laser scanning microscopy. Several staining protocols were examined to yield optimized immunolabeling. Results In vehicle-treated animals, 42% of the trigeminal ganglion neurons were immunopositive for RAMP1 and 41% for CLR. After GTN pretreatment CLR-immunopositivity was unchanged, while there was an increase in RAMP1-immunopositive neurons to 46%. RAMP1 and CLR immunoreactivity was also detected in satellite cells. Neurons immunoreactive for sGC were on average smaller than sGC-immunonegative neurons. The percentage of sGC-immunopositive neurons (51% after vehicle) was decreased after GTN infusion (48%). Conclusions Prolonged infusion of GTN caused increased fractions of RAMP1- and decreased fractions of sGC-immunopositive neurons in the trigeminal ganglion. The observed alterations are likely immunophenotypic correlates of the pathophysiological processes underlying nitrovasodilator-induced migraine attacks and indicate that signalling via CGRP receptors but not sGC-mediated mechanisms may be enhanced through endogenous NO production.

Hydrogen sulfide (H 2 S) emerged recently as an important signaling molecule involved in regulation of blood pressure and neuronal activity. Additionally, H 2 S and its donors possess huge pharmacological potential to prevent ischemia–reperfusion injuries and, as it has been speculated, to induce suspended animation-like state in small animals. However, the real (bio) chemical events behind all these observed phenomena remained elusive. In this talk, physiological effects of H 2 S in context of its interaction with another physiological gassotransmitter, nitric oxide (NO), and its metabolites (peroxynitrite, S-nitrosothiols, nitrite) will be addressed, providing the evidence for the existence of a strong cross-talk of these molecules. Applying state-of-the art analytical techniques, as the products of these reactions, we identify thionitric (HSNO 2 ) and thionitrous acid (HSNO) as well as nitroxyl (HNO), and show on cellular level that they too serve as important signaling molecules. Finally on the animal level we prove our chemistry and show that H 2 S-induced vasodilatory effects are directly NO dependent, identifying therefore completely new signaling cascade for systemic neurovascular control.

TRPA1 receptor channels are activated by environmental irritants and by endogenous mediators released during inflammatory conditions ( McMahon & Wood 2006 ). Activation of TRPA1 receptors causes CGRP release from trigeminal ganglion neurons and increases meningeal blood flow upon nasal stimulation ( Kunkler et al. 2011 ), providing evidence that TRPA1 receptors may be involved in the generation of headaches.

The role of the neuropeptide substance P (SP) in the control of dural arterial blood flow was examined in barbiturate-anaesthetised rats. The parietal skull was trephinised and the blood flow in branches of the medial meningeal artery was monitored with a laser Doppler flowmeter. Electrical stimulation of the dura mater encephali at a parasagittal site with pulses of 0.5 ms (10-20 V, 5-10 Hz, 30 s) caused a transient increase in dural blood flow which was reproducible in size with repetitive stimulation. Neither the basal flow nor the stimulus-evoked flow was significantly changed by topical administration of SP, the SP analog septide, or the NK1 antagonist RP 67580. It is concluded that SP released from dural nerve fibres upon local stimulation does not play an important role in the regulation of dural arterial flow.

Publisher Summary It is generally held that non-corpuscular fine sensory endings (free nerve endings) arise from slowly conducting (group III and group IV) fibers, whereas corpuscular nerve endings are the sensory end formations of fast conducting fibers. The sensory axons of non-corpuscular free endings are not accompanied by other cellular structures than the terminal Schwann cells. The terminal Schwann cells of group III fibers do not produce a myelin sheath. In articular tissues, group III fibers lose their myelin sheath already within the most distal portion of the peripheral nerves. The endings of mechanosensitive afferents are connected to the structures that are able to transmit the mechanical stimuli, for example, stretch. In the knee joint, low-threshold mechanosensory endings are found in loose connective tissue composed of fibroblasts, thin collagenous fibers, and fat cells, whereas the endings of high-threshold (nociceptive) afferents seem to be embedded in dense collagenous tissue such as the fibrous capsule or ligaments. The rigidity of the surrounding tissue and its resistance to stretch may determine the mechanical sensitivity of the sensory endings, and hence the mechanical threshold of the afferent units.

A combination of methods is described to identify and reconstruct corpuscular and non-corpuscular sensory endings of group II and group III nerve fibers following functional examination by electrophysiology. Afferent units activated by electrical stimulation of the medial articular nerve of the cat's knee were analyzed by single fiber recordings and characterized by their responsiveness to mechanical stimuli. The receptive fields of the units were closely demarcated by fine needles when the responses elicited by insertion of the needles were being recorded. After fixation, the tissue around the demarcated field was dissected and histologically processed. Series of semithin sections were cut from the embedded tissue blocks containing the receptive fields. Corpuscular endings of group II fibers and peripheral myelinated group III nerve fibers, presumably corresponding to the characterized units, were identified by light microscopy of semithin sections and localized within the demarcated area. Non-corpuscular endings were identified by electron microscopy of ultrathin sections cut in alternation with, or after re-embedding of, semithin sections. Morphometric analysis of ultrathin section series allowed the measurement of parameters such as the mean axon diameter and the organelle content of the sensory endings. The methods described are appropriate for collecting data that correlate the structural and functional characteristics of sensory endings in deep tissues.

Arthritic hyperalgesia and pain result from an increased activity of nociceptive afferents that may be induced and maintained by inflammatory mediators like bradykinin (BK). The B 1 receptor antagonist des-Arg 9 -Leu 8 -BK and the B 2 receptor antagonists Thi 5,8 -D-Phe 7 -BK and Hoe 140 were used to study the involvement of BK receptors in the generation of ongoing afferent activity in the cat's knee joint that was inflamed by kaolin and carrageenin. After i.a. bolus administration of BK receptor antagonists (26–260 μg) close to the joint, the ongoing activity did not significantly vary in any group III or group IV unit. We conclude that activation of BK receptors by endogenous BK is probably not the mechanism that is responsible for the increased ongoing activity of articular afferents in the inflamed joint.

The effects of beta-phorbol 12,13-dibutyrate (PDBu) on the discharge properties of slowly conducting knee joint afferents (group III and group IV fibers) were studied to determine the role of protein kinase C in nociception. Extracellular single unit recordings were made from small filaments dissected from the medial articular nerve in cats anesthetized with alpha-chloralose. PDBu was applied intra-arterially close to the joint in concentrations of 10(-6) up to 10(-4) M. The afferents were classified as low-threshold and high-threshold units with regard to their sensitivity to passive noxious and innocuous movements of the knee joint. Following PDBu application, an excitation occurred in 28% of the group III and in 40% of the group IV fibers. An enhancement of responses to passive movements of the joint (sensitization) occurred in 37% of group III and 19% of group IV afferents. In summary, 37.5% of the low-threshold and 50% of the high-threshold fibers proved to be sensitive to PDBu. Most of the PDBu-positive units responded also to bradykinin, whereas only a few PDBu-positive units were sensitive to prostaglandin I2 and E2. We conclude from these results that, in a distinct population of slowly conducting joint afferents, protein kinase C is likely to be involved in the process of transduction. Thus, pain and hyperalgesia may be mediated at least partly by intracellular mechanisms that are linked to protein kinase C.

In den letzten Jahren haben zahlreiche neuere Erkenntnisse der klinischen und grundlagenwissenschaftlichen Forschung unser Verstandnis von Nozizeption und Schmerz betrachtlich verandert und erweitert. Nicht nur die Nomenklatur ist praziser, auch die Hypothesen uber Aufnahme, Weiterleitung und Verarbeitung nozizeptiver Signale sind konkreter und die Zusammenhange zwischen Nozizeption und Schmerz sind durchsichtiger geworden. Auf diesem Hintergrund beginnen sich auch neue Behandlungsmethoden abzuzeichnen, deren Einfuhrung in das therapeutische Routinearsenal erhebliche Vorteile, insbesondere bei der Behandlung chronischer, z.B. rheumatischer Schmerzen, erwarten last.

The noncorpuscular endings ("free nerve endings") of thinly myelinated group III and nonmyelinated group IV afferent nerve fibers have been examined in the knee joint capsule of sympathectomized cats by transmission electron microscopy and three-dimensional reconstruction of series of semi- and ultrathin sections. The sensory ending is the most distal part of a group III or IV nerve fiber that consists only of the sensory axon and associated Schwann cells but lacks a myelin sheath and is not surrounded by perineurium. The sensory axon divides into several branches and forms a terminal tree. The branches run either as single fibers or within small Remak bundles in parallel to sensory axons of other endings; they spread along vessel walls and also extend into dense connective tissue. Each sensory axon consists of a series of spindle-shaped thick segments ("beads") connected by waist-like thin segments. Thus all axons of sensory endings have a string-of-beads appearance, which resembles that of efferent sympathetic nerve fibers. The beads of the sensory axon and the end bulb at its tip show the same ultrastructural features which are characteristic of receptive sites: an accumulation of mitochondria and glycogen particles and various vesicles in the axoplasm and "bare" areas of axolemma that are not covered by Schwann cell processes. Group III and group IV sensory endings differ in the length of their branches (up to 200 microM in group III vs. more than 300 microM in group IV), number of beads per 100 microM axon length (about seven vs. nine or ten), mean diameter of axons (0.9-1.5 microM vs. 0.3-0.6 microM), and the presence of a neurofilament core consisting of bundles of parallel microfilaments only in group III. In conclusion, we propose that the sensory part of noncorpuscular "free nerve endings" is formed by the entire terminal tree of group III or group IV nerve fibers and that the beads in the course of the sensory axon represent multiple receptive sites.

In recent years the normal and artificially inflamed knee joint of the cat has become an important model for neurophysiological studies on arthritic pain. The noxious stimuli causing pain are presumably perceived via “free nerve endings”, i.e., non-corpuscular endings of thinly myelinated fine afferent nerve fibers (group III or A ∂-fibers) and of non-myelinated fine afferent group IV or C-fibers (Schaible and Schmidt, 1983a, b, 1985; Grigg et al., 1986). But in comparison with neurophysiological knowledge, little is known about the morphological characteristics of articular “free nerve endings”. This is why we have studied the ultrastructure of “free nerve endings” and fine afferent nerve fibers in the normal knee joint capsule of the cat by serial sectioning and with three-dimensional reconstruction of nerve fibers for up to 300 µm length.

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.

The peripheral and central terminals of primary afferents of the cat's knee joint were studied with light- and electron microscopy (including HRP-transprot methods). The majority of these afferent fibres are unmyelinated or thinly myelinated. In the periphery, they terminate in non-corpuscular endings. A dense network of branching nerve fibres surround the blood vessels to terminate at the vessel walls or in the adjacent dense collagenous tissue. Besides the terminals the fibres show preterminal bulges which are assumed to represent additional transduction sites. This implies that the whole distal fibre portion has a receptive function. The spinal projection is located in lamina I and V–VII of the dorsal horn. This location parallels that of muscular and visceral fibres, pointing to the possibility of a common projection pattern for deep somatic afferent fibres in the spinal cord of the cat.

Activation of TRPA1 receptors has recently been shown to cause release of calcitonin gene-related peptide (CGRP) from trigeminal ganglion neurons and to increase meningeal blood flow in animals ( Kunkler et al. 2011 ), events regarded to be associated with headaches. Meningeal blood flow is also increased by donors of nitric oxide (NO), partly mediated by the release of CGRP from meningeal afferents ( Strecker et al. 2002 ).