Novel insight into atogepant mechanisms of action in migraine prevention.

Recently, we showed that while atogepant - a small-molecule calcitonin gene-related peptide (CGRP) receptor antagonist - does not fully prevent activation of nociceptors, it significantly reduces a cortical spreading depression (CSD)-induced early response probability in C-fibers and late response probability in A™-fibers. The current study investigates atogepant effect on CSD-induced activation and sensitization of high-threshold (HT) and wide dynamic range (WDR) dura-sensitive neurons. In anesthetized male rats, single-unit recordings were used to assess effects of atogepant (5mg/kg) vs vehicle on CSD-induced activation and sensitization of HT and WDR dura-sensitive neurons. Single cell analysis of atogepant pretreatment effects on CSD-induced activation and sensitization of central trigeminovascular neurons in the spinal trigeminal nucleus (STN) revealed ability of this small molecule CGRP receptor antagonist to prevent activation and sensitization of nearly all HT neurons (8/10 vs. 1/10 activated neurons in the control vs. treated groups, p=0.005). In contrast, atogepant pretreatment effects on CSD-induced activation and sensitization of WDR neurons revealed an overall inability to prevent their activation (7/10 vs. 5/10 activated neurons in the control vs. treated groups, p=0.64). Unexpectedly however, in spite of atogepant inability to prevent activation of WDR neurons, it prevented their sensitization (as reflected their responses to mechanical stimulation of the facial receptive field before and after the CSD). Atogepant ability to prevent activation and sensitization of HT neurons is attributed to its preferential inhibitory effects on thinly unmyelinated A™ fibers. Atogepant inability to prevent activation of WDR neurons is attributed to its lesser inhibitory effects on the unmyelinated C fibers. Molecular and physiological processes that govern neuronal activation vs. sensitization can explain how reduction in CGRP-mediated slow but not glutamate-mediated fast synaptic transmission between central branches of meningeal nociceptors and nociceptive neurons in the STN can prevent their sensitization but not activation.

Atogepant - an orally-administered CGRP antagonist - attenuates activation of meningeal nociceptors by CSD.

BACKGROUND: This study investigated the mechanism of action of atogepant, a small-molecule CGRP receptor antagonist recently approved for the preventive treatment of episodic migraine, by assessing its effect on activation of mechanosensitive C- and Aδ-meningeal nociceptors following cortical spreading depression. METHODS: Single-unit recordings of trigeminal ganglion neurons (32 Aδ and 20 C-fibers) innervating the dura was used to document effects of orally administered atogepant (5 mg/kg) or vehicle on cortical spreading depression-induced activation in anesthetized male rats. RESULTS: Bayesian analysis of time effects found that atogepant did not completely prevent the activation of nociceptors at the tested dose, but it significantly reduced response amplitude and probability of response in both the C- and the Aδ-fibers at different time intervals following cortical spreading depression induction. For C-fibers, the reduction in responses was significant in the early phase (first hour), but not delayed phase of activation, whereas in Aδ-fibers, significant reduction in activation was apparent in the delayed phase (second and third hours) but not early phase of activation. CONCLUSIONS: These findings identify differences between the actions of atogepant, a small molecule CGRP antagonist (partially inhibiting both Aδ and C-fibers) and those found previously for fremanezumab, a CGRP-targeted antibody (inhibiting Aδ fibers only) and onabotulinumtoxinA (inhibiting C-fibers only)- suggesting that these agents differ in their mechanisms for the preventive treatment of migraine.

Activation of Peripheral and Central Trigeminovascular Neurons by Seizure: Implications for Ictal and Postictal Headache.

An epileptic seizure can trigger a headache during (ictal) or after (postictal) the termination of the event. Little is known about the pathophysiology of seizure-induced headaches. In the current study, we determined whether a seizure can activate nociceptive pathways that carry pain signals from the meninges to the spinal cord, and if so, to what extent and through which classes of peripheral and central neurons. To achieve these goals, we used single-unit recording techniques and an established animal model of seizure (picrotoxin) to determine the effects of epileptic seizure on the activity of trigeminovascular Aδ-, C-, wide-dynamic range, and high-threshold neurons in male and female rats. Occurrence of seizure activated 54%, 50%, 68%, and 39% of the Aδ-, C-, wide-dynamic range, and high-threshold neurons, respectively. Regardless of their class, activated neurons exhibited a twofold to fourfold increase in their firing, which started immediately (1 min) or up to 90 min after seizure initiation, and lasted as short as 10 min or as long as 120 min. Administration of lidocaine to the dura prevented activation of all neuronal classes but not the initiation or maintenance of the seizure. These findings suggest that all neuronal classes may be involved in the initiation and maintenance of seizure-induced headache, and that their activation patterns can provide a neural substrate for explaining the timing and duration of ictal and possibly postictal headaches. By using seizure, which is evident in humans, this study bypasses controversies associated with cortical spreading depression, which is less readily observed in humans.SIGNIFICANCE STATEMENT This preclinical study provides a neural substrate for ictal and postictal headache. By studying seizure effects on the activity of peripheral (C and Aδ) and central (wide dynamic range and high-threshold) trigeminovascular neurons in intact and anesthetized dura, the findings help resolve two outstanding questions about the pathophysiology of headaches of intracranial origin. The first is that abnormal brain activity (i.e., seizure) that is evident in human (unlike cortical spreading depression) gives rise to specific and selective activation of the different components of the trigeminovascular system, and the second is that the activation of all components of the trigeminovascular pathway (i.e., peripheral and central neurons) depends on activation of the meningeal nociceptors from their receptors in the dura.

Combined onabotulinumtoxinA/atogepant treatment blocks activation/sensitization of high-threshold and wide-dynamic range neurons.

BACKGROUND: OnabotulinumtoxinA and agents that block calcitonin gene‒receptor peptide action have both been found to have anti-migraine effects, but they inhibit different populations of meningeal nociceptors. We therefore tested the effects of combined treatment with onabotulinumtoxinA and the calcitonin gene‒receptor peptide antagonist atogepant on activation/sensitization of trigeminovascular neurons by cortical spreading depression. MATERIAL AND METHODS: Single-unit recordings were obtained of high-threshold and wide-dynamic-range neurons in the spinal trigeminal nucleus, and cortical spreading depression was then induced in anesthetized rats that had received scalp injections of onabotulinumtoxinA 7 days earlier and intravenous atogepant infusion 1 h earlier. The control group received scalp saline injections and intravenous vehicle infusion. RESULTS: OnabotulinumtoxinA/atogepant pretreatment prevented cortical spreading depression-induced activation and sensitization in both populations (control: Activation in 80% of high-threshold and 70% of wide-dynamic-range neurons, sensitization in 80% of high-threshold and 60% of wide-dynamic-range neurons; treatment: activation in 10% of high-threshold and 0% of wide-dynamic-range neurons, sensitization in 0% of high-threshold and 5% of wide-dynamic-range neurons). DISCUSSION: We propose that the robust inhibition of high-threshold and wide-dynamic-range neurons by the combination treatment was achieved through dual blockade of the Aδ and C classes of meningeal nociceptors. Combination therapy that inhibits meningeal C-fibers and prevents calcitonin gene‒receptor peptide from activating its receptors on Aδ-meningeal nociceptors may be more effective than a monotherapy in reducing migraine days per month in patients with chronic migraine.

CSD-Induced Arterial Dilatation and Plasma Protein Extravasation Are Unaffected by Fremanezumab: Implications for CGRP's Role in Migraine with Aura.

Cortical spreading depression (CSD) is a wave of neuronal depolarization thought to underlie migraine aura. Calcitonin gene-related peptide (CGRP) is a potent vasodilator involved in migraine pathophysiology. Evidence for functional connectivity between CSD and CGRP has triggered scientific interest in the possibility that CGRP antagonism may disrupt vascular responses to CSD and the ensuing plasma protein extravasation (PPE). Using imaging tools that allow us to generate continuous, live, high-resolution views of spatial and temporal changes that affect arteries and veins in the dura and pia, we determined the extent to which CGRP contributes to the induction of arterial dilatation or PPE by CSD in female rats, and how these events are affected by the anti-CGRP monoclonal antibody (anti-CGRP-mAb) fremanezumab. We found that the CSD-induced brief dilatation and prolonged constriction of pial arteries, prolonged dilatation of dural arteries and PPE are all unaffected by fremanezumab, whereas the brief constriction and prolonged dilatation of pial veins are affected. In comparison, although CGRP infusion gave rise to the expected dilatation of dural arteries, which was effectively blocked by fremanezumab, it did not induce dilatation in pial arteries, pial veins, or dural veins. It also failed to induce PPE. Regardless of whether the nociceptors become active before or after the induction of arterial dilatation or PPE by CSD, the inability of fremanezumab to prevent them suggests that these events are not mediated by CGRP, a conclusion with important implications for our understanding of the mechanism of action of anti-CGRP-mAbs in migraine prevention.SIGNIFICANCE STATEMENT The current study identifies fundamental differences between two commonly used models of migraine, CSD induction and systemic CGRP infusion. It raises the possibility that conclusions drawn from one model may not be true or relevant to the other. It sharpens the need to accept the view that there is more than one truth to migraine pathophysiology and that it is unlikely that one theory will explain all types of migraine headache or the mechanisms of action of drugs that prevent it. Regarding the latter, it is concluded that not all vascular responses in the meninges are born alike and, consequently, that drugs that prevent vascular dilatation through different molecular pathways may have different therapeutic outcomes in different types of migraine.

Non-Trigeminal Nociceptive Innervation of the Posterior Dura: Implications to Occipital Headache.

Current understanding of the origin of occipital headache falls short of distinguishing between cause and effect. Most preclinical studies involving trigeminovascular neurons sample neurons that are responsive to stimulation of dural areas in the anterior 2/3 of the cranium and the periorbital skin. Hypothesizing that occipital headache may involve activation of meningeal nociceptors that innervate the posterior ⅓ of the dura, we sought to map the origin and course of meningeal nociceptors that innervate the posterior dura overlying the cerebellum. Using AAV-GFP tracing and single-unit recording techniques in male rats, we found that neurons in C2-C3 DRGs innervate the dura of the posterior fossa; that nearly half originate in DRG neurons containing CGRP and TRPV1; that nerve bundles traverse suboccipital muscles before entering the cranium through bony canals and large foramens; that central neurons receiving nociceptive information from the posterior dura are located in C2-C4 spinal cord and that their cutaneous and muscle receptive fields are found around the ears, occipital skin and neck muscles; and that administration of inflammatory mediators to their dural receptive field, sensitize their responses to stimulation of the posterior dura, peri-occipital skin and neck muscles. These findings lend rationale for the common practice of attempting to alleviate migraine headaches by targeting the greater and lesser occipital nerves with anesthetics. The findings also raise the possibility that such procedures may be more beneficial for alleviating occipital than non-occipital headaches and that occipital migraines may be associated more closely with cerebellar abnormalities than in non-occipital migraines.SIGNIFICANCE STATEMENT Occipital headaches are common in both migraine and non-migraine headaches. Historically, two distinct scenarios have been proposed for such headaches; the first suggests that the headaches are caused by spasm or tension of scalp, shoulders, and neck muscles inserted in the occipital region, whereas the second suggests that these headaches are initiated by activation of meningeal nociceptors. The current study shows that the posterior dura overlying the cerebellum is innervated by cervicovascular neurons in C2 DRG whose axons reach the posterior dura through multiple intracranial and extracranial pathways, and sensitization of central cervicovascular neurons from the posterior dura can result in hyper-responsiveness to stimulation of neck muscles. The findings suggest that the origin of occipital and frontal migraine may differ.

Fluorescently-labeled fremanezumab is distributed to sensory and autonomic ganglia and the dura but not to the brain of rats with uncompromised blood brain barrier.

BACKGROUND: The presence of calcitonin gene-related peptide and its receptors in multiple brain areas and peripheral tissues previously implicated in migraine initiation and its many associated symptoms raises the possibility that humanized monoclonal anti-calcitonin gene-related peptide antibodies (CGRP-mAbs) can prevent migraine by modulating neuronal behavior inside and outside the brain. Critical to our ability to conduct a fair discussion over the mechanisms of action of CGRP-mAbs in migraine prevention is data generation that determines which of the many possible peripheral and central sites are accessible to these antibodies - a question raised frequently due to their large size. MATERIAL AND METHODS: Rats with uncompromised and compromised blood-brain barrier (BBB) were injected with Alexa Fluor 594-conjugated fremanezumab (Frema594), sacrificed 4 h or 7 d later, and relevant tissues were examined for the presence of Frema594. RESULTS: In rats with uncompromised BBB, Frema594 was similarly observed at 4 h and 7 d in the dura, dural blood vessels, trigeminal ganglion, C2 dorsal root ganglion, the parasympathetic sphenopalatine ganglion and the sympathetic superior cervical ganglion but not in the spinal trigeminal nucleus, thalamus, hypothalamus or cortex. In rats with compromised BBB, Frema594 was detected in the cortex (100 µm surrounding the compromised BBB site) 4 h but not 7 d after injections. DISCUSSION: Our inability to detect fluorescent (CGRP-mAbs) in the brain supports the conclusion that CGRP-mAbs prevent the headache phase of migraine by acting mostly, if not exclusively, outside the brain as the amount of CGRP-mAbs that enters the brain (if any) is too small to be physiologically meaningful.

Spatial organization of activity evoked by focal stimulation within the rat spinal dorsal horn as visualized by voltage-sensitive dye imaging in the slice.

In a prior study using laser scanning photostimulation, we found a pronounced cell type-specific mediolateral asymmetry in the local synaptic connectivity in the superficial laminae of the spinal dorsal horn (Kosugi M, Kato G, Lukashov S, Pendse G, Puskar Z, Kozsurek M, Strassman AM. J Physiol 591: 1935-1949, 2013). To obtain information on dorsal horn organization that might complement findings from microelectrode studies, voltage-sensitive dye imaging was used in the present study to examine patterns of activity evoked by focal electrical stimulation, in the presence and absence of synaptic blocking agents, at different positions in transverse, parasagittal, and horizontal slices of the dorsal horn of 2- to 3-wk -old male rats. A pronounced difference in responsiveness was found between medial and lateral dorsal horn, in that medial sites in the superficial dorsal horn showed much larger synaptic responses to focal stimulation than lateral sites. This difference appeared to be a result of a difference in the intrinsic elements of the dorsal horn, rather than a difference in the inputs from the white matter, because the stimulus intensities were subthreshold for evoking synaptic responses from stimulation at sites in the white matter, although it is also possible that the greater responsiveness is due, at least in part, to activation of Aß primary afferent fibers that pass through the medial dorsal horn. The results raise the possibility of differences between medial and dorsal horn that need to be taken into account in the interpretation of studies of dorsal horn organization.NEW & NOTEWORTHY We used voltage-sensitive dye imaging to obtain information on spatial aspects of dorsal horn organization that are difficult to examine with single-cell approaches because of the limitations of microelectrode sampling. The most noteworthy finding was a previously unreported, extreme difference between medial and lateral dorsal horn in responsiveness to focal stimulation that appears to result, at least in part, from a greater degree of excitability or local connectivity in medial dorsal horn.

Activation of pial and dural macrophages and dendritic cells by cortical spreading depression.

OBJECTIVE: Cortical spreading depression (CSD) has long been implicated in migraine attacks with aura. The process by which CSD, a cortical event that occurs within the blood-brain barrier (BBB), results in nociceptor activation outside the BBB is likely mediated by multiple molecules and cells. The objective of this study was to determine whether CSD activates immune cells inside the BBB (pia), outside the BBB (dura), or in both, and if so, when. METHODS: Investigating cellular events in the meninges shortly after CSD, we used in vivo two-photon imaging to identify changes in macrophages and dendritic cells (DCs) that reside in the pia, arachnoid, and dura and their anatomical relationship to TRPV1 axons. RESULTS: We found that activated meningeal macrophages retract their processes and become circular, and that activated meningeal DCs stop migrating. We found that CSD activates pial macrophages instantaneously, pial, subarachnoid, and dural DCs 6-12 minutes later, and dural macrophages 20 minutes later. Dural macrophages and DCs can appear in close proximity to TRPV1-positive axons. INTERPRETATION: The findings suggest that activation of pial macrophages may be more relevant to cases where aura and migraine begin simultaneously, that activation of dural macrophages may be more relevant to cases where headache begins 20 to 30 minutes after aura, and that activation of dural macrophages may be mediated by activation of migratory DCs in the subarachnoid space and dura. The anatomical relationship between TRPV1-positive meningeal nociceptors, and dural macrophages and DCs supports a role for these immune cells in the modulation of head pain. Ann Neurol 2018;83:508-521.

Exploring the effects of extracranial injections of botulinum toxin type A on prolonged intracranial meningeal nociceptors responses to cortical spreading depression in female rats.

BACKGROUND: Botulinum neurotoxin type A, an FDA-approved prophylactic drug for chronic migraine, is thought to achieve its therapeutic effect through blocking activation of unmyelinated meningeal nociceptors and their downstream communications with myelinated nociceptors and potentially the vasculature and immune cells. Prior investigations to determine botulinum neurotoxin type A effects on meningeal nociceptors were carried out in male rats and tested with stimuli that act outside the blood brain barrier. Here, we sought to explore the effects of extracranial injections of botulinum neurotoxin type A on activation of meningeal nociceptors by cortical spreading depression, an event which occurs inside the blood brain barrier, in female rats. MATERIAL AND METHODS: Using single-unit recording, we studied myelinated C- and unmyelinated Aδ-meningeal nociceptors' responses to cortical spreading depression 7-14 days after injection of botulinum neurotoxin type A or saline along calvarial sutures. RESULTS: In female rats, responses to cortical spreading depression were typically more prolonged and, in some cases, began at relatively longer latencies post-cortical spreading depression, than had been observed in previous studies in male rats. Extracranial administration of botulinum neurotoxin type A reduced significantly the prolonged firing of the meningeal nociceptors, in the combined sample of Aδ- and C-fiber, but not their response probability. DISCUSSION: The findings suggest that the mechanism of action by which botulinum neurotoxin type A prevents migraine differ from the one by which calcitonin gene-related peptide monoclonal antibodies prevent migraine and that even when the origin of migraine is central (i.e. in the cortex), a peripherally acting drug can intercept/prevent the headache.

Cortical Spreading Depression Closes Paravascular Space and Impairs Glymphatic Flow: Implications for Migraine Headache.

Functioning of the glymphatic system, a network of paravascular tunnels through which cortical interstitial solutes are cleared from the brain, has recently been linked to sleep and traumatic brain injury, both of which can affect the progression of migraine. This led us to investigate the connection between migraine and the glymphatic system. Taking advantage of a novel in vivo method we developed using two-photon microscopy to visualize the paravascular space (PVS) in naive uninjected mice, we show that a single wave of cortical spreading depression (CSD), an animal model of migraine aura, induces a rapid and nearly complete closure of the PVS around surface as well as penetrating cortical arteries and veins lasting several minutes, and gradually recovering over 30 min. A temporal mismatch between the constriction or dilation of the blood vessel lumen and the closure of the PVS suggests that this closure is not likely to result from changes in vessel diameter. We also show that CSD impairs glymphatic flow, as indicated by the reduced rate at which intraparenchymally injected dye was cleared from the cortex to the PVS. This is the first observation of a PVS closure in connection with an abnormal cortical event that underlies a neurological disorder. More specifically, the findings demonstrate a link between the glymphatic system and migraine, and suggest a novel mechanism for regulation of glymphatic flow.SIGNIFICANCE STATEMENT Impairment of brain solute clearance through the recently described glymphatic system has been linked with traumatic brain injury, prolonged wakefulness, and aging. This paper shows that cortical spreading depression, the neural correlate of migraine aura, closes the paravascular space and impairs glymphatic flow. This closure holds the potential to define a novel mechanism for regulation of glymphatic flow. It also implicates the glymphatic system in the altered cortical and endothelial functioning of the migraine brain.

Fremanezumab-A Humanized Monoclonal Anti-CGRP Antibody-Inhibits Thinly Myelinated (Aδ) But Not Unmyelinated (C) Meningeal Nociceptors.

Calcitonin gene-related peptide (CGRP), the most abundant neuropeptide in primary afferent sensory neurons, is strongly implicated in the pathophysiology of migraine headache, but its role in migraine is still equivocal. As a new approach to migraine treatment, humanized anti-CGRP monoclonal antibodies (CGRP-mAbs) were developed to reduce the availability of CGRP, and were found effective in reducing the frequency of chronic and episodic migraine. We recently tested the effect of fremanezumab (TEV-48125), a CGRP-mAb, on the activity of second-order trigeminovascular dorsal horn neurons that receive peripheral input from the cranial dura, and found a selective inhibition of high-threshold but not wide-dynamic range class of neurons. To investigate the basis for this selective inhibitory effect, and further explore the mechanism of action of CGRP-mAbs, we tested the effect of fremanezumab on the cortical spreading depression-evoked activation of mechanosensitive primary afferent meningeal nociceptors that innervate the cranial dura, using single-unit recording in the trigeminal ganglion of anesthetized male rats. Fremanezumab pretreatment selectively inhibited the responsiveness of Aδ neurons, but not C-fiber neurons, as reflected in a decrease in the percentage of neurons that showed activation by cortical spreading depression. These findings identify Aδ meningeal nociceptors as a likely site of action of fremanezumab in the prevention of headache. The selectivity in its peripheral inhibitory action may partly account for fremanezumab's selective inhibition of high-threshold, as a result of a predominant A-δ input to high-threshold neurons, but not wide dynamic-range dorsal horn neurons, and why it may not be effective in all migraine patients.SIGNIFICANCE STATEMENT Recently, we reported that humanized CGRP monoclonal antibodies (CGRP-mAbs) prevent activation and sensitization of high-threshold (HT) but not wide-dynamic range trigeminovascular neurons by cortical spreading depression (CSD). In the current paper, we report that CGRP-mAbs prevent the activation of Aδ but not C-type meningeal nociceptors by CSD. This is the first identification of an anti-migraine drug that appears to be selective for Aδ-fibers (peripherally) and HT neurons (centrally). As the main CGRP-mAb site of action appears to be situated outside the brain, we conclude that the initiation of the headache phase of migraine depends on activation of meningeal nociceptors, and that for selected patients, activation of the Aδ-HT pain pathway may be sufficient for the generation of headache perception.

Selective Inhibition of Trigeminovascular Neurons by Fremanezumab: A Humanized Monoclonal Anti-CGRP Antibody.

A large body of evidence supports an important role for calcitonin gene-related peptide (CGRP) in migraine pathophysiology. This evidence gave rise to a global effort to develop a new generation of therapeutics that inhibit the interaction of CGRP with its receptor in migraineurs. Recently, a new class of such drugs, humanized anti-CGRP monoclonal antibodies (CGRP-mAbs), were found to be effective in reducing the frequency of migraine. The purpose of this study was to better understand how the CGRP-mAb fremanezumab (TEV-48125) modulates meningeal sensory pathways. To answer this question, we used single-unit recording to determine the effects of fremanezumab (30 mg/kg, IV) and its isotype control Ab on spontaneous and evoked activity in naive and cortical spreading depression (CSD)-sensitized trigeminovascular neurons in the spinal trigeminal nucleus of anesthetized male and female rats. The study demonstrates that, in both sexes, fremanezumab inhibited naive high-threshold (HT) neurons, but not wide-dynamic range trigeminovascular neurons, and that the inhibitory effects on the neurons were limited to their activation from the intracranial dura but not facial skin or cornea. In addition, when given sufficient time, fremanezumab prevents the activation and sensitization of HT neurons by CSD. Mechanistically, these findings suggest that HT neurons play a critical role in the initiation of the perception of headache and the development of cutaneous allodynia and central sensitization. Clinically, the findings may help to explain the therapeutic benefit of CGRP-mAb in reducing headaches of intracranial origin such as migraine with aura and why this therapeutic approach may not be effective for every migraine patient.SIGNIFICANCE STATEMENT Calcitonin gene-related peptide (CGRP) monoclonal antibodies (CGRP-mAbs) are capable of preventing migraine. However, their mechanism of action is unknown. In the current study, we show that, if given enough time, a CGRP-mAb can prevent the activation and sensitization of high-threshold (central) trigeminovascular neurons by cortical spreading depression, but not their activation from the skin or cornea, suggesting a potential explanation for selectivity to migraine headache, but not other pains, and a predominantly peripheral site of action.

Cranial dural permeability of inflammatory nociceptive mediators: Potential implications for animal models of migraine.

Background Application of inflammatory mediators to the cranial dura has been used as a method to activate and sensitize neurons in the meningeal sensory pathway in preclinical behavioral studies of headache mechanisms. However, the relatively high concentrations and volumes used in these studies raise the question of whether the applied agents might pass through the dura to act directly on central neurons, thus bypassing the dural afferent pathway. Methods We used a radiolabeling approach to quantify the meningeal permeability of two of the inflammatory mediators, 5-HT and PGE2, when applied to the cranial dura as part of an inflammatory mixture used in preclinical headache models. Results Both agents could be detected in samples taken four hours after dural application in the cerebrospinal fluid (CSF) and, in measurements made only for PGE2, in the central nervous system (CNS) as well. Based on our measurements, we made estimates of the CSF and CNS levels that would be attained with the higher concentrations and volumes of 5HT and PGE2 that were exogenously applied in previous pre-clinical headache studies. These estimated levels were comparable to or larger than normal endogenous levels, potentially large enough to have physiological effects. Conclusions The finding that the cranial meninges are permeable to the two tested inflammatory mediators PGE2 and 5-HT raises some uncertainty about whether the behavioral changes observed in prior pre-clinical headache studies with these as well as other agents can be attributed entirely to the activation of dural nociceptors, particularly when the agents are applied at concentrations several orders of magnitude above physiological levels.

Extracranial injections of botulinum neurotoxin type A inhibit intracranial meningeal nociceptors' responses to stimulation of TRPV1 and TRPA1 channels: Are we getting closer to solving this puzzle?

BACKGROUND: Administration of onabotulinumtoxinA (BoNT-A) to peripheral tissues outside the calvaria reduces the number of days chronic migraine patients experience headache. Because the headache phase of a migraine attack, especially those preceded by aura, is thought to involve activation of meningeal nociceptors by endogenous stimuli such as changes in intracranial pressure (i.e. mechanical) or chemical irritants that appear in the meninges as a result of a yet-to-be-discovered sequence of molecular/cellular events triggered by the aura, we sought to determine whether extracranial injections of BoNT-A alter the chemosensitivity of meningeal nociceptors to stimulation of their intracranial receptive fields. MATERIAL AND METHODS: Using electrophysiological techniques, we identified 161 C- and 135 Aδ-meningeal nociceptors in rats and determined their mechanical response threshold and responsiveness to chemical stimulation of their dural receptive fields with TRPV1 and TRPA1 agonists seven days after BoNT-A administration to different extracranial sites. Two paradigms were compared: distribution of 5 U BoNT-A to the lambdoid and sagittal sutures alone, and 1.25 U to the sutures and 3.75 U to the temporalis and trapezius muscles. RESULTS: Seven days after it was administered to tissues outside the calvaria, BoNT-A inhibited responses of C-type meningeal nociceptors to stimulation of their intracranial dural receptive fields with the TRPV1 agonist capsaicin and the TRPA1 agonist mustard oil. BoNT-A inhibition of responses to capsaicin was more effective when the entire dose was injected along the suture lines than when it was injected into muscles and sutures. As in our previous study, BoNT-A had no effect on non-noxious mechanosensitivity of C-fibers or on responsiveness of Aδ-fibers to mechanical and chemical stimulation. DISCUSSION: This study demonstrates that extracranial administration of BoNT-A suppresses meningeal nociceptors' responses to stimulation of their intracranial dural receptive fields with capsaicin and mustard oil. The findings suggest that surface expression of TRPV1 and TRPA1 channels in dural nerve endings of meningeal nociceptors is reduced seven days after extracranial administration of BoNT-A. In the context of chronic migraine, reduced sensitivity to molecules that activate meningeal nociceptors through the TRPV1 and TRPA1 channels can be important for BoNT-A's ability to act as a prophylactic.

Vascular extracellular signal-regulated kinase mediates migraine-related sensitization of meningeal nociceptors.

OBJECTIVE: To examine changes in the response properties of meningeal nociceptors that might lead to migraine pain and examine endogenous processes that could play a role in mediating them using a clinically relevant model of migraine triggering, namely infusion of the nitric oxide (NO) donor nitroglycerin (NTG). METHODS: Single-unit recordings made in the trigeminal ganglion of rats were used to test changes in the activity and mechanosensitivity of meningeal nociceptors in response to administration of the migraine trigger NTG or another NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) at doses relevant to the human model of migraine headache. Immunohistochemistry and pharmacological manipulations were used to investigate the possible role of meningeal vascular signaling in mediating the responses of meningeal nociceptors to NO. RESULTS: Infusion of NTG promoted a delayed and robust increase in the mechanosensitivity of meningeal nociceptors, with a time course resembling the development of the delayed migraine headache. A similar sensitization was elicited by dural application of NTG and SNAP. NTG-evoked delayed meningeal nociceptor sensitization was associated with a robust extracellular signal-regulated kinase (ERK) phosphorylation in meningeal arteries. Pharmacological blockade of meningeal ERK phosphorylation inhibited the development of NTG-evoked delayed meningeal nociceptor sensitization. INTERPRETATION: The development of delayed mechanical sensitization evoked by the migraine trigger NTG is potentially of great importance as the first finding of a neurophysiological correlate of migraine headache in meningeal nociceptors. The arterial ERK phosphorylation and its involvement in mediating the NTG-evoked delayed sensitization points to an important, yet unappreciated, role of the meningeal vasculature in the genesis of migraine pain.

Three-dimensional organization of local excitatory and inhibitory inputs to neurons in laminae III-IV of the spinal dorsal horn.

Laser scanning photostimulation was used to map the distribution of the synaptic input zones (sites that give local synaptic inputs) for dorsal horn laminae III-IV neurons, in parasagittal and transverse slices of the rat lumbar spinal cord, and examine how these inputs differed for neurons of different morphologies. All neurons received local excitatory and inhibitory synaptic inputs from within laminae III-IV, while a subset of neurons also received excitatory input from the superficial laminae, especially lamina IIi, as well as the II/III border region. Two anatomical properties were found to be predictive of the dorsoventral position of a neuron's input zone relative to its soma: (1) both excitatory and inhibitory input zones were more dorsal for neurons with longer dorsal dendrites, and (2) excitatory, but not inhibitory, input zones were more dorsal (relative to the soma) for more ventral neurons, with the transition between the dorsal input zones of laminae III-IV neurons and the ventral input zones of lamina II neurons occurring at the II/III border. The observed morphophysiological correlations support the idea that interlaminar connectivity is mediated via translaminar dendritic extensions and that, more generally, local connectivity within the dorsal horn is governed by rules relating the position of a neuron's soma and dendrites to the position of the local presynaptic neurons from which it receives inputs, which are specific to the axis and direction (dorsal vs. ventral), whether the input is excitatory or inhibitory, and the laminar position of the postsynaptic neuron.

Subpopulation-specific patterns of intrinsic connectivity in mouse superficial dorsal horn as revealed by laser scanning photostimulation.

The primary goal of this study was to map the transverse distribution of local excitatory and inhibitory synaptic inputs to mouse lamina I spinal dorsal horn neurons, using laser scanning photostimulation. A sample of lamina II neurons was also studied for comparison. Lamina I neurons received excitatory synaptic input from both laminae I-II and the outer part of III-IV, especially the II/III border region, while the inhibitory input zones were mostly confined within I-II. The excitatory synaptic input zones showed a pronounced medial asymmetry, which was correlated with a matching asymmetry in the dendritic fields of the neurons. Inhibitory input from laminae III-IV was found in a subpopulation of neurons occupying a highly restricted zone, essentially one cell layer thick, immediately below the lamina I/II border, with morphological and physiological properties that were distinct from other laminar populations in the superficial dorsal horn, and that suggest a critical role in interlaminar communication. This subpopulation also received excitatory input from laminae III-IV. Within this subpopulation, inhibitory III-IV input was correlated with the presence of long ventral dendrites. Correlations between the distribution of synaptic input zones and dendritic fields support the concept that interlaminar communication is mediated in part via contacts made onto ventrally extending dendrites of superficial laminae neurons. The results point to the presence of cell type specificity in dorsal horn circuitry, and show how the study of connectivity can itself help identify previously unrecognized neuronal populations.

Separate inhibitory and excitatory components underlying receptive field organization in superficial medullary dorsal horn neurons.

Extracellular recording has shown that dorsal horn neurons can have an inhibitory surround outside their excitatory receptive field, but cannot reveal inhibitory inputs within the excitatory field, or show the underlying excitatory and inhibitory synaptic inputs that determine net output. To study the underlying components of receptive field organization, in vivo patch-clamp recording was used to compare the size and distribution of subthreshold, suprathreshold, and inhibitory fields, in neurons in the mouse superficial medullary dorsal horn that were characterized by their responses to noxious and innocuous mechanical facial stimulation. Subthreshold excitatory fields typically extended some distance beyond the borders of the suprathreshold field, and also commonly exhibited broader stimulus selectivity, in that the majority of nociceptive-specific neurons exhibited subthreshold responses to brush. Separate voltage-clamp recording of excitatory and inhibitory inputs using different holding potentials revealed that inhibition could be evoked from both within and outside the excitatory field. In nociceptive neurons, inhibition tended to be maximal at the excitatory receptive field center, and was usually greater for pinch than brush, although the selectivity for pinch versus brush was not as great as with excitatory responses. Based on current data on dorsal horn organization, we propose that the localized peak of inhibition at the excitatory field center could be mediated by local interneurons, while the more widespread surrounding inhibition may depend on supraspinal circuitry.

Mast cell degranulation distinctly activates trigemino-cervical and lumbosacral pain pathways and elicits widespread tactile pain hypersensitivity.

Mast cells (MCs) are tissue resident immune cells that participate in a variety of allergic and other inflammatory conditions. In most tissues, MCs are found in close proximity to nerve endings of primary afferent neurons that signal pain (i.e. nociceptors). Activation of MCs causes the release of a plethora of mediators that can activate these nociceptors and promote pain. Although MCs are ubiquitous, conditions associated with systemic MC activation give rise primarily to two major types of pain, headache and visceral pain. In this study we therefore examined the extent to which systemic MC degranulation induced by intraperitoneal administration of the MC secretagogue compound 48/80 activates pain pathways that originate in different parts of the body and studied whether this action can lead to development of behavioral pain hypersensitivity. Using c-fos expression as a marker of central nervous system neural activation, we found that intraperitoneal administration of 48/80 leads to the activation of dorsal horn neurons at two specific levels of the spinal cord; one responsible for processing cranial pain, at the medullary/C2 level, and one that processes pelvic visceral pain, at the caudal lumbar/rostral sacral level (L6-S2). Using behavioral sensory testing, we found that this nociceptive activation is associated with development of widespread tactile pain hypersensitivity within and outside the body regions corresponding to the activated spinal levels. Our data provide a neural basis for understanding the primacy of headache and visceral pain in conditions that involve systemic MC degranulation. Our data further suggest that MC activation may lead to widespread tactile pain hypersensitivity.