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Suture-line injections of onabotulinumtoxinA alter a neuronal function along a central nervous system pathway involved in the pathophysiology of migraine that is secondary to appearance of SNAP25 in dural nerve fibers., OnabotulinumtoxinA (BoNT-A) is an Food and Drug Administration-approved, peripherally acting preventive migraine drug capable of inhibiting meningeal nociceptors. Expanding our view of how else this neurotoxin attenuates the activation of the meningeal nociceptors, we reasoned that if the stimulus that triggers the activation of the nociceptor is lessened, the magnitude and/or duration of the nociceptors' activation could diminish as well. In the current study, we further examine this possibility using electrocorticogram recording techniques, immunohistochemistry, and 2-photon microscopy. We report (1) that scalp (head) but not lumbar (back) injections of BoNT-A shorten the period of profound depression of spontaneous cortical activity that follows a pinprick-induced cortical spreading depression (CSD); (2) that neither scalp nor lumbar injections prevent the induction, occurrence, propagation, or spreading velocity of a single wave of CSD; (3) that cleaved SNAP25—one of the most convincing tools to determine the anatomical targeting of BoNT-A treatment—could easily be detected in pericranial muscles at the injection sites and in nerve fibers of the intracranial dura, but not within any cortical area affected by the CSD; (4) that the absence of cleaved SNAP25 within the cortex and pia is unrelated to whether the blood–brain barrier is intact or compromised; and (5) that BoNT-A does not alter vascular responses to CSD. To the best of our knowledge, this is the first report of peripherally applied BoNT-A's ability to alter a neuronal function along a central nervous system pathway involved in the pathophysiology of migraine.

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.

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.

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.

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 (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.

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.

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.

Most centrally acting migraine preventive drugs suppress frequency and velocity of cortical spreading depression (CSD). The purpose of the current study was to determine how the new class of peripherally acting migraine preventive drug (ie, the anti-CGRP-mAbs) affect CSD-an established animal model of migraine aura, which affects about 1/3 of people with migraine-when allowed to cross the blood-brain barrier (BBB). Using standard electrocorticogram recording techniques and rats in which the BBB was intentionally compromised, we found that when the BBB was opened, the anti-CGRP-mAb fremanezumab did not prevent the induction, occurrence, or propagation of a single wave of CSD induced by a pinprick, but that both fremanezumab and its isotype were capable of slowing down the propagation velocity of CSD and shortening the period of profound depression of spontaneous cortical activity that followed the spreading depolarization. Fremanezumab's inability to completely block the occurrence of CSD in animals in which the BBB was compromised suggests that calcitonin gene-related peptide (CGRP) may not be involved in the initiation of CSD, at least not to the extent that it can prevent its occurrence. Similarly, we cannot conclude that CGRP is involved in the propagation velocity or the neuronal silencing period (also called cortical recovery period) that follows the CSD because similar effects were observed when the isotype was used. These finding call for caution with interpretations of studies that claim to show direct central nervous system effects of CGRP-mAbs.

Nonsteroidal anti-inflammatory drugs, commonly known as COX-1/COX-2 inhibitors, can be effective in treating mild to moderate migraine headache. However, neither the mechanism by which these drugs act in migraine is known, nor is the specific contribution of COX-1 vs COX-2. We sought to investigate these unknowns using celecoxib, which selectively inhibits the enzymatic activity of COX-2, by determining its effects on several migraine-associated vascular and inflammatory events. Using in vivo 2-photon microscopy, we determined intraperitoneal celecoxib effects on cortical spreading depression (CSD)-induced blood vessel responses, plasma protein extravasation, and immune cell activation in the dura and pia of mice and rats. Compared to vehicle (control group), celecoxib reduced CSD-induced dilatation of dural arteries and activation of dural and pial macrophages significantly, but not dilatation or constriction of pial arteries and veins, or the occurrence of plasma protein extravasation. Collectively, these findings suggest that a mechanism by which celecoxib-mediated COX-2 inhibition might ease the intensity of migraine headache and potentially terminate an attack is by attenuating dural macrophages' activation and arterial dilatation outside the blood-brain barrier, and pial macrophages' activation inside the blood-brain barrier.

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 Aβ 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.

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 STATEMENTOccipital 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.

Converging lines of evidence suggest that the pathophysiology of pain is mediated to a substantial degree via allostatic neuroadaptations in reward- and stress-related brain circuits. Thus, reward deficiency (RD) represents a within-system neuroadaptation to pain-induced protracted activation of the reward circuits that leads to depletion-like hypodopaminergia, clinically manifested anhedonia, and diminished motivation for natural reinforcers. Anti-reward (AR) conversely pertains to a between-systems neuroadaptation involving over-recruitment of key limbic structures (e.g., the central and basolateral amygdala nuclei, the bed nucleus of the stria terminalis, the lateral tegmental noradrenergic nuclei of the brain stem, the hippocampus and the habenula) responsible for massive outpouring of stressogenic neurochemicals (e.g., norepinephrine, corticotropin releasing factor, vasopressin, hypocretin, and substance P) giving rise to such negative affective states as anxiety, fear and depression. We propose here the Combined Reward deficiency and Anti-reward Model (CReAM), in which biopsychosocial variables modulating brain reward, motivation and stress functions can interact in a ‘downward spiral’ fashion to exacerbate the intensity, chronicity and comorbidities of chronic pain syndromes (i.e., pain chronification).

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.

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 STATEMENTRecently, 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.

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 2-photon imaging to identify changes in macrophages and dendritic cells (DC) 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 DC stop migrating. We found that CSD activates pial macrophages instantaneously, pial, subarachnoid and dural DC 6-12 minutes later, and dural macrophages 20 minutes later. Dural macrophages and DC 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 20-30 minutes after aura, and that activation of dural macrophages may be mediated by activation of migratory DC in the SAS and dura. The anatomical relationship between TRPV1-positive meningeal nociceptors, and dural macrophages and dendritic cells support a role for these immune cells in the modulation of head pain.

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.

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. ANN NEUROL 2013;73:741–750

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.

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 novelin vivomethod 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 STATEMENTImpairment 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.

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.

A number of distinct endogenous and exogenous factors have been implicated in migraine precipitation but the exact nature of the triggering process itself and its relationship to the genesis of the headache remains largely speculative. In this article, we examine the potential sites and downstream cascades through which migraine triggers might exert their action to promote the activation of the migraine pain pathway. We further look at the laterality of the headache as a potential indicator for the site of migraine pain initiation and examine the question of triggering factor specificity in relation to the current understanding of migraine pathophysiology.

The spinal dorsal horn exhibits a high degree of intrinsic connectivity that is critical to its role in the processing of nociceptive information. To examine the spatial organization of this intrinsic connectivity, we used laser-scanning photostimulation in parasagittal and transverse slices of lumbar spinal cord to stimulate presynaptic neurons by glutamate uncaging, and mapped the location of sites that provide excitatory and inhibitory synaptic input to neurons of the superficial laminae. Excitatory interneuronal connectivity within lamina II exhibited a pronounced sagittal orientation, in keeping with the somatotopic organization present in the pattern of primary afferent projections. Excitatory inputs to all classes of lamina II neurons arose from a wider rostrocaudal area than inhibitory inputs, whereas both excitatory and inhibitory input zones were restricted mediolaterally. Lamina I–II neurons exhibited cell type-specific patterns in the laminar distribution of their excitatory inputs that were related to their dorsoventral dendritic expanse. All cell types received excitatory input predominantly from positions ventral to that of their soma, but in lamina I neurons and lamina II vertical cells this ventral displacement of the excitatory input zone was greater than in the other cell types, resulting in a more pronounced translaminar input pattern. A previously unknown excitatory input to the superficial dorsal horn from lamina III–IV was identified in a subset of the vertical cell population. These results reveal a specific three-dimensional organization in the local patterns of excitatory and inhibitory connectivity that has implications for the processing of information related to both somatotopy and sensory modality.

Primary headaches such as migraine can be aborted by systemic administration of non-steroidal anti-inflammatory drugs (NSAIDs), potentially through the non-selective inhibition of cyclooxygenase (COX) activity in the intracranial meninges. In this study we have used single and double labeling immunohistochemistry to examine the distribution of the COX-1 and COX-2 isoforms in the intracranial dura mater of the rat and identify cell types that express them. COX-1 immunoreactivity was found in medium and small dural blood vessels and was co-expressed with the endothelial cell markers vimentin and the endothelial isoform of nitric oxide synthase (ecNOS). COX-1 was also found to be present in most dural mast cells. COX-2 was mainly expressed in ED2-positive resident dural macrophages. Constitutive COX-2 expression was also found in some axonal profiles, many of which were co-labeled with the nociceptor peptide marker CGRP. The findings suggest that NSAIDs may abort headache, at least in part, by inhibiting either neuronal or non-neuronal COX activity in the dura mater.

Intracranial headaches such as that of migraine are generally accepted to be mediated by prolonged activation of meningeal nociceptors but the mechanisms responsible for such nociceptor activation are poorly understood. In this study, we examined the hypothesis that meningeal nociceptors can be activated locally through a neuroimmune interaction with resident mast cells, granulated immune cells that densely populate the dura mater. Using in vivo electrophysiological single unit recording of meningeal nociceptors in the rat we observed that degranulation of dural mast cells using intraperitoneal administration of the basic secretagogue agent compound 48/80 (2 mg/kg) induced a prolonged state of excitation in meningeal nociceptors. Such activation was accompanied by increased expression of the phosphorylated form of the extracellular signal-regulated kinase (pERK), an anatomical marker for nociceptor activation. Mast cell - induced nociceptor interaction was also associated with downstream activation of the spinal trigeminal nucleus as indicated by an increase in c-fos expression. Our findings provide evidence linking dural mast cell degranulation to prolonged activation of the trigeminal pain pathway believed to underlie intracranial headaches such as that of migraine.

The substantia gelatinosa (lamina II) of the spinal dorsal horn contains inhibitory and excitatory interneurons that are thought to play a critical role in the modulation of nociception. However, the organization of the intrinsic circuitry within lamina II remains poorly understood. We used glutamate uncaging by laser scanning photostimulation to map the location of neurons that give rise to local synaptic inputs to islet cells, a major class of inhibitory interneuron in lamina II. We also mapped the distribution of sites on the islet cells that exhibited direct (non-synaptic) responses to uncaging of excitatory and inhibitory transmitters. Local synaptic inputs to islet cells arose almost entirely from within lamina II, and these local inputs included both excitatory and inhibitory components. Furthermore, there was a striking segregation in the location of sites that evoked excitatory versus inhibitory synaptic inputs, such that inhibitory presynaptic neurons were distributed more proximal to the islet cell soma. This was paralleled in part by a differential distribution of transmitter receptor sites on the islet cell, in that inhibitory sites were confined to the peri-somatic region while excitatory sites were more widespread. This differential organization of excitatory and inhibitory inputs suggests a principle for the wiring of local circuitry within the substantia gelatinosa.

Single-unit electrophysiological recording studies have examined the activity of sensory neurons in the trigeminal ganglion that innervate the intracranial meninges to better understand their possible role in headache. A key question is whether the meningeal sensory neurons are similar to nociceptive neurons in other tissues or, alternatively, whether they have unique properties that might be of significance for headache pathogenesis and drug therapy. Such studies have indeed found that the intracranial dura is innervated by neurons that exhibit properties characteristic of nociceptors in other tissues, including chemosensitivity and sensitization. This sensitization, consisting of an enhanced responsiveness to mechanical stimuli, might be relevant to symptoms that are characteristic of certain headaches that indicate the presence of an exaggerated intracranial mechanosensitivity. Studies that examined whether the anti-migraine agent sumatriptan might inhibit this sensitization (in addition to its well-known inhibition of neurotransmitter release) found that it had no inhibitory effect but rather produced a calcium-dependent discharge, which might account for the initial worsening of headache that can follow sumatriptan administration. In studies that examined the effects of vasodilator agents, nitroprusside produced mixed effects on mechanosensitivity, whereas calciton gene-related peptide (CGRP) had no effect on either spontaneous or mechanically evoked discharge. These results call into question the role of vasodilation in headache and suggest that the role of CGRP in headache may be through its action as a central neurotransmitter rather than through vasodilation and activation of meningeal nociceptors. In general, studies of meningeal sensory neurons have not found evidence of unique properties that distinguish them from nociceptive neurons in other tissues. Ultimately the distinctive clinical characteristics of headache may prove to be related not so much to any differences in the intrinsic molecular or cellular properties of the meningeal sensory neurons but rather to the distinctive properties of the tissue that they innervate.

Migraine is among the most common types of pain, but its mechanisms are poorly understood. A growing body of evidence points to a critical role of calcitonin gene-related peptide (CGRP) in the pathophysiology of migraine headache. During migraine, CGRP is thought to be released from peripheral endings of perivascular meningeal nociceptors primary and to promote vasodilatation. A current hypothesis suggests that peripheral CGRP and its related meningeal vasodilatation results in activation and sensitization, leading to the generation of migraine headache. However, direct evidence supporting this idea is lacking. Here, using electrophysiological, extracellular, single-unit recording combined with laser-Doppler flowmetry measurements of dural blood flow (DBF), we examined whether CGRP and meningeal vasodilatation promote activation or sensitization of meningeal nociceptors. Changes in (DBF), ongoing discharge, and responsiveness to mechanical stimulation of the dura were studied after either topical administration or intravenous infusion of rat alpha-CGRP in anesthetized rats. Both topical and systemic administration of CGRP caused a significant increase in dural blood flow; however, neither method of CGRP administration resulted in activation or sensitization of meningeal nociceptors. The results of this study suggest that CGRP effects in the meninges, including meningeal vasodilatation, are not sufficient to activate or sensitize meningeal nociceptors.

The role of the nitric oxide (NO)-cGMP signaling cascade in modulation of peripheral nociception is controversial. Although behavioral studies have suggested both pro- and anti-nociceptive effects, little is known about the direct action of this signaling cascade on primary afferent nociceptive neurons that mediate these behaviors. Here, using single-unit recordings, we examined the direct effect of NO-cGMP signaling on spontaneous activity and mechanical responses of nociceptive afferents that innervate the dura mater. We found that the NO donor sodium nitroprusside (SNP), when applied topically to the neuronal receptive field, induced both sensitization and inhibition of the mechanical responses, albeit in different populations of neurons, which could be distinguished based on their baseline mechanical thresholds. SNP, however, did not change the level of spontaneous activity. Administration of the cGMP analogue 8-pCPT-cGMP mimicked only the inhibitory effect. When SNP was co-applied with either an inhibitor of guanylyl cyclase or a cGMP blocker, sensitization never occurred, and the inhibitory effect of SNP could also be blocked. Our findings suggest that NO can either increase or decrease the mechanical responsiveness of nociceptors and that its action might depend, in part, on the baseline level of neuronal excitability. Our results also implicate cGMP in mediating the inhibitory effect of NO.

The anti-migraine agent sumatriptan constricts cerebral blood vessels, and also blocks neuropeptide release from meningeal sensory neurons. We investigated whether sumatriptan can also affect neuronal discharge, by recording the activity of dural primary afferent neurons during dural application of sumatriptan in anesthetized rats. Sumatriptan (0.24-24 microM) induced a concentration-dependent increase in ongoing discharge. The discharge occurred only in the presence of calcium. The suppressive effect of calcium removal was not a result of a general decrease in excitability, because calcium removal produced a pronounced increase in mechanically evoked discharge. The excitatory effect on dural afferent discharge might underlie the initial worsening of the headache that can occur following sumatriptan administration, prior to the subsequent onset of headache relief.

For the past 25 years, it has been hotly debated whether cortical spreading depression (CSD), the underlying mechanism of aura, can trigger migraine headache through activation of the trigeminovascular pain pathway. According to one school of thought, CSD leads to a cascade of molecular events (1) that activate pain fibers in meningeal areas overlying the affected cortices (2). According to another school of thought, the aura (and therefore CSD) and the headache are coincidental events with no causal relationship (3). In the past 2 years, this debate has been put to rest by a wealth of scientific evidence that CSD is a noxious event that activates the trigeminovascular pathway (4,5). The recent publication by Lambert and colleagues opens yet a new debate on whether the activation of the central trigeminovascular neurons by CSD depends on peripheral input from meningeal nociceptors or central input from the cortex (6). In their study, the authors suggest that activation of brainstem trigeminovascular neurons by CSD can occur independently of peripheral input (6). This stands in sharp contrast to a large body of evidence for the critical role of peripheral nociceptors in the activation of central trigeminovascular neurons (4,5,7–11). Settling this issue is therefore critical, as it is relevant to the very biological underpinnings of migraine in general, and to the relationship between the aura and headache in particular. Unfortunately, as experts in the experimental techniques used in this study, we believe that the veracity of the findings are questionable, for a number of reasons. First, the recordings are not single units, as claimed, or even multi-units, but instead appear to reflect background electrical noise (e.g. Figure 5 lower trace; multi-unit discharge appears only after the second arrow, but the rate meter registers high levels of activity before that point, even though no discharge is visible). Even the top panel in Figure 1, which is vastly better than the one shown in Figure 5, shows that firing of several neurons are registered into the data analysis system as coming from only one neuron (6). Second, the blockade of peripheral input to the spinal trigeminal nucleus is incomplete. For the study to be valid, a complete blockade of transmission of all action potentials from the dura to the spinal trigeminal nucleus should have been achieved. Only then could the authors conclude with any kind of certainty that the activation of brainstem trigeminovascular neurons by CSD was partially independent of peripheral input (1). As shown in Figure 2 (6), lignocaine injections in the trigeminal ganglion did not eliminate all neuronal responses to stimulation of the dura and skin. At that point, the study should have been stopped or redesigned as it could no longer refute the possibility that the activation seen in the spinal trigeminal nucleus after the induction of CSD was mediated solely by the peripheral input from meningeal nociceptors, whose activity, by the way, was increased by CSD in a similar manner (5). (Scientifically, there are two ways to explain the incomplete blockade of peripheral input from skin and dura to the spinal trigeminal nucleus: (a) incomplete inhibition of the trigeminal ganglion by the lignocaine injection, especially the difficult-to-reach neurons in the ophthalmic division of the ganglion, and (b) peripheral input from intact sensory neurons in the 2nd and 3rd dorsal root ganglia.) Third, the blockade of peripheral input to the spinal trigeminal nucleus is assessed inadequately. The inhibition of sensory signal transmission in the trigeminal ganglion, as presented in Figure 2, was assessed by skin and dural stimulation that induced responses that lasted 10–40 hundredths of a single second (Figure 1 bottom, histograms) (6). These responses are too short (e.g. shorter than an eye blink reflex) and thus cannot reflect the true effects of any drug intervention. An adequate way to assess the lignocaine effects in the trigeminal ganglion would have been to show a continuous (120 min) recording of single-unit activity in response to quantitative stimulation that induced firing that lasted many seconds or even minutes (e.g. noxious heating of the skin or capsaicin on the dura). Fourth, the exclusive sampling of wide-dynamic range (WDR) neurons introduces sampling bias that leads to type II error. For their conclusions to be meaningful and interpretable, the authors (6) should have studied high-threshold (HT), low-threshold (LT) and WDR neurons in different laminae of the spinal trigeminal nucleus, as well as neurons whose activity is driven by Aδ and, even more importantly, C-fiber input (4). As pointed out by the authors, they only studied WDR neurons in deep laminae (we do not really know which laminae as no histology is provided) whose activity was driven by Aδ fibers (6). For this reason, they cannot refute the possibility that other classes of neurons (HT, LT), especially those that receive input from C-fibers and those located in laminae I–II, are activated solely by the peripheral input from meningeal nociceptors (simply because they did not study them). Fifth, the lack of a time-controlled protocol by which changes in firing of all studied neurons are measured over one time period (e.g. 5 or 10 min) and at the same time points after CSD or lignocaine injection (e.g. 15, 30, 60, 120 min) renders the statistical group analysis (Figure 4) invalid because the comparison is based on the experimenter’s subjective judgment rather than objective predetermined parameters (6). (As stated in the paper (6), ‘CSD was initiated with a cortical KCl micro-injection and recording continued until no further changes in discharge rate of the neuron occurred. At this point, 10 μg lignocaine was injected slowly into the trigeminal ganglion and recording continued until no further changes in discharge rate of the neuron occurred.) Based on these observations, it is difficult to reconcile the results reported in this study (6) with those in the authors’ recent report that CSD does not activate trigeminovascular neurons in the spinal trigeminal nucleus (12). It is likely that sampling error led them to find no activation in the first study and activation that is partially independent of peripheral input in the current study. Finally, the authors (6) cite a study that shows that CSD activates meningeal nociceptors in the trigeminal ganglion (5). Given that the central axons of these neurons terminate in several laminae of the spinal trigeminal nucleus, the rationale for proposing that this functional anatomical circuit does not drive activity in central neurons is unclear. This issue is especially bewildering because the latency for activation by CSD in the spinal trigeminal nucleus was similar to the latency for activation of meningeal nociceptors in the trigeminal ganglion. If the activation of central trigeminovascular neurons by CSD is in fact induced by direct (or even indirect) cortical projections to the spinal trigeminal nucleus, one would have expected it to occur within a second, rather than the 10–20 min shown in this and the Zhang et al. (5) studies. Regarding the last point, we are in full agreement with the attention given by Lambert and colleagues to the possibility that cortical projections to the medullary dorsal horn have an important role in trigeminal nociception (6). In support of this concept, Noseda and colleagues (13) showed recently that laminae I–II neurons in the spinal trigeminal nucleus receive direct facilitatory input from the insular cortex, whereas laminae III–V neurons receive direct inhibitory input from the primary somatosensory cortex. Synthesizing these data with all previous studies that showed activation of central trigeminovascular neurons by CSD, it seems reasonable to propose that the initial activation of these neurons is achieved through incoming signals from (peripheral) meningeal nociceptors, whereas the magnitude and duration are determined by the direct and/or indirect inputs they receive from different cortical and brainstem areas.

Functional magnetic resonance imaging was used to image pain-associated activity in three levels of the neuraxis: the medullary dorsal horn, thalamus, and primary somatosensory cortex. In nine subjects, noxious thermal stimuli (46 degrees C) were applied to the facial skin at sites within the three divisions of the trigeminal nerve (V1, V2, and V3) and also to the ipsilateral thumb. Anatomical and functional data were acquired to capture activation across the spinothalamocortical pathway in each individual. Significant activation was observed in the ipsilateral spinal trigeminal nucleus within the medulla and lower pons in response to at least one of the three facial stimuli in all applicable data sets. Activation from the three facial stimulation sites exhibited a somatotopic organization along the longitudinal (rostrocaudal) axis of the brain stem that was consistent with the classically described "onion skin" pattern of sensory deficits observed in patients after trigeminal tractotomy. In the thalamus, activation was observed in the contralateral side involving the ventroposteromedial and dorsomedial nuclei after stimulation of the face and in the ventroposterolateral and dorsomedial nuclei after stimulation of the thumb. Activation in the primary somatosensory cortex displayed a laminar sequence that resembled the trigeminal nucleus, with V2 more rostral, V1 caudal, and V3 medial, abutting the region of cortical activation observed for the thumb. These results represent the first simultaneous imaging of pain-associated activation at three levels of the neuraxis in individual subjects. This approach will be useful for exploring central correlates of plasticity in models of experimental and clinical pain.

Activation of the cAMP/protein kinase A (PKA) second messenger cascade has been implicated in the induction of mechanical hyperalgesia by inflammatory mediators. We examined the role of this cascade in mechanical sensitization of nociceptive neurons that innervate the meninges, a process thought to be involved in the pathophysiology of headache syndromes such as migraine. Single unit activity was recorded in the trigeminal ganglion from 40 mechanosensitive dural afferents (conduction velocitity: 0.3-6.6 m s(-1)) and nine mechanically insensitive dural afferents (MIAs) (conduction velocitity: 0.3-2.8 m s(-1)) while stimulating the dura with a servo force-controlled stimulator or von Frey monofilaments, respectively. Local application to the dura of dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP, 100 microM), a stable membrane-permeant cAMP analogue, produced mechanical sensitization in the majority of mechanosensitive units (19/29, 66 %). Two distinct patterns of mechanical sensitization were observed. Thirty-eight per cent of the units exhibited only a decrease in threshold (TH group), while 28 % showed only an increase in suprathreshold responses (STH group). dbcAMP also induced mechanosensitivity in the majority of MIA units (6/9, 67 %). dbcAMP-induced sensitization was blocked by the PKA inhibitors, Rp-cAMP (1 mM) and H-89 (100 microM). A mixture of inflammatory mediators induced both components of sensitization in the majority of mechanosensitive units tested. However, in each unit, PKA inhibitors blocked only one of the two effects (either TH or STH). Units that were classified as TH or STH also differed in their baseline stimulus-response slopes, thresholds and conduction velocities. These findings implicate the cAMP-PKA cascade in sensitization of dural mechanonociceptors and suggest that this cascade may produce sensitization through at least two different mechanisms operating in separate neuronal populations.

Electrophysiological evidence for tetrodotoxin-resistant sodium channels in mechanosensitive nerve endings of slowly conducting fibers in the intracranial dura. A tetrodotoxin (TTX)-resistant sodium channel was recently identified that is expressed only in small diameter neurons of peripheral sensory ganglia. The peripheral axons of sensory neurons appear to lack this channel, but its presence has not been investigated in peripheral nerve endings, the site of sensory transduction in vivo. We investigated the effect of TTX on mechanoresponsiveness in nerve endings of sensory neurons that innervate the intracranial dura. Because the degree of TTX resistance of axonal branches could potentially be affected by factors other than channel subtype, the neurons were also tested for sensitivity to lidocaine, which blocks both TTX-sensitive and TTX-resistant sodium channels. Single-unit activity was recorded from dural afferent neurons in the trigeminal ganglion of urethan-anesthetized rats. Response thresholds to mechanical stimulation of the dura were determined with von Frey monofilaments while exposing the dura to progressively increasing concentrations of TTX or lidocaine. Neurons with slowly conducting axons were relatively resistant to TTX. Application of 1 μM TTX produced complete suppression of mechanoresponsiveness in all (11/11) fast A-δ units [conduction velocity (c.v.) 5–18 m/s] but only 50% (5/10) of slow A-δ units (1.5

Headache is one of the most common types of pain, but its causes remain poorly understood. The long-standing idea that some headaches, particularly migraine, might be caused by cerebral or cranial vasodilation has failed to find support in recent studies. Alternative hypotheses have focused on other processes that might be capable of activating or sensitizing sensory nerve fibres that innervate the blood vessels of the intracranial meninges.

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.

The present study compared the peripheral and systemic antinociceptive effect of morphine on formalin-induced facial pain behavior in the rat. Formalin (5%, 50 microliters) was injected subcutaneously into the vibrissal pad of adult rats (250-300 g). Morphine sulfate at doses of 100-1000 micrograms was subcutaneously injected locally (same area) or systemically (in the neck), 30 min before, or simultaneously with, formalin. The typical biphasic face grooming response, consisting of an early phasic phase (0-6 min) and a delayed tonic phase (12-42 min), displayed by control animals, was suppressed by both local and systemic administration of morphine; this effect was dose dependent. However, the suppression of the early phase with local morphine administration 30 min before formalin could be significantly greater (49-52%) than with systemic administration, depending on the dose used. Administration of local morphine simultaneously with formalin produced up to 34% reduction in the early and an additional 32% reduction of the late phases of face grooming, compared to systemic injections. Local injection of naloxone (10 micrograms) almost completely reversed the antinociceptive effect of 1000 micrograms of morphine (early phase 85 +/- 7%, late phase 100 +/- 26% reduction), whereas the same dose of naloxone applied systemically (i.p.) produced only partial reversal (early phase 29 +/- 16%, late phase 36 +/- 1% reduction). This study further indicates that locally administered morphine can exert an analgesic effect superior to systemic administration in the case of inflammatory and non-inflammatory pain through a peripheral site of action. These results support the clinical use of peripheral opioid administration in the treatment of human painful conditions.

Chronic constriction injury to the rat's infraorbital nerve (IoN-CCI) induces asymmetric face grooming directed to the injured nerve territory and, beginning at 7-12 days postoperative, hyperresponsiveness to mechanical stimulation in this territory (B.P. Vos, A.M. Strassman, and R.J. Maciewicz, 1994, J. Neurosci. 14:2708-2723). To examine central mechanisms involved in these behavioral alterations, changes in nonevoked and mechanical stimulation-evoked fos-like immunoreactivity (fos-LI) following IoN-CCI were quantified in the medullary dorsal horn. Following the appearance of hyperresponsiveness in IoN-CCI rats, experimental and matched sham-operated rats were anesthetized with urethane and received either no stimulation or repeated stimulation with either a 2- or 15-g von Frey hair applied to the hairy skin between vibrissae B3-4/C3-4 on the operated side. Unstimulated IoN-CCI rats had increased fos-LI in laminae I-IV of the ipsilateral medullary dorsal horn. In both groups, mechanical stimulation produced a distinct pattern of fos-LI in the ipsilateral medullary dorsal horn, the quantity of which was related to stimulus intensity. For both stimulus intensities, the total amount and the rostrocaudal spread of evoked fos-LI were significantly larger in IoN-CCI rats. In IoN-CCI rats, stimulation-evoked increases in fos-LI were proportionally larger in laminae I-II than in III-IV. This laminar effect was also present in sham-operated rats but only for 15-g stimulation. Neither condition nor stimulus intensity affected fos-LI in the contralateral medullary dorsal horn. Positive correlations were found between the behavioral parameters of increased trigeminal nociceptive activity and the total amount of fos-LI in the ipsilateral medullary dorsal horn. The results demonstrate that IoN-CCI induces significant alterations in the central processing of afferent signals, which may underlie behavioral manifestations of increased nociceptive activity.

Microglia survey and directly contact neurons in both healthy and damaged brain but the mechanisms and functional consequences of these contacts are not yet fully elucidated. Combining two-photon imaging and patch-clamping, we have developed an acute experimental model for studying the role of microglia in CNS excitotoxicity induced by neuronal hyperactivity. Our model allows us to simultaneously examine the effects of repetitive supramaximal stimulation on axonal morphology, neuronal membrane potential, and microglial migration, using cortical brain slices from Iba-1 eGFP mice. We demonstrate that microglia exert an acute and highly localized neuroprotective action under conditions of neuronal hyperactivity. Evoking repetitive action potentials (APs) in individual layer 2/3 pyramidal neurons elicited swelling of axons, but not dendrites, which was accompanied by a large, sustained depolarization of soma membrane potential. Microglial processes migrated to these swollen axons in a mechanism involving both ATP and glutamate release via volume-activated anion channels. This migration was followed by intensive microglial wrapping of affected axons, and in some cases removal of axonal debris, that induced a rapid soma membrane repolarization back to resting potentials. When the microglial migration was pharmacologically blocked, the activity-induced depolarization continued until cell death ensued, demonstrating that the microglia-axon contact served to prevent pathological depolarization of the soma and maintain neuronal viability. This is a novel aspect of microglia surveillance, detecting, wrapping and rescuing neuronal soma from damage due to excessive activity. Significant Statement: Microglia, as immune cells in central nervous system, are highly motile cells, continuously expanding and retracting their processes as they monitor brain parenchyma. They can exert neuro-protective or neuro-toxic effects depending on their activation state. In this paper, we demonstrate that microglia are attracted to over-active axons, directly connecting to these axons to reduce membrane potential and exert neuro-protection. The attraction of microglia processes depends on ATP release through volume activated anion channels (VAACs). Blocking VAACs inhibited microglial attraction to axons and impaired the restoration of membrane potential and axonal survival.

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 vivopatch-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 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.

The distribution of fos-like-immunoreactivity (fos-LI) in the medullary and upper cervical dorsal horn was examined following noxious facial stimulation, in order to evaluate the use of fos as a marker for neuronal activation in trigeminal nociceptive pathways. Control animals that received urethane anesthesia and no facial stimulation showed substantial bilateral labeling in the trigeminal complex that was restricted to one rostrocaudal level, at the transition between the medullary dorsal horn (nucleus caudalis) and nucleus interpolaris. Noxious mechanical stimulation (pinch) of different facial sites produced labeling in the ipsilateral dorsal horn whose distribution varied predictably with the rostrocaudal and dorsoventral position of the facial stimulation site, such that rostral facial sites were represented rostrally in the dorsal horn and dorsal sites were represented ventrolaterally. The cornea was exceptional among the facial stimulation sites in that it had a specific representation at two distinct rostrocaudal levels, in C1 and the interpolaris-caudalis transition region; the position of the rostral peak was somatotopically inappropriate, based on the representation of other facial sites. The proportion of labelling in laminae III-IV relative to laminae I-II was higher with noxious mechanical stimulation than with noxious thermal (55 degrees C) or chemical (subcutaneous injection of capsaicin) stimulation. The proportion of labelling in laminae III-IV produced by electrical stimulation of the infraorbital nerve was no greater than that produced by pinch. The results suggest that fos-LI mapping can be a useful method for the investigation of somatotopy but is subject to serious limitations when used for the investigation of laminar organization. The results also suggest that the interpolaris-caudalis transition region may have properties that are distinct from those of the rest of the trigeminal complex, possibly related to an involvement in autonomic function.

Brainstem trigeminal vascular convergence (TVC) neurons receive an excitatory, nociceptive input from cranial blood vessels as well as the facial skin or cornea. In the present study, a population of cat TVC neurons was electrophysiologically identified and then intracellularly labelled with horseradish peroxidase. One TVC neuron from lamin IV and one from lamina V were processed for electron microscopy. Both cells have extensive axon terminal fields within trigeminal nucleus interpolaris as well as lamina IV and V of trigeminal nucleus caudalis. Analysis of thin sections showed that the soma and dendrites of the TVC neurons are contacted by synaptic terminals of various types. Both cells have myelinated axons and collaterals that give rise to unmyelinated preterminal processes. Within nucleus caudalis, terminals of the TVC cells contain round synaptic vesicles that synapse primarily with dendrites and spines.

Intracranial headaches such as migraine are thought to result from activation of sensory trigeminal pain neurons that supply intracranial blood vessels and the meninges, also known as meningeal nociceptors. Although the mechanism underlying the triggering of such activation is not completely understood, our previous work indicates that the local activation of the inflammatory dural mast cells can provoke a persistent sensitization of meningeal nociceptors. Given the potential importance of mast cells to the pain of migraine it is important to understand which mast cell-derived mediators interact with meningeal nociceptors to promote their activation and sensitization. In the present study, we have used in vivo electrophysiological single-unit recording of meningeal nociceptors in the trigeminal ganglion of anesthetized rats to examine the effect of a number of mast cell mediators on the activity level and mechanosensitivity of meningeal nociceptors. We have found that that serotonin (5-HT), prostaglandin I 2 (PGI 2 ), and to a lesser extent histamine can promote a robust sensitization and activation of meningeal nociceptors, whereas the inflammatory eicosanoids PGD 2 and leukotriene C 4 are largely ineffective. We propose that dural mast cells could promote headache by releasing 5-HT, PGI 2 , and histamine.

The aim of this study was to differentiate the processing of nociceptive information, matched for pain intensity, from capsaicin-induced hyperalgesic vs. control skin at multiple levels in the trigeminal nociceptive pathway. Using an event-related fMRI approach, 12 male subjects underwent three functional scans beginning 1 h after topical application of capsaicin to a defined location on the maxillary skin, when pain from capsaicin application had completely subsided. Brush and two levels of painful heat (low-Thermal-1 and high-Thermal-2) were applied to the site of capsaicin application and to the mirror image region on the opposite side. Temperatures for each side were set to evoke perceptually matched pain (mean temperatures [capsaicin/control]: Thermal-1=38.4/42.8 degrees C; Thermal-2=44.9/47.8 degrees C). We found differences in activation patterns following stimuli to treated and untreated sides in sensory circuits across all stimulus conditions. Across the trigeminal nociceptive pathway, Thermal-2 stimulation of hyperalgesic skin evoked greater activation in trigeminal ganglion and nucleus, thalamus, and somatosensory cortex than the control side. Thus, trigeminal nociceptive regions showed increased activation in the context of perceptually equal pain levels. Beyond these regions, contrast analyses of capsaicin vs. control skin stimulation indicated significant changes in bilateral dorsolateral prefrontal cortex and amygdala. The involvement of these emotion-related regions suggests that they may be highly sensitive to context, such as prior experience (application of capsaicin) and the specific pain mechanism (hyperalgesic vs. normal skin).

Migraine attacks are triggered by a variety of conditions including endogenous and exogenous factors. Evidence suggests that activation and sensitization of primary afferent meningeal nociceptive neurons, the peripheral arm of the trigeminovascular system, constitutes one of the earliest events promoting the intracranial pain of migraine. However, the link between the varied triggering factors and activation of meningeal nociceptive neurons is not completely understood. Local inflammation with release of mediators from local immune/inflammatory cells is thought to play a critical role in such neuronal response. Meningeal mast cells may play such a role by virtue of their proximity both to meningeal blood vessels and nociceptive axons and their ability to release a host of proinflammatory/algesic mediators. This paper reviews data relevant to the hypothesis that mast cells, upon activation by migraine triggers, contribute to the genesis of migraine headache. Epidemiologic findings, clinical data, and observations on anatomical and physiological characteristics of mast cells converge to suggest an important role of these immune cells in the pathogenesis of migraine. Migraine triggers might directly or indirectly promote mediator secretion from meningeal mast cells, and thereby cause inflammation and activation of the trigeminovascular system. While consistent, the evidence supporting mast cell involvement in the genesis of migraine is largely circumstantial to date. Further studies are needed to test directly the nature of mast cell involvement in the pathogenesis of migraine headache.

The field of basic pain research has had an ongoing need to develop better ways of assessing pain in animals, especially ‘‘spontaneous’’ or ongoing pain as opposed to tactile or thermal hypersensitivity (1–7). It has been argued that the lack of adequate behavioral assays for spontaneous pain is a key factor in the failure to develop new drugs for pain treatment, in spite of enormous advances in the understanding of basic mechanisms and the identification of molecular targets. The headache field in particular for many years suffered from the severe limitation of having no behavioral assay whatsoever. One exception was the finding of Malick et al. of a moderate decrease in feeding behavior over a six-hour period following chemical stimulation of the dura in awake rats (8). However, this finding was not pursued further as a behavioral assay, in part because of the relatively long observation time required. A major breakthrough for basic headache research came when Oshinsky and Gomonchareonsiri showed that chemical stimulation of the dura induced facial allodynia in awake rats, providing for the first time an acutely measureable behavioral correlate, albeit indirect, of ‘‘headache’’ in animals (9). However, the problem still remained of finding a behavioral correlate of ongoing or ‘‘spontaneous’’ headache pain, as opposed to the allodynia that can sometimes be an accompaniment to headache (10). This issue of Cephalalgia reports a finding that represents a further breakthrough for the study of headache: a new observation of a change in ‘‘spontaneous’’/ ongoing behavior following chemical stimulation of the dura in chronically prepared awake rats (11). This observation was made possible by exceptionally careful surgical implantation of the extradural cannula and acclimation of the rats to the observation environment, and second-by-second video analysis of ongoing behavior over a 45-minute observation period, made during the awake period of the animal’s circadian cycle. The core finding is a suppression of the exploratory behavior that normally occurs in control animals when placed into the observation chamber, and a concomitant increase in the amount of ‘‘resting’ behavior or quiet wakefulness. This observation might not in itself sound very remarkable. However, it is potentially of great significance as an example of the more general phenomenon of the suppression of an innate behavior/locomotor activity by pain. Such suppression is now being recognized as a new, powerful approach for assessing ‘‘spontaneous’’/ongoing pain, which has been put forth by Andrews and others who are at the forefront of developing new methods for the study of pain behavior in animals (6,7,12–15). This observation of a suppression of exploratory behavior has further significance in view of the long-standing idea that visceral pain is associated with a distinct response pattern, consisting of behavioral as well as autonomic quiescence and withdrawal, in contrast to the arousal and escape behavior evoked by superficial pain (16–19). Future studies could test the hypothesis that this suppression of exploratory behavior or other innate behaviors is greater following stimulation of deep rather than superficial tissues. This finding of Melo-Carillo and Lopez-Avila is in a sense more direct than that of Oshinsky and Gomonchareonsiri in that it is potentially a behavioral correlate of the headache itself, not merely the allodynia that can sometimes accompany headache. It may also prove to be a more sensitive assay, in that allodynia is present only in a subset of migraine attacks, whereas headache pain is (by definition) present in all attacks. The dural stimulus also induced a brief initial period of ipsilateral facial ‘‘grooming’’ or rubbing that

Neurophysiological studies have characterized the sensory responses of primary afferent nociceptors that innervate the intracranial dura. The present study used anatomical methods to examine in greater detail the axonal trajectories within the dura, as well as the axonal size distribution of the dural innervation. Immunostaining for CGRP in dural wholemounts revealed a network of fibers extending across the entire dura, with an especially dense plexus running along the borders of the transverse and superior sagittal sinuses. The plexus along the caudal border of the transverse sinus partially overlapped the dural area that shows the greatest density of mast cells. Visualization of axon bundles by DiI application in formalin-fixed tissue revealed two separate systems of fibers in the dura that could be distinguished by the orientation of their trajectories: one that runs parallel to the middle meningeal artery (MMA), and another with a more or less orthogonal orientation that runs rostromedially from the transverse sinus across the MMA. Axons traversed large distances across the dura, but the majority of the branching and arborization was usually concentrated in the distal part of the trajectory. In separate animals, measurement of myelinated axon diameters with electron microscopy showed that approximately one-third of the myelinated axons in the nerves supplying the dura (nervus spinosus and tentorial nerves) could be classified as A-beta, since they were comparable in size to the majority of axons in the trochlear nerve and the upper end of the size range in the trigeminal nerve (i.e., >5 μm). J. Comp. Neurol. 473:364–376, 2004. © 2004 Wiley-Liss, Inc.

The intracranial dura receives a small-fiber sensory innervation from the trigeminal ganglion that is thought to be involved in some types of headaches, including migraine. Mechanical response properties of dural afferent neurons were examined to investigate variation across the population in the properties of threshold, slope, adaptation, and incidence of mechanosensitivity. Dural afferent neurons were recorded in the trigeminal ganglion of urethan-anesthetized rats and were identified by their constant-latency response to dural shock. Neurons were classified as fast A (>5 m/s), slow A (5 ≥ conduction velocity (CV) ≥ 1.5 m/s), or C (