Inhibition of receptor signalling in trigeminal ganglion cells for the treatment of migraine

Abstract

The present invention relates to reducing an activation of a trigeminal ganglion neuron or cell and related uses in treating or preventing migraine.

Description

[0001] Brain Protein Targets for The Treatment of Migraine

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing date of U.S. Provisional Patent No. 63 / 658,300, filed on June 10, 2024, which is hereby incorporated by reference in its entirety.

[0004] GOVERNMENT INTERESTS

[0005] This invention was made with government support under AG057575, and NS128613 awarded by the National Institutes of Health, and W911NF-19-1-0280 awarded by the Army Research Laboratory - Army Research Office. The government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The contents of the electronic sequence listing (161118.08201SeqList.xml; Size: 160,534 bytes; and Date of Creation: May 23, 2025) is herein incorporated by reference in its entirety.

[0008] FIELD OF THE INVENTION

[0009] This invention relates to reducing an activation of a trigeminal ganglion neuron or cell and related uses in treating or preventing migraine.

[0010] BACKGROUND

[0011] Migraine is a chronic, painful, neurovascular disorder that affects an estimated 12% of the population (Stewart W, et cd.. Prevalence of migraine headache in the United States. Relation to age, income, race, and other sociodemographic factors. JAMA. 1991;267:64-69). Sensitization and activation of trigeminal ganglion nerves are thought to play a central role in the underlying pathology of migraine and other orofacial diseases. Migraine is a significant source of both medical costs and lost productivity. It has been estimated that migraine is the costliest neurological disorder in the European Community, costing more than €27 billion per year. In the United States, direct costs have been estimated at $17 billion, while indirect costs- such as missed or decreased ability to work- is estimated at $15 billion. There is a need for new treatments of migraine.

[0012] SUMMARY OF THE INVENTION

[0013] This disclosure addresses the need mentioned above in a number of aspects.

[0014] In one aspect, the disclosure provides a method of reducing an activation of a trigeminal ganglion neuron or cell. The method comprises inhibiting an action of a receptor on or in said neuron or cell. The receptor may be selected from the group consisting of Tlr4, Lepr, Lrpl, Tshr, Itga3, Mtnrla, Calcr, Calcrl, Rampl, Ramp2, Ramp3, Itgav, Scarfl, Cntnl, Cntnap2, Egfr, Epha4, Ephb2, Ephb3, Ephb4, Pdgfirb, Itgb3, Itgal, Itgbl, Plxncl, Cd44, Itga4, and Plaur.

[0015] In a second aspect, the disclosure provides a method of reducing an activation of a trigeminal ganglion neuron or cell. The method comprises blocking the binding of a ligand to a receptor on or in said neuron or cell, wherein (I) the ligand is selected from the group consisting of S100A8, S100A9, Apod, Calr, Cntn2, Efempl, Efnb3, Mfge8, Sema7a, Sppl, Vtn, and Calca, or (II) the receptor is selected from the group consisting of Tlr4, Lepr, Lrpl, Tshr, Itga3, Mtnrla, Calcr, Calcrl, Rampl, Ramp2, Ramp3, Itgav, Scarfl, Cntnl, Cntnap2, Egfr, Epha4, Ephb2, Ephb3, Ephb4, Pdgfirb, Itgb3, Itgal, Itgbl, Plxncl, Cd44, Itga4, and Plaur. In some embodiments, the ligand is selected from the group consisting of S100A8, S100A9, Sema7a, Sppl, and Vtn.

[0016] For each of the methods described above, the receptor can be selected from the group consisting of Tlr4, Plxncl, Cd44, and Plaur. In some embodiments, the ligand and the receptor are a pair selected from the group consisting of (a) S100A8 and TLR4, (b) S100A9 and TLR4, (c) SPP1 and CD44, (d) VTN and PLAUR, and (e) SEMA7A and PLXNCL In each of the methods described, the inhibiting can be conducted by a process comprising contacting the neuron or cell with an agent that specifically binds to the receptor. The blocking can be conducted by a process comprising contacting the neuron or cell with a substance that specifically binds to the ligand. In some embodiments, the agent or substance is a small molecule compound, a peptide, or a protein. In one embodiment, the protein is an antibody or antigen-binding fragment thereof. The trigeminal ganglion neuron or cell can be in a subject. In some embodiments, the agent or substance is one selected from monoclonal antibody Ab45, monoclonal antibody 5.5, monoclonal antibody A22124D, monoclonal antibody 1F8, anti- S100A8 polyclonal antibody (PA5-82881), Paquinimod (ABR-215757), Laquinimod (ABR- 215062), Tasquinimod (ABR-215050), ABR-238901, Midostaurin, Eritoran, Peptide3A5, Divalent Peptide3A5, Tetravalent ILVIK, a peptide-Fc fusion protein, PIM447, GDC-0339, TAK-242 (Resatorvid), Eritoran (E5564), CLL095, FP7, IAXO-101, C34, VX-15 / 2503, VIPER peptide, TLR4 inhibitory peptide 1 (TIPI), NI-0101, TLR4 monoclonal antibody (clone HTA125), TLR4 siRNA, TLR4 antisense oligonucleotide, monoclonal antibody 6B4, monoclonal antibody MAC387, monoclonal antibody H5, S100A9 Antibody Blocking Peptide (LS-E31467), S100A9 Peptide (AAP36677), S100A9 Peptide -N-terminal region (AAP36676), monoclonal antibody MEM-150, monoclonal antibody D-4, polyclonal anti- Sema7a antibody ab23578, polyclonal anti-Sema7a antibody A03832, SEMA7A Antibody Blocking Peptide (LS-E23467), monoclonal antibody 1A7, monoclonal antibody 1D7, monoclonal antibody 1F5, monoclonal antibody h2Kl, monoclonal antibody c2Kl, SPP1 Blocking Peptide (SPP1-BP), SPP1 Peptide (AAP36677), SPP1 Peptide - N-terminal region (AAP36676), SPP1 Antibody Blocking Peptide (LS-E31467), SPP1 Aptamer (SPP1-APT), Cilengitide (EMD121974), MK-0429, ATN-161 (Ac-PHSCN-NEE), an RGD peptide, Volociximab (M200), 8E6 (LJ8) monoclonal antibody, Vitronectin (VTN) siRNA, Vitronectin antisense oligonucleotides, PLXNC1 siRNA, PLXNC1 shRNA, small molecule RG7356, SB- 525334, PEP-1, A6 peptide, HABP (Hyaluronic Acid Binding Peptide), Bivatuzumab mertansine, antibody RG7356, antibody IM7, antibody H4C4, CD44 siRNA, CD44 shRNA, UK-371804, WX-360, AE105, UP ARANT (UP AR antagonist peptide), ATN-658, HuATN- 658, PLAUR siRNA, and PLAUR shRNA. The antibody can be a chimeric antibody, a humanized antibody, a human antibody, a single chain antibody, or a nanobody.

[0017] In a third aspect, the disclosure features a method for treating or preventing a migraine in a subject in need thereof. The method comprises: (I) inhibiting an action of a receptor on or in a trigeminal ganglion neuron or cell in the subject according to the method described above; or (II) blocking the binding of a ligand to a receptor on or in said neuron or cell according to the method described above. In some embodiments, the method comprises comprising administering to the subject an effective amount of the agent or substance described herein.

[0018] The details of one or more embodiments of the invention are set forth in the description below. Other features, objectives, and advantages of the invention will be apparent from the description and from the claims. The examples below and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entireties.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIGS. 1A-1D demonstrate that solutes in cerebrospinal fluid reaches and activates trigeminal ganglion cells. FIG. 1A shows that a GRIN lens in TRPVl :GCaMP6f animals allowed for simultaneous imaging of neuronal activity and CSF tracer (1 % capsaicin, dextran 70 kDa) in the right trigeminal ganglion in vivo. Images display examples of GRIN lens recordings during baseline and infusion (scalebar, 50 pm). FIG. IB is an orange heatmap that shows GcaMP6f Af / fo in one representative animal during baseline and infusion. Blue heatmap displays CSF tracer concentration in the trigeminal ganglion in the same animal. The bar graphs display area under the curve (AUC) of the calcium signal from trigeminal TRP VI -lineage cells (orange) and CSF tracer signal (blue) during baseline and infusion (n=6 animals, N=86 cells). P<0.001, Wilcoxon signed rank test. FIG. 1C is a histogram of trigeminal cell activations during baseline and infusion. FIG. ID shows immunohistochemical cross sections of trigeminal ganglion in mice with CSF tracer (70 kDa). CSF tracer (cyan) pervades all compartments of the ganglion and is accumulating in connective tissue (collagen-1, col-1) that enwraps the ganglion (epineurium) and nerve fascicles (perineurium), as well as in extracellular space between sensory neuron somas (microtubule associated protein (MAP), (scale bar, 50 pm)) and between axons (myelin basic protein (MBP), neurofilament heavy chain (NFH), (scale bars, 25 pm)).

[0021] FIGS. 2A-2D show that lack of a tight barrier allows CSF influx at the root of the trigeminal ganglion. FIG. 2A is a trigeminal ganglion heatmap (stippled outline) showing contrast arrival time after contrast enhanced-MRI (CE-MRI) imaging where gadobutrol (0.6 kDa) was delivered to the cerebrospinal fluid (CSF) by cistema magna injection. Scalebar, 1 mm. FIG. 2B is a section of mouse head that shows trigeminal ganglion (black box) in basis cranii, hematoxylin and eosin staining. Scalebar, 5 mm. FIG. 2C shows immunohistochemical sections. Dura mater (aquaporin-1 positive, AQP1) and the arachnoid barrier membrane (claudin-11 positive) runs in close apposition and are dissociated from the trigeminal ganglion at the proximal third before it fuses with the ganglion. The subarachnoid lymphatic-like membrane (SLYM, co-expressing PROX1-GFP, CRABP2 and PDPN) runs beneath the dura- arachnoid barrier membrane, and is dissociated from the ganglion at the proximal third. SLYM fuses with dura mater and the arachnoid barrier membrane just before their insertion upon the ganglion. Pia mater (positive for PDPN and CRABP2, negative for PROX1-GFP) is fused directly to the trigeminal ganglion everywhere. The border between the central (CNS) and peripheral nervous system (PNS) lies roughly one fourth into the ganglion (astrocytes in CNS express aquaporin-4, AQP4). Scalebars, 500 pm. FIG. 2D is a schematic of trigeminal ganglion membranes. Solutes in CSF can (1) enter the trigeminal ganglion by transport from the inner subarachnoid space, or from (2) transport from the CNS. This constitutes a novel CSF flow route compared to the known CSF egress along nerve sheaths towards cervical lymphatics (3).

[0022] FIGS. 3A-3C illustrate that cortical spreading depression increases washout of extracellular solutes. FIG. 3A shows the efflux path of gadobutrol injected into visual cortex that was mapped using dynamic contrast enhanced-MRI. Line graphs depict signal enhancement ratio (SER) at ipsilateral cerebrospinal fluid (CSF) space around the trigeminal ganglion (TG) and in the parasagittal dural space (PSD). Bar graphs plot the area under the curve (AUC) of the line graphs and tracer arrival time. FIG. 3B shows CSF tracer was delivered to Thyl-ChR2 mice and cortical spreading depression (CSD) was induced optogenetically. Images display CSF tracer in dorsal cortex at various timepoints. Line graph displays the CSF tracer concentration (mean pixel intensity, MPI) and influx rate (AMPI / min) in the two hemispheres. Bar graph displays concentration of cortical CSF tracer after CSD. Wilcoxon signed rank test, P < 0.05. FIG. 3C shows extracellular space tracer that was delivered by intracortical injection and multiple CSDs were evoked with IM KC1. Cortical tracer concentration was monitored over Ih. Pictures display extracellular tracer at various timepoints in one representative control and CSD animal. Line graph displays MPI of cortical tracer between groups. Bar graph shows mean reduction in MPI. P<0.05, Mann-Whitney test (n=12, 6 CSD animals, 6 control animals). *P < 0.05, **P < 0.01.

[0023] FIGS. 4A-4G illustrate that cerebrospinal fluid collected after cortical spreading depression provokes activity in trigeminal neurons. FIG. 4A is a volcano plot of mass spectrometry proteomic analysis of cerebrospinal fluid (CSF) from animals with and without exposure to cortical spreading depression (CSD). Black circles represent a CSF protein with ligand capabilities to receptors in the trigeminal ganglion (TG). CSF ligands significantly upregulated in the CSD proteome are labelled by gene name. FIG. 4B is a dot plot of RNA levels of receptors matching to significantly upregulated CSF ligands. Left Y-axis: cell clusters, lower X-axis: receptor genes, top X-axis: ligand genes paired to the receptor gene, right Y-axis bar graph: total cluster cell counts. FIG. 4C shows CSD CSF that was transferred to exposed trigeminal ganglions in TRPVl-cre:GCaMP6f mice. FIG. 4D is a ganglion overview. Inserts showing GCaMP6f signals (scale bar, 200 pm). FIG. 4E shows GCaMP6f Af / fo heatmaps. FIG. 4F shows area under the curve (AUC) of GCaMP signal. Repeated measures ANOVA with post hoc Tukey’s test (n=5, N=107 cells). FIG. 4G shows AUC of GCaMP signal during endogenous CSF application. Kruskal-Wallis test with Dunn’s post hoc analysis (n= 13 animals; 4 exposed to naive CSF, 5 exposed to CSD CSF Ih, and 4 exposed to CSD CSF 2.5h, N=247 cells). *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant. Single cell RNA sequence data in B from Yang et al. (44)

[0024] FIGS. 5A-5D demonstrate expression rates of cerebrospinal fluid proteins in cortex and their interaction proteins in the trigeminal ganglion. FIG. 5A shows gene ontology enrichment analysis of the upregulated and downregulated proteins in CSF after CSD. The top 10 most significant terms are displayed. FIG. 5B shows gene ontology enrichment analysis of the cerebrospinal fluid ligands and corresponding receptors in the trigeminal ganglion. The top 10 most significant terms are displayed. FIG. 5C is a Sankey plot illustrates the communication pathways between cortical cell clusters and trigeminal cell clusters established by CSF flow to the trigeminal ganglion for the significantly upregulated CSF ligands. The relative size of each connection is based on the cell clusters RNA level mean. FIG. 5D is a dot plot showing RNA levels of receptors across cell clusters in the brain cortex of all identified CSF ligands. Left Y- axis: cell clusters, lower X-axis: ligand genes, bar graphs on right Y-axis represents total cluster cell counts. RNA figures based on publicly available single cell sequencing data from Yang et al. (44) and Zeisel et al. (86)

[0025] FIG. 6 shows that receptor expression rates of all cerebrospinal fluid ligands. Dot plot shows RNA levels of receptors across cell clusters in the trigeminal ganglion of all CSF ligands identified; also those without significant changes in concentration. Left Y-axis: trigeminal cell clusters, lower X-axis: receptor genes, top X-axis: ligand genes paired to the receptor gene, bar graphs on right Y-axis: total cluster cell counts. RNA data based on publicly available single cell sequencing data from Yang et al. (44).

[0026] FIGS. 7A-7F demonstrate that CSD increases activity in the trigeminal ganglion likely by way of calcitonin gene-related peptide (CGRP). FIG. 7A shows that cortical spreading depression (CSD) was evoked with KC1 in animals with GRIN lens implanted over right trigeminal ganglion. Inserts show GCaMP6f signals. GCaMP6f Af / fo heatmap shows activity from one representative animal during baseline and CSD. Graphs display from right to left: Boxplot shows area under the curve (AUC) of entire recording for each cell, second boxplot shows number of active cells per animal (active cell defined as cell with at least one calcium peak above 3 standard deviations), bar graph shows cell activations per minute per animal. Wilcoxon signed-rank test (n=6, N=73 cells). Histogram displays number of cell activations over time during baseline and CSD. FIG. 7B is a bar graph that shows potassium levels in naive and CSD CSF. Mann-Whitney U test, (n=15, 9 controls, 6 CSD animals). FIG. 7C shows 0.1 mM CGRP in artificial cerebrospinal fluid (aCSF) applied to exposed trigeminal ganglion in TRPVl-cre:GCaMP6f mice. (D) Ganglion with GCaMP6f maximum projections of insert. FIG. 7E shows GCaMP6f Af / fo heatmaps. FIG. 7F shows the AUC of the stimuli conditions for each cell. Repeated measures ANOVA with post hoc Tukey’s test (n=7,N=180). *P < 0.05, ***P < 0.001, ns = non-significant. FIGS. 8A and 8B demonstrate that S100A8 application to trigeminal ganglion induces sensory nerve activation. FIG. 8A is a heatmap showing AF / F of GCAMP signal in sensory nerves in the trigeminal ganglion during baseline, aCSF and S100A8 application. FIG. 8B is a bar graph displaying area under the curve of AF / F of GCAMP signal during the three conditions. Kruskal -Wallis test with Dunn’s post hoc analysis (n=14).

[0027] FIGS. 9A and 9B demonstrate that osteopontin application to trigeminal ganglion induces sensory nerve activation. FIG. 9A is a heatmap showing AF / F of GCAMP signal in sensory nerves in the trigeminal ganglion during baseline, a CSF and osteopontin application (opn). FIG. 9B is a bar graph displaying area under the curve of AF / F of GCAMP signal during the three conditions. Kruskal-Wallis test with Dunn’s post hoc analysis (n =12).

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] Migraine with aura patients experience classical attacks of headache preceded by aura and transient neurological deficits, associated with cortical spreading depression (CSD). It is not currently understood how a pathological event in cortex activate peripheral sensory neurons outside the brain. This disclosure demonstrates that cerebrospinal fluid (CSF) flow freely into the trigeminal ganglion and establish non-synaptic signaling between brain and trigeminal cells. After CSD, ~5% of the CSF proteome is altered, with upregulation of proteins that directly activate receptors in trigeminal ganglion. CSF collected from animals exposed to CSD activates trigeminal neurons in naive mice in part due to calcitonin gene-related peptide. This disclosure defines a novel interface between the central and peripheral nervous system, explaining how CSD activate trigeminal afferents in migraine with aura.

[0030] For thousands of years, scientists and doctors have speculated about the origin of migraine headache (1). Clinical evidence largely suggests that onset of migraine headache as well as site of pharmacological rescue is outside the central nervous system (CNS) (2-4). Yet, for a third of migraine patients, headache is preceded by an aura and transient neurological deficits, associated with cortical spreading depression (CSD), a pathological depolarization of cortical tissue (5). How can such an event in the CNS trigger the activation of peripheral nociceptors (6-9)?

[0031] Current evidence suggests that migraine headache is driven by activation of sensory nerve endings in the dura mater (10-14). However, efflux of cortical solutes to dura mater is limited by the arachnoid barrier layer, and only pass into dura mater along bridging veins feeding into the major venous sinuses (15-19), thus restricting the contact between CSF solutes and afferents in dura mater. In this disclosure, provided is a novel interaction between the CNS and peripheral nervous system (PNS), mediated by the transit of CSF -borne solutes to the trigeminal ganglion. This disclosure shows that CSF transports CGRP and other solutes released from the cortex during CSD to the trigeminal ganglion extracellular space and instigates trigeminal activation in a mouse model of classical migraine (5, 10, 20, 21).

[0032] The data described herein indicates that CSF transports solutes from the cortex to the trigeminal ganglion, and by so doing establishes a non-synaptic route of communication between the central and peripheral nervous systems that underlies the pathogenesis of classical migraine. The cortex to ganglion flow is rapid and retains considerable laterality, so that solutes released into cortical extracellular space in one hemisphere flow primarily to the ipsilateral trigeminal ganglion. This signaling route may therefore account for the unilaterality of migraine headache, as well as the typical delay between aura and headache onset.

[0033] The CSF transport of signaling molecules has been shown to drive hyperalgesia (51), but it has not hitherto been linked to migraine pathophysiology. It was found that CSD induces substantial changes in CSF composition, with 10% of the CSF proteome being significantly altered in concentration, and 5% with a more than two-fold increase. These dramatic changes are likely driven by the cascade of perturbances driven by CSD, including changes in cerebral blood flow, neuronal firing, tissue oxygenation and CSF flow (9, 10, 52-54). CSD upregulates RNA levels of inflammatory mediators (42, 55), and it was found that the changes in hemodynamics and CSF transport instigated by CSD, increased the washout of cortical extracellular solutes, further explaining the dramatic proteomic changes. ~5 % of the CSF proteins were ligands to receptors in the trigeminal ganglion, including peptides that provoke migraine in humans and which antagonists are migraine abortive, z.e., pituitary adenylate- cyclase-activating polypeptide (PACAP, FC = 1.2, ns) and CGRP (56, 57), as well as several other pro-nociceptive signaling molecules (58, 59). CGRP doubled in concentration, consistent with prior reports of elevated tissue RNA and extracellular CGRP levels after CSD (60, 61). Leptomeningeal afferent release of CGRP into the CSF likely also contributes to elevated CGRP levels (62). Past studies have demonstrated that the key signaling target of CGRP is outside of the CNS: the peptide administered IV provokes migraine, while effective CGRP blockers do not cross the blood-brain-barrier, and neither does CGRP itself (2, 4, 63-65). Transport of CGRP from the cortex to the trigeminal ganglion thus explains how centrally derived CGRP exerts its effect peripherally. Interestingly, CGRP has also been found elevated in the CSF of migraine patients without aura, suggesting that in these patients as well, the trigeminal CSF pathway could drive headache (66). Though the observations described herein indicate that the trigeminal CSF uptake drives the immediate migraine headache, it was also found that CSF composition quickly normalizes, suggesting that other processes might drive headache at later phases. These processes might include ongoing CGRP-stimulated signaling intrinsic to the ganglion, as well as the efflux of CSF to dura mater with both direct and indirect sensory afferent activation, such as by mast cell degranulation (10, 67, 68). The initial CSF transport of ligands into the trigeminal ganglion could instigate several of these processes downstream. As such, defining the role of the ligand-receptor pairs identified in the CSD proteome may enable the discovery of new pharmacological targets, to the benefit of the large portion of patients not responding well to currently available therapies.

[0034] The trigeminal nerve arises from the pons and traverses the basal subarachnoid space in its intracranial portion (FIG. 2B). Serial sections of the trigeminal nerve with intact meninges revealed that dura mater and the arachnoid barrier layer (labeled with claudin-11) (32) formed a coherent membrane, that in the proximal third of trigeminal nerve was physically separable from the nerve, while the two membranes fused with the trigeminal ganglion connective tissue sheath in the distal two thirds of the ganglion (FIG. 2C). The subarachnoidal lymphatic-like membrane (SLYM) - defined antigenically as expressing PROXI, CRABP2 and PDPN (33) - was dissociated from the dura and arachnoid membranes, as well as from the proximal third of the trigeminal ganglion; in contrast, it fused with the dura mater and arachnoid membranes just prior to the membranous insertion on the ganglion forming the perineural sheath (33). Thus, at the proximal third of the ganglion, an inner CSF space is delineated by SLYM as the roof, and the permeable pial membrane (labeled with PDPN) attached to the ganglion as its floor, allowing free passage of CSF and solutes into the ganglion (34). At its distal two thirds, the dura mater, the arachnoid barrier layer and SLYM fuse with the connective tissue sheath around the trigeminal ganglion, creating an impermeable barrier to the surrounding CSF.

[0035] Treatment Methods

[0036] Provided is a method of reducing an activation of a trigeminal ganglion neuron or cell, comprising inhibiting an action of a receptor on or in said neuron or cell. In some embodiments, the wherein the receptor is selected from the group consisting of Tlr4, Lepr, Lrpl, Tshr, Itga3, Mtnrla, Calcr, Calcrl, Rampl, Ramp2, Ramp3, Itgav, Scarfl, Cntnl, Cntnap2, Egfr, Epha4, Ephb2, Ephb3, Ephb4, Pdgfirb, Itgb3, Itgal, Itgbl, Plxncl, Cd44, Itga4, and Plaur. In some embodiments, the receptor has at least 80% sequence identity to a receptor described in Table 1 such as at least 80%, at least 85, at least 90%, at least 95%, 100% identity or any percent therebetween. Also provided is a method of reducing an activation of a trigeminal ganglion neuron or cell, comprising blocking the binding of a ligand to a receptor on or in said neuron or cell, wherein

[0037] (I) the ligand is selected from the group consisting of S100A8, S100A9, Apod, Calr, Cntn2, Efempl, Efnb3, Mfge8, Sema7a, Sppl, Vtn, and Calca, or

[0038] (II) the receptor is selected from the group consisting of Tlr4, Lepr, Lrpl, Tshr, Itga3, Mtnrla, Calcr, Calcrl, Rampl, Ramp2, Ramp3, Itgav, Scarfl, Cntnl, Cntnap2, Egfr, Epha4, Ephb2, Ephb3, Ephb4, Pdgfirb, Itgb3, Itgal, Itgbl, Plxncl, Cd44, Itga4, and Plaur. In some embodiments, the ligand has at least 80% sequence identity to a ligand described in Table 2 such as at least 80%, at least 85, at least 90%, at least 95%, 100% identity or any percent therebetween. In some embodiments, the ligand is selected from the group consisting of S100A8, S100A9, Sema7a, Sppl, and Vtn. In some embodiments, the receptor is selected from the group consisting of Tlr4, Plxncl, Cd44, and Plaur. In some embodiments, the ligand and the receptor are a pair selected from the group consisting of (a) S100A8 and TLR4, (b) S100A9 and TLR4, (c) SPP1 and CD44, (d) VTN and PLAUR, and (e) SEMA7A and PLXNCL

[0039] In one embodiment, the inhibiting is conducted by a process comprising contacting the neuron or cell with an agent that specifically binds to the receptor. In one embodiment, the blocking is conducted by a process comprising contacting the neuron or cell with a substance that specifically binds to the ligand. In some embodiments, wherein the agent or substance is a small molecule compound, a peptide, or a protein. In one embodiment, the protein is an antibody or antigen-binding fragment thereof.

[0040] Examples of agents that inhibit the receptors and ligands described herein are provided in Tables 3 and 4, respectively.

[0041] In one embodiment, the trigeminal ganglion neuron or cell is in a subject. In one embodiment, the subject is a human.

[0042] Provided is a method for treating or preventing a migraine in a subject in need thereof, comprising: (I) inhibiting an action of a receptor on or in a trigeminal ganglion neuron or cell in the subject according to the method described herein; or (II) blocking the binding of a ligand to a receptor on or in said neuron or cell according to the method described herein. In one embodiment, the method comprises administering to the subject an effective amount of the agent or substance.

[0043] In some embodiments, the methods described herein reduce the number of migraine attacks experienced by the subject. Table 1. Exemplary receptors.

[0044] Table 2. Exemplary ligands.

[0045] Table 3. Exemplary inhibitory agents against the indicated receptors.

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] Table 4. Exemplary inhibitory agents against the indicated ligands.

[0052]

[0053] Some of the agents or materials are small molecule compounds. The derivatives, variants, and analogues of these compounds can also be used. As used herein, the terms "derivative," "variant," and "analogue" are used interchangeable to refer to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein and one whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary derivatives include salts (preferably pharmaceutically acceptable salt), esters (preferably physiologically cleavable ester), amides, salts of esters or amides, and N-oxides of a parent compound. Examples also include prodrugs.

[0054] The term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al.. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemi sulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N+(CI-4 alkyljy salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0055] The term "physiologically cleavable ester" refers to a derivative of the hydroxyl of a compound and an acid or acid derivative, wherein the product is cleaved in the body to give the compound or an active metabolite. Such a physiologically cleavable ester can be viewed as a "prodrug." Such a "prodrug" is valuable if it increases the bioavailability of the corresponding hydroxyl compound when such a pro-drug is administered to a subject. For example, a "prodrug" administered intranasally may be more readily absorbed into the blood, may facilitate the delivery of the parent compound to a biological compartment of the subject such as the brain or lymphatic, which may also have more favorable patient acceptance, safety profiles and / or pharmacokinetics for specific tailoring to subjects for use in the intended indication. A general overview of pro-drugs is provided in (1) "Pro-drugs As Novel Delivery Systems," Vol. 14 of the ACS Symposium Series, by T. Higuchi and V. Stella, and (2) "Bioreversible Carriers in Drug Design," American Pharmaceutical Association, Pergam on Press, 1987, Edward B. Roche, Ed.

[0056] "Prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host after administration to form the compound of the present disclosure. The present disclosure includes, within its scope, prodrugs of the compounds described herein. Such examples include, but are not limited to, choline ester derivatives and the like, N-alkylmorpholine esters and the like. Other derivatives of the compounds described herein have activity in both their acid and acid derivative forms, but in the acid sensitive form often offer advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see, Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well known to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters, amides, and anhydrides derived from acidic groups pendant on the compounds described herein are particular prodrugs. In some cases, it is desirable to prepare double ester type prodrugs such as (acyloxy)alkyl esters or ((alkoxy carbonyl)oxy)alkylesters. C1-C8 alkyl, C2- C8 alkenyl, C2-C8 alkynyl, aryl, C7-C12 substituted aryl, and C7-C12 arylalkyl esters of the compounds described herein may be preferred. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs" Ed. H. Bundgaard, Elsevier, 1985..

[0057] Pharmaceutical Compositions

[0058] The present disclosure provides a pharmaceutical composition, or medicament, for treating or preventing a migraine in a subject in need thereof. Such a pharmaceutical composition comprises one or more of the agents or substance described in, such as RNA molecules, polynucleotides, expression cassettes, expression vectors (e.g., viral vector genome, expression vector, rAAV vector), proteins, and small molecule compounds.

[0059] In some embodiments, the compositions of the disclosure are formulated with a pharmaceutically acceptable carrier. In alternative embodiments, the pharmaceutical compositions and formulations can be administered parenterally, topically, orally, or by local administration, such as by aerosol or transdermally. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa. (“Remington's”).

[0060] An agent can be administered alone or as a component of a pharmaceutical formulation (composition). The agent may be formulated for administration, in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0061] Formulations of the compositions of this disclosure may include those suitable for intradermal, inhalation, oral / nasal, topical, parenteral, rectal, and / or intravaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the agent which produces a therapeutic effect.

[0062] Pharmaceutical formulations of this disclosure can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.

[0063] Pharmaceutical formulations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores. Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen. Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

[0064] Aqueous suspensions can contain an active agent in admixture with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injections. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol monooleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity. In one embodiment, oil-based pharmaceuticals are used for administration of nucleic acid sequences or small molecule compounds disclosed herein. Oil-based suspensions can be formulated by suspending an active agent in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g., U.S. Pat. No. 5,716,928 describing using essential oils or essential oil components for increasing bioavailability and reducing inter- and intra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also U.S. Pat. No. 5,858,401). The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto et al.., J. Pharmacol. Exp. Ther. 1997 281 :93-102.

[0065] Pharmaceutical formulations of this disclosure can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent. In alternative embodiments, these injectable oil-in-water emulsions of the invention comprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitan monooleate and / or an ethoxylated sorbitan trioleate.

[0066] In practicing the method disclosed herein, the pharmaceutical compounds can also be administered by in intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see e.g., Rohatagi J. Clin. Pharmacol. 1995 35: 1187-1193; Tjwa etal.., Ann. Allergy Asthma Immunol. 1995 75: 107-111). Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug. Such materials are cocoa butter and polyethylene glycols.

[0067] In some embodiments, the pharmaceutical agent can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. In some embodiments, the pharmaceutical agent can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao J. Biomater Sci. Polym. Ed. 1995 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao Pharm. Res. 1995 12:857-863; or, as microspheres for oral administration, see, e.g., Eyles J. Pharm. Pharmacol. 1997 49:669-674.

[0068] In some embodiments, the pharmaceutical compounds can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or directly into a trigeminal ganglion. These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3- butanediol. The administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).

[0069] The pharmaceutical compounds and formulations of this disclosure can be lyophilized. The disclosure provides a stable lyophilized formulation comprising a composition of the disclosure, which can be made by lyophilizing a solution comprising a pharmaceutical of the disclosure and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof. The compositions and formulations of the disclosure can be delivered by the use of liposomes. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Pat. Nos. 6,063,400; 6,007,839; Al-Muhammed J. Microencapsul. 1996 13:293-306; Chonn Curr. Opin. Biotechnol. 1995 6:698-708; Ostro Am. J. Hosp. Pharm. 1989 46: 1576-1587.

[0070] The formulations of the disclosure can be administered for prophylactic and / or therapeutic treatments. In alternative embodiments, for therapeutic applications, compositions are administered to a subject in need of treating migraine or its complications.

[0071] The amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose. The dosage schedule and amounts effective for this use, z.e., the dosing regimen, will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.

[0072] The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, z.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones J. Steroid Biochem. Mol. Biol. 1996 58:611-617; Groning Pharmazie 199651 :337-341; Fotherby Contraception 1996 54:59-69; Johnson J. Pharm. Sci. 1995 84: 1144-1146; Rohatagi Pharmazie 1995 50:610-613; Brophy Eur. J. Clin. Pharmacol. 1983 24: 103-108; the latest Remington's, supra. The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, z.e., dose schedule and dosage levels, administered practicing the methods of the invention are correct and appropriate.

[0073] Single or multiple administrations of formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient. The formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms, e.g., migraine.

[0074] In some embodiments, pharmaceutical formulations for oral administration are in a daily amount of between about 1 to 100 or more mg per kilogram of body weight per day. Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a trigeminal ganglion. Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation. Actual methods for preparing parenterally or non-parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's, supra.

[0075] To aid in understanding the detailed description of the compositions and methods according to the disclosure, a few express definitions are provided to facilitate an unambiguous disclosure of the various aspects of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0076] As used herein, a “trigeminal ganglion” is the largest cranial ganglion that transmits sensory information from the jaws and face to the brain.

[0077] As used herein, the terms “reducing,” or “reduced” refers to a decrease in a measured amount, size, or number.

[0078] As used herein, a "subject" or "individual" means a human or animal. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, sheep, goats, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a human or a non -human mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disorders. The terms, "individual," "patient" and "subject" can be used interchangeably herein. A subject can be male or female. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental, non-human animal or animal suitable as a disease model.

[0079] A "subject in need" of treatment for a particular condition or disorder can be a subject having that condition or disorder, diagnosed as having that condition or disorder, or at risk of developing that condition or disorder.

[0080] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a migraine or a condition associated with a migraine. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a migraine or a condition associated with a migraine. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of the migraine is reduced. That is, "treatment" includes not just the improvement of symptoms or markers, but also a slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of a migraine, stabilized (i.e., not worsening) state of a migraine or a condition associated with a migraine, and / or delay or slowing of migraine progression. The term "treatment" of a migraine or a condition associated with a migraine also includes providing relief from the symptoms or side-effects of the migraine or condition. Symptoms that can be ameliorated or treated with the methods described herein, but are not limited to, vasomotor symptoms (e.g. hot flashes, facial flushing, sweating, and night sweats), photophobia (sensitivity to light), phonophobia (sensitivity to sound), sensitivity to smells, vertigo, dizziness, nausea, vomiting, and headache pain.

[0081] The terms “prevent,” “preventing,” “prevention” and the like are used interchangeably herein to mean inhibit, hinder, retard, reduce or otherwise delay the development of and / or progression of a migraine, a condition associated with a migraine or symptom thereof, in a subject. In the context of the present disclosure, the term “prevent” and variations thereof does not necessarily imply the complete prevention of the specified event. Rather, the prevention may be to an extent, and / or for a time, sufficient to produce the desired effect. Prevention may be inhibition, retardation, reduction or otherwise hindrance of the event, activity or function. Such preventative effects may be in magnitude and / or be temporal in nature.

[0082] As used herein, the term “migraine attack” refers to an episode of any migraine headache as defined herein. A migraine attack that is interrupted by sleep or temporarily remits and then recurs within 48 hours is considered to be a single attack. Similarly, a migraine attack that is successfully treated with acute migraine-specific medication but relapses within 48 hours is also considered to be a single attack. In some embodiments, the number of migraine attacks is reduced in the patient by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 75% following administration of an agent that that specifically binds to a receptor as described herein.

[0083] The terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0084] The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion. Alternatively, a therapeutic agent described herein can be administered via a non- parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically.

[0085] The term "antibody" as referred to herein includes whole antibodies and any antigenbinding fragment or single chains thereof. Whole antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CHI, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain variable region CDRs and FRs are HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, HFR4. The light chain variable region CDRs and FRs are LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, LFR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0086] Accordingly, the terms "antibody" and "antibodies" include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH regions or VL regions. Examples of antibodies include monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain-antibody heavy chain pair, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), scFv-Fcs, camelid antibodies (e.g., llama antibodies), camelized antibodies, affybodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti -Id antibodies), and antigen-binding fragments of any of the above. In certain embodiments, antibodies disclosed herein refer to polyclonal antibody populations. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgGi, IgG2, IgGs, IgG4, IgAi or IgA2), or any subclass (e.g., IgG2aor IgG2b) of immunoglobulin molecule. In certain embodiments, antibodies disclosed herein are IgG antibodies, or a class (e.g., human IgGi or IgG4) or subclass thereof. In a specific embodiment, the antibody is a humanized monoclonal antibody.

[0087] The term "antigen-binding fragment or portion" of an antibody (or simply "antibody fragment or portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment or portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is essentially an Fab with part of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3rded. 1993)); (iv) a Fd fragment consisting of the VH and CHI domains; (v) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (vi) a dAb fragment (Ward et al.., (1989) Nature 341 :544-546), which consists of a VH domain; (vii) an isolated CDR; and (viii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be j oined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv or scFv); see e.g., Bird et al.. (1988) Science 242:423-426; and Huston etal.. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment or portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0088] An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to a specific antigen is substantially free of antibodies that specifically bind antigens other than the specific antigen). An isolated antibody can be substantially free of other cellular material and / or chemicals.

[0089] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0090] The term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0091] The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0092] The term "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes. The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen."

[0093] The term "human antibody derivatives" refers to any modified form of the human antibody, e.g., a conjugate of the antibody and another agent or antibody. The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences.

[0094] The term "chimeric antibody" is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody. The term can also refer to an antibody in which its variable region sequence or CDR(s) is derived from one source (e.g., an IgAl antibody) and the constant region sequence or Fc is derived from a different source (e.g., a different antibody, such as an IgG, IgA2, IgD, IgE or IgM antibody). "Single chain antibodies" or "scFvs" are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding region. scFvs are discussed in detail in WO 88 / 01649 and U.S. Pat. No. 4,946,778 and No. 5,260,203, the disclosures of which are incorporated by reference.

[0095] A "domain antibody" or "single chain immunoglobulin" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. Examples of domain antibodies include Nanobodies®. In some instances, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.

[0096] As used herein, the term "antibody mimetic" refers to any molecule which mimics the function or effect of an antibody and which binds specifically and with high affinity to their molecular targets. In some embodiments, antibody mimetics may be monobodies, designed to incorporate the fibronectin type III domain (Fn3) as a protein scaffold (U.S. Pat. Nos. 6,673,901 and 6,348,584, the contents of each of which are herein incorporated by reference in their entirety). In some embodiments, antibody mimetics may include those known in the art including, but not limited to Adnectins, Affibodies, Affilins, Affimers, Affitins, Alphabodies, Anticalins, Aptamers, Armadillo repeat protein, based scaffolds, Atrimers, Avimers, Centyrins, DARPins, Fynomers, Knottins, Kunitz domain peptide, Monobodics, and Nanofitin. In other embodiments, antibody mimetics may include one or more non-peptide region.

[0097] "Antigenic agent," "antigen," or "immunogen" means a substance that induces a specific immune response in a host animal. It can be a molecule containing one or more epitopes (either linear, conformational or both) that elicit an immunological response. The term "epitope" refers to the basic element or smallest unit of recognition by an individual antibody, B-cell receptor, or T-cell receptor, and thus the particular domain, region or molecular structure to which said antibody or T-cell receptor binds. An antigen may consist of numerous epitopes while a hapten, typically, may possess few epitopes.

[0098] In general, an antigen binding protein or antibody is said to "specifically bind" its target antigen when the antigen binding protein exhibits essentially background binding to non-target molecules. An antigen binding protein that specifically binds to a target polypeptide may, however, cross-react with the polypeptides from different species or strains. Typically, an antigen binding protein specifically binds its target when the dissociation constant (KD) is < 10'7M as measured via a surface plasmon resonance technique (e.g., BIACORE, GE- HEALTHCARE, Uppsala, Sweden) or KINETIC EXCLUSION ASSAY (KINEXA, Sapidyne, Boise, Id.). An antigen binding protein specifically binds to its target with "high affinity" when the KD is < 5xl0'9M, and with "very high affinity" when the KD is < 5xlO'10, as measured using methods described.

[0099] As used herein, the term “affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0100] The term "epitope" as used herein refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term "epitope" also refers to a site on an antigen to which B and / or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and / or specific charge characteristics. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody (z.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immune-precipitation assays, wherein overlapping or contiguous peptides from a protein are tested for reactivity with a given antibody. Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g. , Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)). The term "epitope mapping" refers to the process of identification of the molecular determinants for antibody-antigen recognition.

[0101] The term "binds to an epitope" or "recognizes an epitope" with reference to an antibody or antibody fragment refers to continuous or discontinuous segments of amino acids within an antigen. Those of skill in the art understand that the terms do not necessarily mean that the antibody or antibody fragment is in direct contact with every amino acid within an epitope sequence.

[0102] An "effective amount" generally means an amount that provides the desired effect on treating a migraine or a condition associated with a migraine. For example, an effective amount is an amount sufficient to effectuate a beneficial or desired clinical result. The effective amounts can be provided all at once in a single administration or in fractional amounts that provide the effective amount in several administrations. The precise determination of what would be considered an effective amount may be based on factors individual to each subject, including their size, age, and severity of migraine being treated. One skilled in the art will be able to determine the effective amount for a given subject based on these considerations which are routine in the art. As used herein, "effective dose" means the same as "effective amount."

[0103] A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of the disease state, or reducing the likelihood of the onset (or reoccurrence) of the disease state or associated symptoms. The full therapeutic or prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

[0104] Generally, doses of the agent of the present disclosure would be from about 0.01 mg / kg per day to about 2000 mg / kg per day. In one embodiment, 0.01, 0.05, 0.1, 0.5, 1, 3, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1700, 1800, 1900, 2000 mg of an agent is administered to a subject. Effective doses range from about 0.01 mg / kg per day to about 2000 mg / kg per day, where the bottom of the range is any integer between 0.01 and 1999, and the top of the range is any integer between 0.02 and 1000. It is expected that doses ranging from about 5 to about 2000 mg / kg will be suitable — depending on the specific agent used. Lower doses will result from certain forms of administration, such as intravenous administration and pharmaceutical. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of a composition disclosed herein.

[0105] As used herein, the term “pharmaceutically acceptable carrier or excipient” refers to a carrier medium or an excipient which does not interfere with the effectiveness of the biological activity of the active ingredient(s) of the composition and which is not excessively toxic to the host at the concentrations at which it is administered. In the context of the present invention, a pharmaceutically acceptable carrier or excipient is suitable for topical formulation. The term includes, but is not limited to, a solvent, a stabilizer, a solubilizer, a tonicity enhancing agent, a structure-forming agent, a suspending agent, a dispersing agent, a chelating agent, an emulsifying agent, an anti-foaming agent, an ointment base, an emollient, a skin protecting agent, a gel-forming agent, a thickening agent, a pH adjusting agent, a preservative, a penetration enhancer, a complexing agent, a lubricant, a demulcent, a viscosity enhancer, a bioadhesive polymer, or a combination thereof. The use of such agents for the formulation of pharmaceutically active substances is well known in the art (see, for example, "Remington 's Pharmaceutical Sciences", E. W. Martin, 18th Ed., 1990, Mack Publishing Co.: Easton, PA, which is incorporated herein by reference in its entirety).

[0106] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (z.e., % homology=# of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the nonlimiting examples below.

[0107] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0108] The terms “substantial identity” and “substantially identical” indicate that a polypeptide or nucleic acid comprises a sequence with between 55-100% sequence identity to a reference sequence, with at least 55% sequence identity, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity or any percentage of value within the range of 55-100% sequence identity relative to the reference sequence. The percent sequence identity may occur over a specified comparison window. Optimal alignment may be ascertained or conducted using the homology alignment algorithm of Needleman and Wunsch, supra.

[0109] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[0110] As used herein, the terms “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted. As used herein, the phrases “in one embodiment,” “in various embodiments,” “in some embodiments,” and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may unless the context dictates otherwise.

[0111] As used herein, the terms “and / or” or “ / ” means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0112] As used herein, the word “substantially” does not exclude “completely,” e.g., a composition that is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

[0113] As used herein, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.

[0114] As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Unless indicated otherwise herein, the term “about” is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.

[0115] As disclosed herein, a number of ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0116] The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0117] All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. In regard to any of the methods provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, the steps may occur in any order, unless noted otherwise. In cases in which a method comprises a combination of steps, each and every combination or subcombination of the steps is encompassed within the scope of the disclosure, unless otherwise noted herein.

[0118] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent that it is not inconsistent with the present disclosure. Publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0119] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

[0120] EXAMPLES

[0121] Example 1. Materials and Methods

[0122] This Example describes the materials and methods used in Examples 2-10.

[0123] Animals

[0124] All experiments were approved by the University Committee on Animal Resources of the University of Rochester and the Danish Animal Experiments Inspectorate. Male C57BL / 6 mice (Charles River) were used as wild type mice. TRPVl-cre (or B6.129-TRPVltml(cre)Bbm / J) and Ai95d (or B6J.Cg-Gt(ROSA)26Sortm95 1(CAG'GCaMP6f)Hze / MwarJ) (Jackson Laboratories) were bred as homozygotes to produce offspring TRPVl-cre: GCaMP6F (het / het). Thyl-ChR2-YFP (or B6.Cg-Tg(Thyl-COP4 / EYFP)18Gfng / J) (Jackson Laboratories) were bred and used as homozygotes. In all experiments, unless otherwise noted, young animals of both genders were used. Animals were anesthetized with ketamine / xylazine intraperitoneal injection (100 mg / kg and 10 mg / kg). Procedures were performed in accordance with the European directive 2010 / 63ZEU, with due care to minimize the number of animals included in the study. Cerebrospinal fluid injections and cistema magna catheters

[0125] Tracers and drugs were delivered to cerebrospinal fluid (CSF) by a cisterna magna cannula as previously described (Xavier et al., Cannula Implantation into the Cisterna Magna of Rodents. J. Vis. Exp. (135): 57378 (2018)). A 30G needle connected to PE10 tubing was placed in cistema magna after surgical exposure and piercing of the atlantooccipital membrane. Tubing was connected to a syringe (Hamilton syringe GASTIGHT®, 1700 series, 1710TLL, volume 100 pL, PTFE Luer lock, Reno, NV, USA ) that was placed into a pump (LEGATO® 130 Syringe pump, KD Scientific, Holliston, MA, USA). 10 pl of a 1 % w / v dilution of fluorescent CSF tracer in artificial CSF (aCSF) was delivered at 2 pl / min.

[0126] HEPES buffered aCSF

[0127] A HEPES buffered aCSF with 160 mM NaCl, 6 mM KC1, 13 mM Glucose, 13 mM HEPES, 2.5 mM CaC12, 2.5 mM MgC12 was used. pH was adjusted with NaOH granules until a pH of 7.2 was reached.

[0128] GRIN lens implantation

[0129] Mice were anesthetized with ketamine / xylazine, placed on a heating pad set to 37 °C and head was tightly fixed in a stereotaxic surgery table. At predefined coordinates relative to lambda (Anterior: 2.62mm, Lateral: 1.76 mm, Depth: 5.85 mm) a GRIN lens (14mm long, 1mm diameter, Grintech GmbH) was inserted over the right trigeminal ganglion without penetrating the ganglion. The steps in the protocol by Resendez et al. were followed (Resendez et al., Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses. Nat. Protoc. 11, 566-597 (2016)). First, a craniotomy was made over right dorsal cortex of 4-5mm diameter. Then, 3-4 mm of cortex was carefully removed with a suction pipette attached to a light vacuum, while irrigating the tissue with HEPES buffered aCSF and carefully stopping any bleeding with gel foam (Pfizer). When all bleeding had stopped, the GRIN lens was inserted using a stereotaxic arm with a bulldog serrefine holding the lens, and the lens was inserted at a rate of 0.02mm / 10 seconds until above-mentioned depth was reached. The aspiration of tissue and slow insertion of the GRIN lens prevents tissue pressure and thus aid in reducing damage and inflammation from the procedure. The GRIN lens was then fixed to the skull with super glue, and a custom-built head plate was mounted around it, and dental cement was used to fix the headplate to the skull. Mice recovered for one hour, and were then transferred to a head holding device for imaging (MAG-1, Narishige).

[0130] Epifluorescence GRIN lens imaging of trigeminal CSF flow

[0131] TRPVl-cre:GCaMP6f animals with a GRIN lens implanted over right trigeminal ganglion and a cisterna magna catheter was imaged on a macroscope (Leica, M205FA) with a complementary metal oxide semiconductor (CMOS) camera (Hamamatsu, Orca-Flash 4.0, Cl 1440). 10 pL CSF tracer of 0.125 w / v % of 70 kDa tetramethylrhodamine dextran (Thermo Fisher Scientific) and 0.12 mg / ml capsaicin (Capsicum sp., M2028, Sigma Aldrich) was injected over 5 minutes. Capsaicin was diluted from stock solution (30 mg / ml in EtOH). 16bit 1024x1024 images were obtained interchangeably with standard GFP and mCherry filter cubes on the macroscope with 200ms exposure and 2x2 binning, resulting in a cycletime of 2.7 seconds / frame. A 3- minute baseline recording was performed without CSF tracer injection. Then, a 30- minute CSF injection recording was done where CSF tracer was injected during the first 5 minutes.

[0132] Epifluorescence GRIN lens imaging of CSD

[0133] TRPVl-cre:GCaMP6f animals with a GRIN lens implanted over right trigeminal ganglion and a cisterna magna catheter were imaged on a macroscope (Leica, M205FA) with a complementary metal oxide semiconductor (CMOS) camera (Hamamatsu, Orca-Flash 4.0, Cl 1440). A 5-minute baseline recording was done before CSD was inducted in a craniotomy over the right frontal cortex with IM KC1. The occurrence of CSD was verified with a laser doppler probe placed over the right middle cerebral artery. Imaging was performed with a standard GFP filtercube. Images were acquired at 16bit 1024x1024, 2x2 binning, exposure time of 190 ms, resulting in an acquisition rate of 5hz. Trigeminal ganglion imaging preparation

[0134] Mice were anesthetized with ketamine / xylazine, placed on a heating pad set to 37 °C and head was tightly fixed in a stereotaxic frame. A custom build headplate was mounted on the exposed skull with super glue and dental cement. After, a large craniotomy of 8mm diameter centered around bregma was made with a dental drill. The forebrain was then carefully removed following the surgical procedure published earlier (Ghitani et al., Specialized Mechanosensory Nociceptors Mediating Rapid Responses to Hair Pull. Neuron 95, 944-954. e4 (2017)). With a glass suction pipette attached to a light vacuum, brain tissue was removed while intermittently irrigating the tissue with HEPES buffered aCSF, until basis cranii with right and left trigeminal ganglions was exposed. Bleeding was stopped using gel foam. The mouse was then transferred to a head-holding device for imaging (MAG-1, Narishige). GCaMP6f epifluorescence imaging of trigeminal ganglion and topical application of fluids

[0135] Animals prepared for trigeminal ganglion imaging were placed under a macroscope (Leica, M205FA) with a complementary metal oxide semiconductor (CMOS) camera (Hamamatsu, Orca-Flash 4.0, Cl 1440). 1024x1024 images with no binning were obtained with a standard GFP filtercube, 190ms exposure time, resulting in a framerate of 5hz. Imaging was done in 2-minute epochs, where 5 pL of either aCSF, various endogenous CSF (CSD CSF, Naive CSF, CSD CSF 2.5h), or aCSF with O. lmM calcitonin Gene related peptide (CGRP, C0292, Sigma Aldrich) was applied with a micropipette at the root portion of the visible part of the right trigeminal ganglion. In between epochs the ganglion was rinsed with aCSF and excess fluid was drained away with a tissue (Kimwipes).

[0136] AAV injections

[0137] Cortex was transfected with CAG-GFP-P2A-Igk-mCherry AAV which resulted in transfected cells expressing a cytosol-bound GFP and a secreted IgK-mCherry, thus allowing for identification of the anatomical pathway of secreted molecules from the transfected cells. Right dorsal cortex was transfected. Animals were anesthetized with isoflurane in atmospheric air and placed in a surgical stereotax and a central craniotomy in the right parietal bone (2mm diameter) was made with a dental drill. A pulled glass pipette attached to a syringe (10 pL Hamilton™ 700 / 1700 Series Microliter / Gastight™ Syringes) was inserted 1mm deep into cortex and a 3 pl 1 : 1 dilution of virus in saline was injected over 10 minutes (300 nl / min). After injection was done, the needle was left in the tissue for 10 minutes, before being removed slowly over 10 minutes to minimize the risk of any backflush of virus.

[0138] Cerebrospinal fluid drainage

[0139] Mice were anesthetized with ketamine / xylazine, placed on a heating pad set to 37 °C and head was tightly fixed in a stereotaxic frame. Patients with migraine with aura develop headache between 5-60 minutes after CSD, and the production of certain inflammatory mediators of CSD also increases at various time points some of them within the 1st hour (Takizawa et al., Noninvasively triggered spreading depolarizations induce a rapid pro- inflammatory response in cerebral cortex. J. Cereb. Blood Flow Metab. 40, 1117-1131 (2020)). To ensure that proteins were collected that are putative headache triggers, the aim was to collect proteins both released immediately after CSD as well as those released in a short timespan after CSD. Thus, a paradigm was performed where 6 CSDs were induced over Ih. For animals in the Ih CSD group, 6 CSDs were provoked with IM KC1 in a right frontal bone craniotomy spaced 10 minutes apart. KC1 was washed away with aCSF as soon as CSD was detected. CSF was drained 10 minutes after last CSD. For the 2.5 h CSD group, 6 CSDs were provoked over Ih, but CSF was only drained 1.5 hours after the last CSD (2.5 hours after the first). When animals were ready for CSF drainage the atlantooccipital membrane was exposed surgically. With a 30G needle a puncture into the underlying cerebrospinal fluid chamber called Cistema Magna was made. Using a pipette CSF was drained over 15- 20 minutes until a volume of 20- 30 pL was obtained. CSF samples were placed in 0.5 mL tubes (Eppendorf) and stored at -80 °C until use.

[0140] Cortical CSF influx during cortical spreading depression epifluorescence imaging

[0141] Thyl-ChR2 animals received a cistema magna catheter and dorsal skull was exposed before they were placed under an epifluorescence macroscope. A laser optic probe (Thorlabs) was placed over the right frontal bone, and a laser doppler probe was placed over the right middle cerebral artery. 10 pL of 2000 kDa tetramethylrhodamine dextran 1% w / v in aCSF was injected during the first 5 minutes of a 15-minute baseline recording. After 15 minutes, cortical spreading depression was evoked with laser light (MBL-FN-473-200mW, CNI laser) with output set to 5, and applied through the skull (473 nm, 10 Hz (50 ms)). Stimulation periods were 3 seconds in total, except for one animal where CSD was not evoked at first attempt, but after a 6 second stimulation period CSD was successfully evoked. Occurrence of CSD was monitored with the hemodynamic response in the right MCA observed by the laser doppler probe. CSF tracer was delivered before CSD induction to allow the tracer to distribute around the circle of Willis so that the CSD-induced vasoconstriction can drive CSF tracer up along the cerebral arteries. Dispersion of tracer within cortex after CSD is reduced as shown by Schain et al. (Schain et al., Cortical Spreading Depression Closes Paravascular Space and Impairs Glymphatic Flow: Implications for Migraine Headache. J. Neurosci. 37, 2904-2915 (2017)), likely reflecting that the increase in brain water content transiently suppress CSD as shown in Du etal. (Du etal., Cerebrospinal fluid is a significant fluid source for anoxic cerebral oedema. Brain 145, 787-797 (2022).

[0142] Cortical extracellular tracer epifluorescence imaging

[0143] Wild type animals received two craniotomies, one over the right parietal bone of 4mm diameter, and one over the right frontal bone of 2mm diameter. A pulled glass pipette attached to a syringe (10 pL Hamilton™ 700 / 1700 Series Microliter / Gastight™ Syringes) was inserted 1mm deep into cortex in the parietal bone craniotomy and 1.5 pl of a 1% w / v 3 kDa FITC dextran in aCSF was injected over 15 minutes. Animals were then moved under a macroscope (Leica, M205FA) with a complementary metal oxide semiconductor (CMOS) camera (Hamamatsu, Orca-Flash 4.0, Cl 1440). Images were done with 200ms exposure, 1024x1024 and 16bits, at 1 frame / minute. A 10-minute baseline recording was done for all groups before a Ih long recording was done where one group of animals was exposed to IM KC1 in the frontal bone craniotomy and the control group received aCSF. Occurrence of CSD was measured with laser doppler flow through an optic probe placed over the right middle cerebral artery.

[0144] Cortical CSF influx monitoring after cortical spreading depression CE-MRI

[0145] C57BL6 animals received a cistema magna cannula (a 30G copper cannula; outer diameter 0.32 mm; Nippon Tokushukan, Mfg, Tokyo, Japan) and dorsal skull was exposed under ketamine / dexmedetomidine anesthesia (i.p. K / Dex: 75 / 1 mg / kg). The animals received a craniotomy over the right frontal bone of 2mm diameter and topical aCSF was applied to the craniotomy. Animals were then moved into the MRI scanner. MRI was performed in a 9.4 T preclinical scanner (BioSpec 94 / 30 USR, Paravision 6.0.1 software, Bruker BioSpin, Ettlingen, Germany) equipped with a IH volume Tx / Rx coil (40 mm inner diameter, Bruker) and a 240 mT / m gradient coil (BGA-12S, Bruker). A T2-weighted structural image was conducted using 3D constructive interference steady-state (3D-CISS). Each 3D-CISS image was calculated as a maximum intensity projection of 4 realigned 3D TrueFISP volumes with 4 orthogonal phase encoding directions (TR / TE 3.5 / 1.75 ms, Nex 1, FA 50°, FOV 19.2 x 19.2 x 16 mm, Matrix 192 x 192 x 160). For dynamic contrast-enhanced MRI (DCE-MRI), T1 -weighted imaging was acquired using a 3D-FLASH sequence (TR / TE 5 / 1.4 ms, Nex 1, FA 10°, FOV 19.2 x 19.2 x 16 mm, Matrix 96 x 96 x 80). First, three baseline DCE-MRI scans (3 min) was acquired. Then IM KC1 was applied in the frontal bone craniotomy and left for 2 minutes, before it was washed away with aCSF. Ten minutes after the KC1 administration, Tl-enhancing contrast agent gadobutrol (15mM; Gadovist, Bayer Pharma AG, Leverkusen, Germany) was injected into cistema magna (1 pL / min for 10 min). The follow-up scans continued over 120 minutes (1 scan / min).

[0146] Implantation of visual cortex cannula

[0147] Isoflurane anesthetized wild-type mice (3% under induction, 1-2% for maintenance) were fixed on a stereotaxic frame. The skin was incised exposing the skull. A small burr hole was drilled at AP: -3.0mm; ML -3.0mm relative to bregma and a 30G needle was used to ensure breakage of meningeal layers. The guide cannula (C315GS-4 / SP GUIDE 26GA 38834, 4.5MM below PED) with its dummy (C315DCNS-4 / SPC DUMM .008 (NYLON) FIT 4MM PED, PlasticsOne, Roanoke, VA) was then inserted at DV - 1 ,7mm (visual cortex) relative to bregma. The cannula was secured using dental cement (AgnThos,7508) and the incision was closed. The animals recovered for 2 days before CE-MRI scanning. Visual cortex drainage pathways CE-MRI scanning

[0148] Animals were anesthetized with ketamine / dexmedetomidine (i.p. K / Dex: 75 / 1 mg / kg) and were placed in the prone position in the MRI scanner. The dummy cannula was replaced by an inner cannula (33G, C315ESP, 0.1 mm projection), connected to PEI 0 tubing filled with contrast agent (gadobutrol, 20 mM; Gadovist, Bayer Pharma AG, Leverkusen, Germany), and connected to a gastight syringe in a microinfusion pump. T2-weighted structural reference image was conducted using 3D constructive interference steady-state (3D-CISS). Each 3D- CISS image was calculated as a maximum intensity projection of 2 realigned 3D TrueFISP volumes with 2 phase encoding directions (TR / TE 3.5 / 1.75 ms, Nex 1, FA 50°, FOV 19.2 x 19.2 x 16 mm, Matrix 192 x 192 x 160). Whole mouse brain pre- and post-contrast Tl- weighted DCEMRI was acquired with 1 min temporal and 200 pm isotropic spatial resolutions using 3D-FLASH sequence (TR / TE 4.8 / 1.4 ms, Nex 1, FA 10°, FOV 19.2 x 19.2 x 16 mm, Mtx 96 x 96 x 80), and a 1H volume Tx / Rx (40 mm inner diameter, Bruker) and a 240 mT / m gradient coils (BGA-12S, Bruker). DCE-MRI continued over 180 minutes (1 scan / min), and T1 -enhancing contrast agent was infused into the right visual cortex (0.1 pL / min for 10 min) after the first three baseline scans (i.e., after 3 min). After DCEMRI, contrast enhanced MR venography (CE-MRV; 3D-FLASH: TR / TE 3.6 / 1.8 ms, Nex 1, FA 15°, FOV 19.2 x 19.2 x 16 mm, Matrix 192 x 192 x 160, 100 mM gadobutrol, infusion in a step-down manner following the procedure published earlier in Ku et al., Assessment of Blood Brain Barrier Leakage with Gadolinium -Enhanced MRI. Methods Mol. Biol. 1718, 395-408 (2018) was used for the venous sinus visualization and segmentation.

[0149] Cortical spreading depression

[0150] In all animals except Thyl-ChR2 animals CSD was evoked by application of IM KC1 in a frontal bone craniotomy of 2-3mm diameter. The occurrence of CSD was observed by monitoring the blood flow in the ipsilateral middle cerebral artery with a laser doppler probe. When the first sign of CSD associated hemodynamic response was observed, the craniotomy was washed with aCSF to limit the exposure to dura of KC1. Histology and immunohistochemistry of basis cranii sections

[0151] Wildtype mice and CAG-GFP-P2A-Igk-mCherry AAV transfected mice were transcardially perfused with first phosphate buffered saline (PBS, pH 7.4, SigmaAldrich, MO, USA), and then 4 % paraformaldehyde (PF A, Sigma-Aldrich) in PBS. Basis cranii sections of TG in situ was then prepared following the protocol described by Ahn et al., Meningeal lymphatic vessels at the skull base drain cerebrospinal fluid. Nature 572, 62-66 (2019). Basis cranii was left in EDTA 0.5 M, pH 7,4 overnight. Tissue was sectioned in 50 pm sections on a cryostat (Cryocut microtome, Leica) without prior dehydration and placed on glass slides. After PBS washes, tissue was left in blocking solution for 2h at RT (3 % NGS, 0.1 % Triton, 1 % BSA, 0.05 % Tween20 in PBS), and then left with primary antibodies overnight at RT. All primary antibodies were diluted 1 :200 in antibody solution ( 1% BSA and 0.1% Triton X-100 in PBS). Next day, tissue was washed in PBS again, and secondary antibodies coupled to fluorophores were diluted 1 : 1000 in the antibody solution and left for 2h at RT. After a final PBS wash slides were mounted with ProLong™ Gold Antifade Reagent (Invitrogen / Thermo Fisher Scientific, Carlsbad, CA, USA). The slides were imaged on a confocal microscope (Nikon Eclipse Ti, Tokyo, Japan) using Plan Fluor 20X / 0.75, 40X / 1.30 and 60x / 1.4 oil objectives. The primary and secondary antibodies used were the following: anti-myelin basic protein MBP), anti-neurofilament heavy chain (NFH), anti-microtubule associated protein, anti-mCherry, biotinylated Ib4 and streptavidin to label vasculature.

[0152] Histology and immunohistochemistry of trigeminal root sections

[0153] Wildtype and PROX-1 GFP mice were perfused as described above. Whole heads were decalcified over three weeks with 10% EDTA in Tris buffer (pH 7) at RT, prior to paraffin embedding and serial sectioning. Sections were cut on a microtome. Protocol for immunohistochemistry was as described above. The primary and secondary antibodies used were the following: anti-aquaporin-1 (AQP1) to label dura mater as described in Weller etal., The meninges as barriers and facilitators for the movement of fluid, cells and pathogens related to the rodent and human CNS. ActaNeuropathol. 135, 363-385 (2018), anti-claudin 11 to label the arachnoid barrier membrane as described in Brochner et al., The meninges as barriers and facilitators for the movement of fluid, cells and pathogens related to the rodent and human CNS. Acta Neuropathol. 135, 363-385 (2018), anti-GFP, anti-CRABP2, anti PDPN to label SLYM and pia mater as described in Mollgard et al., A mesothelium divides the subarachnoid space into functional compartments. Science 379, 84-88 (2023), and anti-aquaporin 4 (anti- AQP4) to label the central nervous system. The images presented in figure 2 are representative of 6 animals / 12 trigeminal ganglions (n=6, N=12). Besides immunohistochemistry paraffin sections were counterstained with Mayer’s hematoxylin, dehydrated in graded alcohols, and cover-slipped with Pertex mounting medium. All slides were imaged with bright field microscopy.

[0154] A healthy male volunteer of 35 years of age was used for example images of the trigeminal ganglion. Magnetic resonance images were acquired on a 3T General Electric Signa Premier MRI scanner. Images were done with isotropic 3D T2 fast spin echo sequences. Potassium concentration of CSF

[0155] For [K ]e analysis, a cation chromatography system (Dionex 1100, Thermo Scientific™) consisting of a CS12A 4 mm analytical and a Dionex lonPac CG12A 4 mm guard column set. 34 mmol / L MSA was used as eluent and was sonicated for 20 min followed by degassing with nitrogen for an additional 10 min prior to IC. The sample was eluted for 20 min with isocratic 34 mmol / L methanesulfonic acid. [K+] was determined from standard curves. Unknown sample [K+] concentrations were determined based on the linear fit. Software used was Thermo Scientific™ Chromeleon™ Chromatography Data System software version 7.2. Protein digestion and Evotips loading of CSF

[0156] Sample preparation was performed on an Agilent Bravo Liquid Handling Platform and the protocol was optimized based on the previously published methods described in Bader et al. (Bader et al. Proteome profiling in cerebrospinal fluid reveals novel biomarkers of Alzheimer’s disease. Mol. Syst. Biol. 16, e9356 (2020); Geyer et al., Proteomics reveals the effects of sustained weight loss on the human plasma proteome. Mol. Syst. Biol. 12, 901 (2016)). Briefly, CSF samples were aliquoted into a 96-well format plate and introduced to the Bravo Robot. 20 pl CSF sample was mixed with 30 pl PreOmics Lysis buffer (P.O. 00001, PreOmics GmbH) and incubated at 95 °C for 10 min in order to denature proteins, reduce disulfide bridges and alkylate cysteines (Kulak etal., Minimal, encapsulated proteomic- sample processing applied to copy-number estimation in eukaryotic cells. Nat. Methods 11, 319-324 (2014)). After cooling the sample for 15 min at room temperature, trypsin and LysC were added in a ratio of 1 pg enzyme to 100 pg proteins and the mixture incubated at 37°C for 4 h. The peptides mixtures were diluted in 100 pl 99% isopropanol, 1% TFA and desalted using two- gauge SDB-RPS stage-tips. Afterwards, the stage-tips were washed using 200 pl 99% isopropanol, 1% TFA, followed by 200 pl 0.2%TFA. The purified peptides were eluted using 80% acetonitrile (ACN) containing 1% ammonia and subsequently dried down. Peptides were resuspended in solvent A and loaded onto Evotips (Evosep Biosystem, Denmark) according to the manufacturer’s recommendations. The Evotips were wetted with isopropanol for 5 min, activated with 20 pl solvent B (99%ACN, 0.1% FA) and centrifuged at 700xg for 1 min. 20 pl of buffer A was then added to equilibrate the tips followed by sample loading. Finally, 20 pl Buffer A was used to wash the Evotip and 100 pl was added to avoid drying.

[0157] Liquid chromatography and mass spectrometry analysis

[0158] The samples were injected to an Exploris 480 Thermo Fischer Scientific system using an Evosep One instrument (Evosep Biosystem). A preset chromatographic method was used corresponding to 60 samples per day. The peptides were separated on an 8 cm Pepsep (Marslev, Denmark) column (150 m ID 1.5 pm bead size Reprosil -Pur Cl 8 beads ) at 1 pL / min flow rate with a 21 min gradient. The heated capillary temperature was set to 275, the spray voltage to 2300 V and the funnel radiofrequency to 40. The mass spectrometer was operated in a data- independent mode (DIA) with a full MS range from 350 to 1650 at a resolution of 60000 at 200 m / z. The AGC target was set to 300 % with an injection time of 50 ms. The AGC value of the targeted MS2 experiment was set to 1000%. Twenty -two windows of variable sizes were defined for target MS2 (tMS2) acquisition and subj ected to high-energy collisional dissociation (HCD) fragmentation with a normalized collision energy at 30%. Each tMS2 scan was acquired at a resolution of 30000 with a maximum ion injection time (IT) of 28 ms for a scan range of m / z 349.5 to 1650.5.

[0159] Mass spectrometry data analysis

[0160] The MS raw files were processed with Spectronaut version 15 (Biognosys, Zurich, Switzerland). A previously generated CSF spectral library was imported from (MaxQuant software analyses). The library contained 2733 protein groups and 17301 peptides. DIA files were searched against the library using default parameters except for the normalization which was set to local. Dynamic mass and retention time tolerances (for both MSI and MS2) were applied. Qvalue cutoff was set to 1% both at precursor and protein level using a mutated decoy method (Bruderer et al., Optimization of Experimental Parameters in Datalndependent Mass Spectrometry Significantly Increases Depth and Reproducibility of Results. Mol. Cell. Proteomics 16, 2296-2309 (2017)). The calibration was performed based on a local regression model (Callister et al., Normalization approaches for removing systematic biases associated with mass spectrometry and label-free proteomics. J. Proteome Res. 5, 277-286 (2006)). GCaMP imaging analysis

[0161] Images were transformed to .tiff and imported to Imaged, and stacks were aligned using a custom script. Cell region of interests (ROIs) was drawn around the cell perimeter with a semiautomatic segmentation tool in Imaged. ROIs were chosen if the cell was displaying activity in any of the experimental setups. Mean pixel intensity (MPI) of each ROI in each stimuli condition was exported to .csv file format and uploaded to a custom python script. GCaMP AF / F0 was calculated with Fo set to the mean of the 10 frames with the lowest MPI over the experiment. Quality control was performed eliminating ROIs with spontaneous activity during baseline and cells with a mean GCaMP AF / Fo outside the interquartile range. Area under the curve (AUC) was calculated relative to the length of the recording. AUC was then normalized to baseline mean. Lastly, peaks in GCaMP signals were also counted using a custom peak tracing algorithm on the AF / Fo trace, and the cumulative number of cells with peaks was compared between conditions. Active cells were then defined as cells where at least one peak was detected by the peak tracing algorithm during baseline or CSD recording.

[0162] MRI processing and analysis

[0163] All acquired MR images were visually inspected and no significant artifacts affecting morphological and functional assessment were found. Further processing pipelines were applied, including motion correction, bias field correction, spatial co-registration, and automatic or semi-automatic pre- and post-processing. For each animal, the coregistration process of 3 different multi-resolution images (CISS, MRA, DCE) was repeated twice using rigid body (6 df.) to ensure accuracy. The fixed image for the registration was 3D-T0F-MRA, therefore the final resolution of all 3 images was 65 pm x 67 pm x 65 pm. For monitoring cortical CSF influx after CSD and visual cortex drainage pathways, the fixed image for the registration was 3D-CISS, and therefore the final resolution of DCE-MRI and CE-MRV was 100 pm x 100 pm x 100 pm. For 3D-CISS, all acquired 3D-TrueFISP volumes were motion corrected prior to calculation as MIP, and the image bias field was removed with Advanced Normalization Tools (ANTs) (Avants etal., The Insight ToolKit image registration framework. Front. Neuroinform. 8, 44 (2014); Tustison etal., N4ITK: Improved N3 bias correction. IEEE Trans. Med. Imaging 29, 1310-1320 (2010)), resulting in an image of removed banding artifacts. Time-series of acquired 3D-FISP or 3D-FLASH volumes were motion corrected and spatially normalized with ANTs and co-registered to the respective baseline image on a subjectwise basis. To normalize the image signal in each time-series their voxel intensities were subjected to Gaussian normalization using the first 3D-FISP volume or 3DFLASH. The resulting images were smoothed with a 3 x 3 x 3 voxel kernel of [0.2, 1,0.2] weights along each axis, to reduce the influence of possible artifacts after the automatic registration and subtracting the baseline volume. Voxel-based percentage enhancement of contrast from baseline (signal enhancement ratio, SER) was calculated as equation of SER = (St-So) / Sox100. The ROI segmentation and SER measurements were performed in Analytics Preclinical software (ver. 3.1.1.0, GremselT GmbH, Aachen, Germany) (Gremse et al., Analytics Preclinical: Interactive Analysis of Biomedical Volume Data. Theranostics 6, 328- 341 (2016)). The tracer arrival time of the contrast agent in the different ROIs was defined as the initial time that the SER was greater than 2.5%. AUC of the SER linegraphs was calculated using the trapz function in the python module numpy.

[0164] CSF tracer cortical influx during CSD analysis

[0165] Images were transformed to .tiff and imported to Imaged, and ROIs were created for left and right hemisphere, and MPI for each ROI for each time frame was exported to .csv and loaded into a custom python script. In each animal all recordings were normalized to the end- of-baseline MPI to ensure that the relative changes instigated by CSD could be compared between animals. For comparison of AMPI during CSD and baseline, the MPI increase from CSD induction and 10 minutes after, was compared to a 10-minute-long baseline recording after the infusion pump had delivered the tracer to cisterna magna. To measure the influx rate (AMPI / min) the derivative of the MPI traces from CSD and baseline recordings were calculated with a custom python script, and the peak in influx rate in the CSD exposed right hemisphere ROI was detected with a signal peak algorithm and the width of the peak was calculated. Comparisons of influx rate were done between baseline recording, the CSD recording during the detected peak, and in the CSD recording after the detected peak.

[0166] Cortical extracellular tracer during CSD analysis

[0167] Images were transformed to .tiff and imported to Imaged, and ROIs were created for left and right hemisphere, and MPI for each ROI for each time frame was exported to .csv and loaded into a custom python script. In each animal all recordings were normalized to the MPI of the first frame to ensure that changes in subsequent changes in MPI could be compared between animals. Comparisons between CSD animals and control animals were made by calculating the percentage difference in start MPI and MPI after Ih.

[0168] Diffusion model

[0169] Diffusion along the line of regions of interest into the nerve extracellular spaces can be described by modified diffusion law based on Fick’s law of diffusion, de = D* d2c dt dx2, where c is tracer concentration, t is time, x is distance to the CSF space and D* the effective diffusion coefficient (assuming constant diffusivity, and with d representing the partial derivative) (Sykova et al., Diffusion in brain extracellular space. Physiol. Rev. 88, 1277-1340 (2008)). The effective diffusion is related to the free Stokes-Einstein diffusion coefficient D of the tracer, the extracellular volume fraction a and the tortuosity of the extracellular space A according to

[0170] D* = aD.

[0171] A2, where A is typically measured around A = 1.5 ± 0.2 and a = 0.23 ± 0.02 for the anesthetized brain state (the latter being included due to the MRI measurements including intracellular spaces) (Sykova et al., Diffusion in brain extracellular space. Physiol. Rev. 88, 1277-1340 (2008); Xie et al., Sleep drives metabolite clearance from the adult brain. Science 342, 373- 377 (2013)). The concentration at the interface with CSF (where x=0) varies in time as h(t), while no tracer reaches the far end of the line of regions of interest, implying CH> 0 when XH> co. With these boundary conditions the evolution of tracer concentration is governed by the known solution (Wikipedia: en.wikipedia.org / wiki / Heat_equation#Homogeneous_heat_equation)

[0172] Note that the diffusion model and its effective parameter is independent of concentration scale but not translation, requiring the assumption that the (background-subtracted) pixel intensities cdata are proportional to tracer concentration. A root-mean-square prediction error is used for this model against the data defined by where NROI and NT are the number of regions of interest along the line and number of observation timepoints respectively, and the subscripts i and j index each of these respectively. To compute c(x,t) and find the error-minimizing D*, first interpolate the boundary concentrations h(t) from the data, integrate using quadrature, and minimize using the Golden section search technique, all using the scipy library. All code for these steps and visual plotting is available with the DOI: 10.5281 / zenodo.10844173.

[0173] Comparison with measured diffusion coefficients

[0174] The molecule Gd-DTPA (550 kDa) is similar to gadobutrol (605 kDa) and was measured by Gordon et al. using an MRI-based method to have an effective diffusion coefficient of acdD*Gd = 2.6 * 1010± 0.3 m2s1= 0.936 ± 0.1 mm2h1(Gordon et al., Measurement of Gd-DTPA diffusion through PVA hydrogel using a novel magnetic resonance imaging method. Biotechnol. Bioeng. 65, 459-467 (1999)). Their 10% hydrogel corresponds to acd = 0.9. To compare with the estimates of gadobutrol in the trigeminal nerve, the tortuosity and volume space fraction (A = 1.5 ± 0.2 and a = 0.23 ± 0.02) are used from Xie et al., Sleep drives metabolite clearance from the adult brain. Science 342, 373-377 (2013). The resulting expected distribution of the effective diffusion coefficient aligns with the average estimate, though there is a much greater variance with the method described herein. This is likely due to the reduced spatial and temporal resolution of the MR images deep in the tissue.

[0175] Cortex, CSF proteome and trigeminal ganglion interaction analysis

[0176] Single cell RNA sequencing (SC RNA seq) data from public repositories were used for analysis. Sensory cortex RNA sequence data were published by Zeisel et al. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 1138-1142 (2015), and trigeminal ganglion sequencing by Yang et al. Human and mouse trigeminal ganglia cell atlas implicates multiple cell types in migraine. Neuron 110, 1806-1821. e8 (2022). For methodological details, see the original publications. Processed data was used from Zeisel et al. and the raw data was obtained from Yang et al. In the raw data set, quality control was performed as per Heumos et al., see (sc-bestpractices.org / preprocessing_visualization / quality_control.html) (Heumos etal., Single-cell Best Practices Consortium, H. B. Schiller, F. J. Theis, Best practices for single-cell analysis across modalities. Nat. Rev. Genet. 24, 550-572 (2023).Next a ligandreceptor database was manually curated using the Omnipath database of Ttirei et al. OmniPath: Guidelines and gateway for literature-curated signaling pathway resources. Nat. Methods 13, 966-967 (2016). Ligand-receptor pairs were included in the database if the interaction was categorized as “resource specific,” and if the number of references describing the interaction was above 4. This generated a database of 1214 ligandreceptor interactions. Genes identified in the CSF proteome were then matched to ligands in the database, and upon a match, the receptor gene was searched in the trigeminal ganglion SC RNA seq dataset. This resulted in 186 ligand-receptor pairs between the CSF and trigeminal ganglion. Among these 186 ligand-receptor pairs, CSF proteins significantly elevated after CSD were identified, as were CSF proteins significantly elevated more than 2-fold. The RNA level mean of the identified receptor pairs were then plotted using Scanpy (Wolf et al., SCANPY: Large-scale single-cell gene expression data analysis. Genome Biol. 19, 15 (2018)) across the annotated trigeminal cell clusters described in the original publication by Yang et al. RNA level mean of the identified CSF ligands in the cortical SC RNA seq data by Zeisel et al. was plotted in a similar way. Network Sankey plots were made based on protein ligandreceptor pairs using the Sankey plot function in the python module Plotly. The plots show relative production of CSF proteins in cortical cells, and receptor proteins in trigeminal cells, as estimated by RNA level mean pr cluster. To simplify the figure, cell clusters that produced less than 10 % of the total RNA of each protein were excluded. Gene ontology enrichment analysis of protein groups was done using the python module GOATOOLS (Klopfenstein et al., GOATOOLS: A Python library for Gene Ontology analyses. Sci. Rep. 8, 10872 (2018)). Table 5. Antibodies.

[0177] Example 2. Cerebrospinal fluid has direct access to the trigeminal ganglion.

[0178] Studies of CSF egress pathways have earlier documented that CSF efflux along cranial nerve sheaths (22-25). Extracranial nerves have a blood-nerve barrier composed of tight junctions in the nerve sheath, which blocks external solutes from entering the extracellular space freely; however, little is known about the barriers that serve the border zones between CNS and PNS. To investigate whether CSF around the trigeminal ganglion freely enters the extracellular space in the living animal, a novel preparation was developed for trigeminal ganglion imaging in vivo. The trigeminal ganglion is situated beneath the brain at the skull base, making optical imaging of the ganglion difficult. This problem was circumvented by identifying the coordinates of the ganglion, and then inserting a GRIN lens over the right trigeminal ganglion. This was done in TRPVl-cre:GCaMP6f mice, which express the calcium sensitive fluorophore GcaMP6f in several classes of sensory neurons including low-threshold mechanoreceptors, AS and C fibers which has previously been implicated in migraine (13, 26- 28). With this preparation, CSF inflow in the ganglion can be imaged simultaneously with sensory nerve activity. Capsaicin, an agonist of the TRPV1 ion-channel, was then injected into the CSF compartment together with a 70 kDa fluorescent dextran tracer to verify that CSF borne solutes can reach and activate trigeminal cells. Real-time imaging revealed that the CSF tracer appeared in the ganglion 3.8+0.4 min after injection, and that CSF capsaicin drove a sharp increase in calcium activity in the ganglion at 5.80+2.04 min (FIGS. 1A-1D). It was next validated that this increase in calcium activity occurred due to CSF flow in the trigeminal ganglion, using contrast enhanced-magnetic resonance imaging (CE-MRI), which showed that CSF contrast agent levels (gadobutrol, 0.6 kDa) were 8.7 times higher in the trigeminal ganglion CSF cisterns than at dural CSF egress sites. Thus, CSF transports solutes directly into the trigeminal ganglion, and activate receptors on trigeminal cells. An immunohistochemical analysis of animals injected with the same 70 kDa fluorescent dextran into the CSF in cistema magna revealed that CSF disperses through all compartments of the ganglion, including the nerve sheath and connective tissue (collagen- 1 positive), and surrounding both neuronal somata (identified by microtubule associated protein-2 immunoreactivity), and axons (immunolabeled with neurofilament heavy chain and myelin basic protein) (Figure ID). To confirm that solutes produced in the brain would also reach the trigeminal ganglion, cells in the dorsal cortex were transfected with an AAV driving the expression of a secretable fluorescent protein (mCherry- IgK, 40 kDa). Subsequent histological analysis revealed that cortically produced protein finds its way to the trigeminal ganglion . Thus, CSF transport comprises a humoral signaling pathway between the brain and the trigeminal ganglion; as such, this flow route effectively permits fluid- borne communication between the central and peripheral nervous systems.

[0179] Example 3. MRI tracer in CSF rapidly enters the root of the trigeminal nerve.

[0180] To delineate - in a minimally invasive way - the flow pathways by which CSF enters the trigeminal ganglion, CE-MRI was used. In live mice, anatomical Ti and T2-weighted scans and time-of-flight angiography revealed close apposition of the trigeminal ganglion with the basal cisterns and basal cerebral arteries. The trigeminal ganglion was directly in contact with CSF in all scanned animals. Similarly, MRI scans of the human trigeminal ganglion and nerve shows that in humans the ganglion sits in a CSF filled cave, and that its sensory rootlets are bathed in CSF, as in mice.

[0181] When a contrast agent (gadobutrol, 0.6 kDa) was injected into the mouse CSF via the cisterna magna, the signal increased in the ganglion immediately. The highest tracer concentrations were found in the proximal part of the ganglion. A multiple linear regression analysis revealed that the tracer concentration in the proximal ganglion was positively associated with CSF cistern volume, and inversely related to its distance from the surrounding CSF cisterns, consistent with tracer influx from the CSF cisterns into the ganglion. This association was not found in the distal end, pointing towards that CSF influx occurs in the proximal ganglion. A diffusion model based on the boundary CSF tracer concentrations determined a diffusion coefficient of 0.13 mm2 / h ±0.02 (SEM) of CSF influx from the surrounding cisterns into the ganglion, analogous to tracer diffusion coefficients measured in the brain extracellular space (Sykova etal. Diffusion in brain extracellular space. Physiological reviews 88, 1277-1340 (2008)). However, in four of eight ganglia the diffusion models underestimated the rate of influx, suggesting that other factors, such as convection, may contribute to influx. As noted, the trigeminal ganglion lies in close apposition to the circle of Willis, and so it is plausible that vascular pulsations generate waves that propel CSF flow in the basal cisterns, thereby facilitating tracer influx into the ganglion (Kedarasetti et al., Arterial vasodilation drives convective fluid flow in the brain: a poroelastic model. Fluids and barriers of the CNS 19, 34 (2022); Mestre et al., Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension. Nat Commun 9, 4878 (2018)). Overall then, in vivo dynamic imaging shows that a small CSF tracer rapidly enters the proximal portion of the trigeminal ganglion.

[0182] Example 4. The trigeminal ganglion root lacks a perineural sheet with tight junctions.

[0183] Extracranial peripheral nerves generally have connective tissue sheaths with tight junctions that bars entry of external solutes into the nerve extracellular space equivalent to the arachnoid barrier cell layer around the CNS (Rasmussen et al., Fluid transport in the brain. Physiological reviews 102, 1025-1151 (2022)). To understand the structural basis for the observed lack of a barrier around the ganglion, immunohistochemistry was next used to dissect the organization of the meningeal membranes surrounding the ganglion and basal cisterns. It was found that the tight barrier in the connective tissue around the trigeminal ganglion only materialized in the distal two thirds of the ganglion when dura mater, the arachnoid barrier layer (labeled with claudin-11) (Brochner et al., Outer brain barriers in rat and human development. Front Neurosci 9, 75 (2015)) and the subarachnoidal lymphatic-like membrane (SLYM - defined antigenically as expressing PROXI, CRABP2 and PDPN) (Mollgard et al., A mesothelium divides the subarachnoid space into functional compartments. Science 379, 84- 88 (2023)) fused with the trigeminal ganglion. At the proximal third of the ganglion, these three membranes were physically separable from the nerve, and thus forming a CSF cistern delineated by SLYM as the roof, and the permeable pial membrane (labeled with PDPN) attached to the ganglion as its floor (Mestre etal., Periarteriolar spaces modulate cerebrospinal fluid transport into brain and demonstrate altered morphology in aging and Alzheimer's disease. Nat Commun 13, 3897 (2022)), allowing free passage of CSF and solutes into the ganglion at the trigeminal root (FIGS. 2A-2D). The border between CNS and PNS in the trigeminal ganglion was identified by immunostaining for astrocytic aquaporin-4, which revealed that the proximal portion of the trigeminal nerve is an extension of CNS (FIG. 2C). In MRI images of the human trigeminal nerve, that insertion of dura mater occurred at the ganglion, and since the arachnoid barrier membrane and dura are usually fused, it is plausible that also in humans the trigeminal rootlets lack a tight barrier. Thus, the histological analysis supported the CE-MRI analysis, showing that the trigeminal ganglion lacks a barrier to CSF over its proximal third. As such, CSF tracer influx into the trigeminal ganglion can take two paths, either 1) flowing from the inner subarachnoid space surrounding the root of the nerve, where no barriers are present, or 2) via the brainstem parenchyma, via the pons and the trigeminal nerve to the ganglion (FIG. 2D). These routes differ from previously reported nerve sheet CSF transport around the trigeminal nerve, which functions as a CSF egress pathway towards the cervical lymphatic system (Rasmussen et al., Fluid transport in the brain. Physiological reviews 102, 1025-1151 (2022); Yoon et al., Nasopharyngeal lymphatic plexus is a hub for cerebrospinal fluid drainage. Nature 625, 768-777 (2024)).

[0184] Example 5. Visual cortex extracellular solutes primarily flow towards trigeminal ganglion.

[0185] To establish the distribution of cortical solute flow to the trigeminal ganglion and other putative sites of trigeminal activation in migraine with aura, the solute drainage pathways were compared from the visual cortex, which is the most common site of migraine aura (Thomsen et al., Clinical features of migraine with aura: a REFORM study. J Headache Pain 25, 22 (2024)). Using CE-MRI, the dispersal of gadobutrol injected into visual cortex was followed. In real time, it was observed that cortical extracellular solutes overwhelmingly drained to the basal cisterns around the trigeminal ganglion; this yielded a 4 times higher CSF tracer accumulation around the ipsilateral trigeminal ganglion, relative to the parasagittal dural spaces (FIG. 3A) (Ringstad et al., Cerebrospinal fluid tracer efflux to parasagittal dura in humans. Nat Commun 11, 354 (2020)). This flow pattern resulted in much higher tracer signal in the trigeminal ganglion compared to dura mater extracellular space traditionally considered an important site for trigeminal activation in migraine. The contrast agent appeared at the trigeminal ganglion CSF at 33.4+4.4 min, suggesting a travel time between visual cortex and the ganglion consistent with the typical 5-60 minute delay between migraine aura and the onset of headache (Viana et al., Migraine aura symptoms: Duration, succession and temporal relationship to headache. Cephalalgia: an international journal of headache 36, 413-421 (2016); Viana et al., The typical duration of migraine aura: a systematic review. Cephalalgia: an international journal of headache 33, 483-490 (2013)). It is also within the same timeframe as the delay observed between CSD induction and increased sensory neuron activity previously reported after CSD in rodents (Zhao et al., Modulation of intracranial meningeal nociceptor activity by cortical spreading depression: a reassessment. J Neurophysiol 113, 2778-2785 (2015); Zhang et al., Activation of meningeal nociceptors by cortical spreading depression: implications for migraine with aura. The Journal of neuroscience: the official journal of the Society for Neuroscience 30, 8807-8814 (2010)). The CSF concentration in the ganglion ipsilateral to the injection was 5.2 times higher than the contralateral ganglion. Together, these data indicate that CSF efflux from the visual cortex drains basally to the ipsilateral trigeminal ganglion, implicating this route as a more effective waterway for signal transmission from the CNS to the PNS than the pathway towards afferent endings in dura mater.

[0186] Example 6. Increased cortical washout of extracellular solutes during migraine with aura.

[0187] Having established a pathway for humoral signaling between the cortex and trigeminal ganglion, it was next asked if this pathway is active in migraine with aura pathophysiology. Multiple lines of evidence suggest that CSD provides the etiologic basis for the reversible neurological deficits known as migrainous aura (Hadjikhani et al., Mechanisms of migraine aura revealed by functional MRI in human visual cortex. Proceedings of the National Academy of Sciences of the United States of America 98, 4687-4692 (2001); Lauritzen et al., Changes in regional cerebral blood flow during the course of classic migraine attacks. Ann Neurol 13, 633-641 (1983)). In animal models, CSD has been shown to instigate increased activity in trigeminal sensory neurons, though by an unknown mechanism (Zhao et al., Modulation of intracranial meningeal nociceptor activity by cortical spreading depression: a reassessment. J Neurophysiol 113, 2778-2785 (2015); Zhang et al., Activation of central trigeminovascular neurons by cortical spreading depression. Ann Neurol 69, 855-865 (2011); Zhang et al., Activation of meningeal nociceptors by cortical spreading depression: implications for migraine with aura. The Journal of neuroscience : the official journal of the Society for Neuroscience 30, 8807-8814 (2010); Fioravanti et al., Evaluation of cutaneous allodynia following induction of cortical spreading depression in freely moving rats. Cephalalgia : an international journal of headache 31, 1090-1100 (2011)). To interrogate the effects of CSD on brain fluid dynamics, CSD was induced optogenetically in Thyl-Chr2 mice over right frontal cortex after a CSF tracer had been delivered (Houben et al., Optogenetic induction of cortical spreading depression in anesthetized and freely behaving mice. J Cereb Blood Flow Metab 37, 1641-1655 (2017)). Immediately after CSD was induced, a more than 4-fold increase in tracer influx was observed in the CSD hemisphere lasting 5.3+0.3 minutes (FIG. 3B). This resulted in a -60% increase in CSF tracer signal in the hemisphere exposed to CSD, compared to its contralateral control. It was next asked whether this increased cortical CSF influx boosted the washout of extracellular solutes. To that end, a fluorescent tracer was injected into the cortex, and then induced multiple waves of CSD. Over the hour thereafter, the animals exposed to multiple CSDs (4.8+0.8 CSDs) displayed a tracer signal decline of 20.5+4.4%, compared to a 2.4+6.0% fall in the controls (FIG. 3C). Together, these data indicate that CSD increases cortical CSF inflow while augmenting the washout of extracellular solutes, thereby increasing the CSF content of solutes released by cortical cells during CSD.

[0188] Example 7. CSD upregulates inflammatory CSF proteins that can activate trigeminal neurons.

[0189] CSD is known to upregulate the transcription of inflammatory mediators (Karatas et al., Spreading Depression Triggers Headache by Activating Neuronal Panxl Channels. Science 339, 1092-1095 (2013); Takizawa et al., Non-invasively triggered spreading depolarizations induce a rapid pro-inflammatory response in cerebral cortex. Journal of Cerebral Blood Flow and Metabolism, (2019)), so it was next asked if the increased cortical solute washout upregulated nociceptive molecules in CSF after CSD. Bottom-up mass spectrometry on CSF obtained from adult mice with and without exposure to CSD detected proteins from 1425 different genes. After CSD, the expression levels of 155 of these proteins (11%) significantly changed, and of these 67 changed by more than 2-fold (21 upregulated, 46 downregulated) (FIGS. 4A and 5A)

[0190] It was next sought to evaluate if humoral agents within the CSD proteome could potentially drive headache by activating receptive cells in the trigeminal ganglion. To this end, the entire CSF proteome was first run through a curated ligand-receptor database (Turei et al., OmniPath: guidelines and gateway for literature-curated signaling pathway resources. Nat Methods 13, 966-967 (2016)); the RNA levels of putative receptors in the trigeminal ganglion were then estimated, based on publicly available single-cell RNA sequencing data of the murine trigeminal ganglion, published by Yang et al. Human and mouse trigeminal ganglia cell atlas implicates multiple cell types in migraine. Neuron 110, 1806-1821 el808 (2022). By this means, 65 unique CSF proteins were identified as ligands to 143 unique receptors expressed in the trigeminal ganglion (FIG. 6). Of all the ligand-receptor interactions, 94 were classified functionally; of these, 98 % were stimulatory, 1% inhibitory, and 1% classified as both, suggesting that the CSF -borne signals to the trigeminal ganglion largely stimulate trigeminal cells. Within the upregulated CSD proteome, 12 protein ligands pairing with 28 unique receptors in the ganglion were identified (FIG. 4B). Among these ligands was the neuropeptide calcitonin-gene related peptide (CGRP), which doubled in concentration (fold-change 2.03, p < 0.05). CGRP is encoded by the gene Calca, and the CSF transport of CGRP to the trigeminal ganglion could be directly involved in the development of migraine headache. Another 6 ligands (Sppl, Efnb3, Cntn2, Efempl, S100a8, S100a9) also increased in concentration by more than two-fold. Several of the corresponding receptors of these ligands had previously been shown to be involved in processes such as hyperalgesia and inflammation (Ferrari etal., CD44 Signaling Mediates High Molecular Weight Hyaluronan-Induced Antihyperalgesia. The Journal of neuroscience : the official journal of the Society for Neuroscience 38, 308-321 (2018); Liu et al., Effect of Sppl on nerve degeneration and regeneration after rat sciatic nerve injury. BMC Neurosci 18, 30 (2017); Zhang et al., Activation of spinal ephrin-B3 / EphBs signaling induces hyperalgesia through a PLP-mediated mechanism. Fundam Clin Pharmacol 36, 262-276 (2022); Kim et al., Early Blockade of EphA4 Pathway Reduces Trigeminal Neuropathic Pain. J Pain Res 13, 1173-1183 (2020)). The receptors were expressed on sensory neurons, including peptidergic neurons, but also on vascular cells, fibroblasts, and Schwann cells, indicating an extensive signaling matrix among cells of the brain and trigeminal ganglion (FIGS. 5A-5D) (Chamessian et al., Pain regulation by non-neuronal cells and inflammation. Science 354, 572-577 (2016)). In summary, these data show that CSD leads to significant changes in the CSF proteome, with the upregulation of several ligands to trigeminal receptors, including CGRP.

[0191] Example 8. Changes in CSF composition after CSD induce activity in trigeminal sensory neurons.

[0192] The observed changes to the CSF proteome after CSD made us hypothesize that the CSF transport of noxious mediators to the trigeminal ganglion might be sufficient to drive trigeminal activity. To test this, the response to CSD in the trigeminal ganglion of TRPV1- cre:GCaMP6f mice (Ghitani etal., Specialized Mechanosensory Nociceptors Mediating Rapid Responses to Hair Pull. Neuron 95, 944-954. e944 (2017)) was mapped using a GRIN lens inserted over the trigeminal ganglion. CSD increased ongoing activity by 5.5-fold in trigeminal TRPVllmneurons with a mean latency from CSD induction to first activation of 11.2+1.0 min (FIG. 7A). Roughly 74 % of the trigeminal cells observed in the GRIN lens were activated after CSD (73 / 99 cells in 6 animals). This result compares to that of a recent study measuring the activation of dural sensory nerve endings, which noted increased activity in only 10% of dural afferents (Blaeser et al., Sensitization of meningeal afferents to locomotion-related meningeal deformations in a migraine model. eLife 12, (2024)), consistent with the role of the trigeminal ganglion as the main signaling site in the aftermath of CSD. To test whether the CSD-associated activity of trigeminal TRPVllmneurons was a consequence of compounds released to the CSF, CSF was collected from mice exposed to CSD for 1 hr (CSD CSF). The CSF was topically applied to exposed trigeminal ganglia in naive TRPVl-cre:GCaMP6f animals (FIG. 4C) (Ghitani et al., Specialized Mechanosensory Nociceptors Mediating Rapid Responses to Hair Pull. Neuron 95, 944-954. e944 (2017)). The CSD CSF triggered a 2.37+0.26 fold increase in calcium activity in the TRPVllmcells compared to control aCSF (FIG. 4F), and a 1.45+0.39 increase compared to CSF of naive animals (FIG. 4G). This effect was time dependent, in that CSD CSF collected 2.5h after the first CSD did not lead to an increase in calcium activity (FIG. 4G). This suggests that the CSD-triggered changes in CSF proteome are short lasting, and as such likely limited to migraine initiation, while downstream processes maintain the headache. Since CSD is known to cause the significant and abrupt release of potassium, CSD CSF K+concentrations were assessed and similar levels between CSD and naive mice were observed (FIG. 7B). Thus, the increased neuronal activity in the trigeminal ganglion upon exposure to CSD CSF is likely driven by signaling solutes released by CNS during CSD. CGRP was prominent among those potential ligands identified and differentially upregulated in the CSD proteome, and given its role in migraine initiation in humans, it was asked if its delivery to CSF might increase trigeminal neuronal activity. To that end, CGRP in aCSF was topically applied to the exposed trigeminal ganglia of TRPV1- cre:GCaMP6f mice (FIG. 7C), and it was found that CGRP increased calcium activity by 3.42+0.34 fold relative to baseline - i.e., to a level similar to that achieved by the application of CSD CSF. The analysis shows that CSD increases ongoing calcium activity in TRPVllmcells in the trigeminal ganglion, and that a similar increase in ongoing activity can be provoked by the application of CSD CSF or CGRP directly to the ganglion.

[0193] Example 9. S100A8 causes activation of trigeminal ganglion neurons.

[0194] A study was performed to determine if S 100 A8 causes activation of trigeminal ganglion neurons. A heatmap showing AF / AF of GCAMP signal in sensory nerves in the trigeminal ganglion treated with aCSF (control) or S100A8 indicated a significant increase in GCAMP signal following treatment with S100A8 (FIGS. 8A and 8B). Thus, inhibiting the binding of S100A8 to TLR4 on trigeminal ganglion neurons can serve as a treatment for migraines. Example 10. Osteopontin (SPP1) causes activation of trigeminal ganglion neurons.

[0195] A study was performed to determine if SPP1 causes activation of trigeminal ganglion neurons. A heatmap showing AF / AF of GCAMP signal in sensory nerves in the trigeminal ganglion treated with aCSF (control) or SPP1 indicated a significant increase in GCAMP signal following treatment with SPP1(FIGS. 9A and 9B). Consequently, inhibiting the binding of SPP1 to CD44 on trigeminal ganglion neurons can serve as a treatment for migraines.

[0196] References

[0197] 1. British Museum., C. E. Wright, Bald's Leechbook (British Museum Royal manuscript 12D. xvii). Early English manuscripts in facsimile, (Rosenkilde and Bagger; Johns Hopkins Press, Copenhagen, Baltimore,, 1955), pp. 30, 32 p., facsim.

[0198] 2. L. H. Lassen et aL, CGRP may play a causative role in migraine. Cephalalgia : an international journal of headache 22, 54-61 (2002).

[0199] 3. L. Edvinsson, K. A. Haanes, K. Warfvinge, D. N. Krause, CGRP as the target of new migraine therapies - successful translation from bench to clinic. Nat Rev Neurol 14, 338-350 (2018).

[0200] 4. L. Edvinsson, E. Nilsson, I. Jansen-Olesen, Inhibitory effect of BIBN4096BS, CGRP(8-37), a CGRP antibody and an RNA-Spiegelmer on CGRP induced vasodilatation in the perfused and non-perfused rat middle cerebral artery. Br J Pharmacol 150, 633-640 (2007).

[0201] 5. N. Hadjikhani et aL, Mechanisms of migraine aura revealed by functional MRI in human visual cortex. Proceedings of the National Academy of Sciences of the United States of America 98, 4687-4692 (2001).

[0202] 6. A. Melo-Carrillo et al. , Fremanezumab-A Humanized Monoclonal Anti-CGRP Antibody-Inhibits Thinly Myelinated (Adelta) But Not Unmyelinated (C) Meningeal Nociceptors. The Journal of neuroscience : the official journal of the Society for Neuroscience 37, 10587-10596 (2017).

[0203] 7. J. Zhao, D. Levy, Modulation of intracranial meningeal nociceptor activity by cortical spreading depression: a reassessment. J Neurophysiol 113, 2778-2785 (2015).

[0204] 8. M. A. Moskowitz, K. Nozaki, R. P. Kraig, Neocortical spreading depression provokes the expression of c-fos protein-like immunoreactivity within trigeminal nucleus caudalis via trigeminovascular mechanisms. The Journal of neuroscience : the official journal of the Society for Neuroscience 13, 1167-1177 (1993).

[0205] 9. H. Karatas et al. , Spreading Depression Triggers Headache by Activating Neuronal Panxl Channels. Science 339, 1092-1095 (2013).

[0206] 10. H. Bolay et al.. Intrinsic brain activity triggers trigeminal meningeal afferents in a migraine model. Nature medicine 8, 136-142 (2002).

[0207] 11. F. E. Pul ous et al. , Cerebrospinal fluid can exit into the skull bone marrow and instruct cranial hematopoiesis in mice with bacterial meningitis. Nat Neurosci 25, 567-576 (2022).

[0208] 12. D. Levy, M. A. Moskowitz, Meningeal Mechanisms and the Migraine Connection. Annu Rev Neurosci, (2023).

[0209] 13. A. M. Strassman, S. A. Raymond, R. Burstein, Sensitization of meningeal sensory neurons and the origin of headaches. Nature 384, 560-564 (1996).

[0210] 14. N. Hadjikhani et al., Extra- Axial Inflammatory Signal in Parameninges in Migraine with Visual Aura. Ann Neurol 87, 939-949 (2020). J. Rustenhoven et al., Functional characterization of the dural sinuses as a neuroimmune interface. Cell, 1-17 (2021). J. A. Mapunda et al., VE-cadherin in arachnoid and pia mater cells serves as a suitable landmark for in vivo imaging of CNS immune surveillance and inflammation. Nat Commun 14, 5837 (2023). L. C. D. Smyth et al., Identification of direct connections between the dura and the brain. Nature, (2024). R. Cai et al., Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull -meninges connections. Nat Neurosci 22, 317-327 (2019). G. Ringstad, P. K. Eide, Cerebrospinal fluid tracer efflux to parasagittal dura in humans. Nat Commun 11, 354 (2020). M. Lauritzen, T. Skyhoj Olsen, N. A. Lassen, O. B. Paulson, Changes in regional cerebral blood flow during the course of classic migraine attacks. Ann Neurol 13, 633- 641 (1983). X. Zhang et al. , Activation of central trigeminovascular neurons by cortical spreading depression. Ann Neurol 69, 855-865 (2011). Q. Ma, B. V. Ineichen, M. Detmar, S. T. Proulx, Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice. Nat Commun 8, 1434 (2017). M. K. Rasmussen, H. Mestre, M. Nedergaard, Fluid transport in the brain. Physiological reviews 102, 1025-1151 (2022). H. Quincke, Zur physiologic der cerebrospinalflussigkeit. Arch Anat Physiol Wiss Med, 153-177 (1872). Key, Retzius, Studien in der Anatomie des Nervensystems und des Bindesgewebe . (Samson and Wallin, Stockholm, Sweden, ed. 1, 1876). N. Ghitani et al., Specialized Mechanosensory Nociceptors Mediating Rapid Responses to Hair Pull. Neuron 95, 944-954. e944 (2017). D. Fontaine et al., Dural and pial pain-sensitive structures in humans: new inputs from awake craniotomies. Brain 141, 1040-1048 (2018). L. J. von Buchholtz, R. M. Lam, J. J. Emrick, A. T. Chesler, N. J. P. Ryba, Assigning transcriptomic class in the trigeminal ganglion using multiplex in situ hybridization and machine learning. Pain 161, 2212-2224 (2020). E. Sykova, C. Nicholson, Diffusion in brain extracellular space. Physiological reviews 88, 1277-1340 (2008). R. T. Kedarasetti, P. J. Drew, F. Costanzo, Arterial vasodilation drives convective fluid flow in the brain: a poroelastic model. Fluids and barriers of the CNS 19, 34 (2022). H. Mestre et al. , Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension. Nat Commun 9, 4878 (2018). C. B. Brochner, C. B. Holst, K. Mollgard, Outer brain barriers in rat and human development. Front Neurosci 9, 75 (2015). K. Mollgard et al. , A mesothelium divides the subarachnoid space into functional compartments. Science 379, 84-88 (2023). H. Mestre et al., Periarteriolar spaces modulate cerebrospinal fluid transport into brain and demonstrate altered morphology in aging and Alzheimer's disease. Nat Commun 13, 3897 (2022). J. H. Yoon et al., Nasopharyngeal lymphatic plexus is a hub for cerebrospinal fluid drainage. Nature 625, 768-777 (2024). A. V. Thomsen et al., Clinical features of migraine with aura: a REFORM study. J Headache Pain 25, 22 (2024). M. Viana et aL, Migraine aura symptoms: Duration, succession and temporal relationship to headache. Cephalalgia : an international journal of headache 36, 413- 421 (2016). M. Viana, T. Sprenger, M. Andelova, P. J. Goadsby, The typical duration of migraine aura: a systematic review. Cephalalgia : an international journal of headache 33, 483-490 (2013). X. Zhang et al., Activation of meningeal nociceptors by cortical spreading depression: implications for migraine with aura. The Journal of neuroscience : the official journal of the Society for Neuroscience 30, 8807-8814 (2010). B. Fioravanti et al., Evaluation of cutaneous allodynia following induction of cortical spreading depression in freely moving rats. Cephalalgia : an international journal of headache 31, 1090-1100 (2011). T. Houben et al., Optogenetic induction of cortical spreading depression in anesthetized and freely behaving mice. J C er eb Blood Flow Metab 37, 1641-1655 (2017). T. Takizawa et al., Non-invasively triggered spreading depolarizations induce a rapid pro-inflammatory response in cerebral cortex. Journal of Cerebral Blood Flow and Metabolism, (2019). D. Turei, T. Korcsmaros, J. Saez-Rodriguez, OmniPath: guidelines and gateway for literature-curated signaling pathway resources. Nat Methods 13, 966-967 (2016). L. Yang et al., Human and mouse trigeminal ganglia cell atlas implicates multiple cell types in migraine. Neuron 110, 1806-1821 el808 (2022). L. F. Ferrari, E. V. Khomula, D. Araldi, J. D. Levine, CD44 Signaling Mediates High Molecular Weight Hyaluronan-Induced Antihyperalgesia. The Journal of neuroscience : the official journal of the Society for Neuroscience 38, 308-321 (2018). X. Liu et al., Effect of Sppl on nerve degeneration and regeneration after rat sciatic nerve injury. BMC Neuro sci 18, 30 (2017). P. Zhang et al., Activation of spinal ephrin-B3 / EphBs signaling induces hyperalgesia through a PLP-mediated mechanism. Fundam Clin Pharmacol 36, 262-276 (2022). M. J. Kim, J. Y. Son, J. S. Ju, D. K. Ahn, Early Blockade of EphA4 Pathway Reduces Trigeminal Neuropathic Pain. J Pain Res 13, 1173-1183 (2020). R. R. Ji, A. Chamessian, Y. Q. Zhang, Pain regulation by non-neuronal cells and inflammation. Science 354, 572-577 (2016). A. S. Blaeser et al., Sensitization of meningeal afferents to locomotion-related meningeal deformations in a migraine model. eLife 12, (2024). M. T. Kronschlager et al. , Gliogenic LTP spreads widely in nociceptive pathways. Science 354, 1144-1148 (2016). T. Takano et al., Cortical spreading depression causes and coincides with tissue hypoxia. Nature neuroscience 10, 754-762 (2007). H. Mestre et al., Cerebrospinal fluid influx drives acute ischemic tissue swelling. Science 367, (2020). O. Windmuller et al., Ion changes in spreading ischaemia induce rat middle cerebral artery constriction in the absence of NO. Brain 128, 2042-2051 (2005). E. Eising et al., Cortical Spreading Depression Causes Unique Dysregulation of Inflammatory Pathways in a Transgenic Mouse Model of Migraine. Mol Neurobiol 54, 2986-2996 (2017). R. B. Lipton et al., Safety, tolerability, and efficacy of zavegepant 10 mg nasal spray for the acute treatment of migraine in the USA: a phase 3, double-blind, randomised, placebo-controlled multicentre trial. Lancet Neurol 22, 209-217 (2023). N. B. Rasmussen et aL, The effect of Lu AG09222 on PACAP38- and VIP-induced vasodilation, heart rate increase, and headache in healthy subjects: an interventional, randomized, double-blind, parallel-group, placebo-controlled study. J Headache Pain 24, 60 (2023). Z. Guan et al., Injured sensory neuron-derived CSF1 induces microglial proliferation and DAP12-dependent pain. Nat Neurosci 19, 94-101 (2016). J. A. Coull et aL, BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. Nature 438, 1017-1021 (2005). A. Tozzi et al.. Critical role of calcitonin gene-related peptide receptors in cortical spreading depression. Proceedings of the National Academy of Sciences of the United States of America 109, 18985-18990 (2012). Y. Wang et al., Induction of calcitonin gene-related peptide expression in rats by cortical spreading depression. Cephalalgia : an international journal of headache 39, 333-341 (2019). J. Hoffmann, S. Wecker, L. Neeb, U. Dirnagl, U. Reuter, Primary trigeminal afferents are the main source for stimulus-induced CGRP release into jugular vein blood and CSF. Cephalalgia : an international journal of headache 32, 659-667 (2012). C. J. Schankin et al., Ictal lack of binding to brain parenchyma suggests integrity of the blood-brain barrier for HC-dihydroergotamine during glyceryl trinitrate-induced migraine. Brain 139, 1994-2001 (2016). P. J. Goadsby, L. Edvinsson, R. Ekman, Vasoactive peptide release in the extracerebral circulation of humans during migraine headache. Ann Neurol 28, 183-187 (1990). J. M. Hansen, A. W. Hauge, J. Olesen, M. Ashina, Calcitonin gene-related peptide triggers migraine-like attacks in patients with migraine with aura. Cephalalgia : an international journal of headache 30, 1179-1186 (2010). R. M. van Dongen et al., Migraine biomarkers in cerebrospinal fluid: A systematic review and meta-analysis. Cephalalgia : an international journal of headache 37, 49- 63 (2017). K. Messlinger, A. F. Russo, Current understanding of trigeminal ganglion structure and function in headache. Cephalalgia : an international journal of headache 39, 1661-1674 (2019). Z. Zhang, C. S. Winborn, B. Marquez de Prado, A. F. Russo, Sensitization of calcitonin gene-related peptide receptors by receptor activity-modifying protein- 1 in the trigeminal ganglion. The Journal of neuroscience : the official journal of the Society for Neuroscience 27, 2693-2703 (2007). A. L. R. Xavier et al., Cannula Implantation into the Cisterna Magna of Rodents. Journal of visualized experiments : JoVE, (2018). S. L. Resendez et al., Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses. Nat Protoc 11, 566-597 (2016). A. J. Schain, A. Melo-Carrillo, A. M. Strassman, R. Burstein, Cortical Spreading Depression Closes Paravascular Space and Impairs Glymphatic Flow: Implications for Migraine Headache. The Journal of neuroscience : the official journal of the Society for Neuroscience 37, 2904-2915 (2017). T. Du et al., Cerebrospinal fluid is a significant fluid source for anoxic cerebral oedema. Brain 145, 787-797 (2022). M. C. Ku, S. Waiczies, T. Niendorf, A. Pohlmann, Assessment of Blood Brain Barrier Leakage with Gadolinium-Enhanced MRI. Methods in molecular biology 1718, 395- 408 (2018). J. H. Ahn et aL, Meningeal lymphatic vessels at the skull base drain cerebrospinal fluid. Nature 572, 62-66 (2019). R. O. Weller, M. M. Sharp, M. Christodoulides, R. O. Carare, K. Mollgard, The meninges as barriers and facilitators for the movement of fluid, cells and pathogens related to the rodent and human CNS. Acta Neuropathol 135, 363-385 (2018). J. M. Bader et aL, Proteome profiling in cerebrospinal fluid reveals novel biomarkers of Alzheimer's disease. Mol Syst Biol 16, e9356 (2020). P. E. Geyer et al., Proteomics reveals the effects of sustained weight loss on the human plasma proteome. Mol Syst Biol 12, 901 (2016). N. A. Kulak, G. Pichler, I. Paron, N. Nagaraj, M. Mann, Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods 11, 319-324 (2014). R. Bruderer et al., Optimization of Experimental Parameters in Data-independent Mass Spectrometry Significantly Increases Depth and Reproducibility of Results. Mol Cell Proteomics 16, 2296-2309 (2017). S. J. Callister et al., Normalization approaches for removing systematic biases associated with mass spectrometry and label-free proteomics. J Proteome Res 5, 277- 286 (2006). B. B. Avants et al., The Insight ToolKit image registration framework. Front Neuroinform 8, 44 (2014). N. J. Tustison et al., N4ITK: improved N3 bias correction. IEEE Trans Med Imaging 29, 1310-1320 (2010). F. Gremse et al., Analytics Preclinical: Interactive Analysis of Biomedical Volume Data. Theranostics 6, 328-341 (2016). L. Xie et al., Sleep drives metabolite clearance from the adult brain. Science 342, 373- 377 (2013). M. J. Gordon et al., Measurement of Gd-DTPA diffusion through PVA hydrogel using a novel magnetic resonance imaging method. Biotechnol Bioeng 65, 459-467 (1999). A. Zeisel et al., Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 1138-1142 (2015). L. Heumos et al., Best practices for single-cell analysis across modalities. Nat Rev Genet 24, 550-572 (2023). F. A. Wolf, P. Angerer, F. J. Theis, SCANPY: large-scale single-cell gene expression data analysis. Genome Biol 19, 15 (2018). D. V. Klopfenstein et al., GOATOOLS: A Python library for Gene Ontology analyses. Sci Rep f 10872 (2018).

Claims

CLAIMSWhat is claimed is:

1. A method of reducing an activation of a trigeminal ganglion neuron or cell, comprising inhibiting an action of a receptor on or in said neuron or cell, wherein the receptor is selected from the group consisting of Tlr4, Lepr, Lrpl, Tshr, Itga3, Mtnrla, Calcr, Calcrl, Rampl, Ramp2, Ramp3, Itgav, Scarfl, Cntnl, Cntnap2, Egfr, Epha4, Ephb2, Ephb3, Ephb4, Pdgfrb, Itgb3, Itgal, Itgbl, Plxncl, Cd44, Itga4, and Plaur.

2. A method of reducing an activation of a trigeminal ganglion neuron or cell, comprising blocking the binding of a ligand to a receptor on or in said neuron or cell, wherein(I) the ligand is selected from the group consisting of S100A8, S100A9, Apod, Calr, Cntn2, Efempl, Efnb3, Mfge8, Sema7a, Sppl, Vtn, and Calca, or(II) the receptor is selected from the group consisting of Tlr4, Lepr, Lrpl, Tshr, Itga3, Mtnrla, Calcr, Calcrl, Rampl, Ramp2, Ramp3, Itgav, Scarfl, Cntnl, Cntnap2, Egfr, Epha4, Ephb2, Ephb3, Ephb4, Pdgfrb, Itgb3, Itgal, Itgbl, Plxncl, Cd44, Itga4, and Plaur.

3. The method of claim 2, wherein the ligand is selected from the group consisting of S100A8, S100A9, Sema7a, Sppl, and Vtn.

4. The method of any one of claims 1-3, wherein the receptor is selected from the group consisting of Tlr4, Plxncl, Cd44, and Plaur.

5. The method of any one of claims 2-4, wherein the ligand and the receptor are a pair selected from the group consisting of (a) S100A8 and TLR4, (b) S100A9 and TLR4, (c) SPP1 and CD44, (d) VTN and PLAUR, and (e) SEMA7A and PLXNCL6. The method of any one of claims 1 and 4-5, wherein the inhibiting is conducted by a process comprising contacting the neuron or cell with an agent that specifically binds to the receptor.

7. The method of any one of claims 2-6, wherein the blocking is conducted by a process comprising contacting the neuron or cell with a substance that specifically binds to the ligand.

8. The method of claim 6 or 7, wherein the agent or substance is a small molecule compound, a peptide, or a protein.

9. The method of claim 8, wherein the protein is an antibody or antigen-binding fragment thereof.

10. The method of any one of claims 1-9, wherein the trigeminal ganglion neuron or cell is in a subject.

11. The method of claim 6 or 7, wherein the agent or substance is one selected from monoclonal antibody Ab45, monoclonal antibody 5.5, monoclonal antibody A22124D, monoclonal antibody 1F8, anti-S100A8 polyclonal antibody (PA5-82881), Paquinimod (ABR-215757), Laquinimod (ABR-215062), Tasquinimod (ABR-215050), ABR-238901, Midostaurin, Eritoran, Peptide3A5, Divalent Peptide3A5, Tetravalent ILVIK, a peptide-Fc fusion protein, PIM447, GDC-0339, TAK-242 (Resatorvid), Eritoran (E5564), CLI-095, FP7, IAXO-101, C34, VX-15 / 2503, VIPER peptide, TLR4 inhibitory peptide 1 (TIPI), NI-0101, TLR4 monoclonal antibody (clone HTA125), TLR4 siRNA, TLR4 antisense oligonucleotide, monoclonal antibody 6B4, monoclonal antibody MAC387, monoclonal antibody H5, S100A9 Antibody Blocking Peptide (LS-E31467), S100A9 Peptide (AAP36677), S100A9 Peptide -N- terminal region (AAP36676), monoclonal antibody MEM-150, monoclonal antibody D-4, polyclonal anti-Sema7a antibody ab23578, polyclonal anti-Sema7a antibody A03832, SEMA7A Antibody Blocking Peptide (LS-E23467), monoclonal antibody 1 A7, monoclonal antibody 1D7, monoclonal antibody 1F5, monoclonal antibody h2Kl, monoclonal antibody c2Kl, SPP1 Blocking Peptide (SPP1-BP), SPP1 Peptide (AAP36677), SPP1 Peptide - N- terminal region (AAP36676), SPP1 Antibody Blocking Peptide (LS-E31467), SPP1 Aptamer (SPP1-APT), Cilengitide (EMD121974), MK-0429, ATN-161 (Ac-PHSCN-NBL), an RGD peptide, Volociximab (M200), 8E6 (LJ8) monoclonal antibody, Vitronectin (VTN) siRNA, Vitronectin antisense oligonucleotides, PLXNC1 siRNA, PLXNC1 shRNA, small molecule RG7356, SB-525334, PEP-1, A6 peptide, HABP (Hyaluronic Acid Binding Peptide), Bivatuzumab mertansine, antibody RG7356, antibody IM7, antibody H4C4, CD44 siRNA, CD44 shRNA, UK-371804, WX-360, AE105, UP ARANT (UPAR antagonist peptide), ATN- 658, HuATN-658, PLAUR siRNA, and PLAUR shRNA.

12. A method for treating or preventing a migraine in a subject in need thereof, comprising: (I) inhibiting an action of a receptor on or in a trigeminal ganglion neuron or cell in the subject according to the method of any one of claims 1 and 4-9; or (II) blocking the binding of a ligand to a receptor on or in said neuron or cell according to the method of any one of claims 2-9.

13. The method of claim 12, comprising administering to the subject an effective amount of the agent or substance.94