Research Paper

Modulation of nociceptive dural input to the trigeminocervical complex through GluK1 kainate receptors Anna P. Andreoua,b, Philip R. Hollandc, Michele P. Lasalandraa, Peter J. Goadsbya,c,*

Abstract Migraine is a common and disabling neurologic disorder, with important psychiatric comorbidities. Its pathophysiology involves activation of neurons in the trigeminocervical complex (TCC). Kainate receptors carrying the glutamate receptor subunit 5 (GluK1) are present in key brain areas involved in migraine pathophysiology. To study the influence of kainate receptors on trigeminovascular neurotransmission, we determined the presence of GluK1 receptors within the trigeminal ganglion and TCC with immunohistochemistry. We performed in vivo electrophysiologic recordings from TCC neurons and investigated whether local or systemic application of GluK1 receptor antagonists modulated trigeminovascular transmission. Microiontophoretic application of a selective GluK1 receptor antagonist, but not of a nonspecific ionotropic glutamate receptor antagonist, markedly attenuated cell firing in a subpopulation of neurons activated in response to dural stimulation, consistent with selective inhibition of postsynaptic GluK1 receptor–evoked firing seen in all recorded neurons. In contrast, trigeminovascular activation was significantly facilitated in a different neuronal population. The clinically active kainate receptor antagonist LY466195 attenuated trigeminovascular activation in all neurons. In addition, LY466195 demonstrated an N-methyl-d-aspartate receptor–mediated effect. This study demonstrates a differential role of GluK1 receptors in the TCC, antagonism of which can inhibit trigeminovascular activation through postsynaptic mechanisms. Furthermore, the data suggest a novel, possibly presynaptic, modulatory role of trigeminocervical kainate receptors in vivo. Differential activation of kainate receptors suggests unique roles for this receptor in pro- and antinociceptive mechanisms in migraine pathophysiology. Keywords: Kainate receptor, Migraine, Trigeminovascular activation, Microiontophoresis

1. Introduction Migraine is the most common cause of neurologic disability worldwide.41 It is costly,42 and although its pathophysiology is incompletely understood, it is thought to involve activation of the trigeminal afferents.22 Glutamate, the major excitatory neurotransmitter in the central nervous system, has been implicated in migraine pathophysiology through experimental work,7,23,39 human biomarker studies,19 and by results of clinical trials.1,45 Given well-recognized side effects of actions at the glutamate N-methyld-aspartate (NMDA) receptor, attention has moved to other targets. The kainate receptor is a member of the ionotropic glutamate receptor (iGluR) family, consisting of the GluK1-5 subunits,15 which can form multimeric assemblies giving rise to functional Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article. a

Headache Group, Department of Neurology, University of California, San Francisco, CA, USA, b Headache Research-Section of Anaesthetics, Pain Medicine and Intensive Care Section, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, UK, c Headache Group, Basic and Clinical Neurosciences, King’s College London, London, United Kingdom *Corresponding author. Address: NIHR–Wellcome Trust King’s Clinical Research Facility, Kings College Hospital, London SE5 9PJ, United Kingdom. E-mail address: [email protected] (P. J. Goadsby). PAIN 156 (2015) 439–450 © 2014 International Association for the Study of Pain http://dx.doi.org/10.1097/01.j.pain.0000460325.25762.c0

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receptor–channels.14 In vitro experiments have demonstrated that kainate receptors function as mediators and modulators of synaptic transmission and plasticity by regulating postsynaptic currents11 and presynaptic neurotransmitter release.31 Increasing evidence has shown activation and modulation of kainate receptors by nociceptive stimuli.46 The presence of functional kainate receptors in dorsal root ganglia,2 trigeminal ganglion (TG),47 and in both preand postsynaptic sites in superficial laminae of the spinal cord and caudal brain stem,24,35,37 indicates their potential importance. Nonselective a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonists have been shown to produce antinociceptive effects in both animal39 and human studies.48 Competitive GluK1-selective receptor antagonists were effective in blocking trigeminal activation after stimulation of the TG.20,50 In a double-blind controlled study, the GluK1 receptor antagonist LY466195 was effective in relieving pain in acute migraine.28 Topiramate, a partial kainate receptor antagonist,17 is a clinically effective migraine preventive12 and has been shown to reduce trigeminovascular and thalamic activation and to inhibit cortical spreading depression in the mammalian cortex.3,4,6 Here, we aimed to examine the role of GluK1 kainate receptors in trigeminovascular nociceptive processing. We used microiontophoresis to investigate the effects of GluK1 agents directly on second-order trigeminocervical neurons, using the GluK1receptor antagonist UBP302 and the clinically active GluK1 receptor antagonist LY466195.50 We further examined the effects of intravenous LY466195 on responses of neurons in www.painjournalonline.com

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the trigeminocervical complex (TCC) to mimic the peripheral administration of the drug clinically.

(WDR) neurons responding to both innocuous and noxious stimulation of the ophthalmic dermatome.

2. Methods

2.3. Drugs

All experiments were conducted under the UK Home Office Animals (Scientific Procedures) Act (1986) and approved by the University of California San Francisco Institutional Animal Care and Use Committee.

Freshly prepared solutions of LY466195 (50, 100, and 200 mg/kg) were intravenously infused over 1 minute at least 10 minutes after 3 consistent baseline responses to electrical stimulation of the MMA. Seven-barrel carbon-fiber electrodes were used to deliver microiontophoretically freshly prepared solutions of glutamate receptor agents using a current generator (Dagan 6400; Dagan Corporation, MN). Micropipette barrels were filled with 25 mM of the GluK1 receptor antagonist (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxybenzyl)pyrimidine-2,4-dione (UBP302; Tocris Cookson Ltd, Bristol, United Kingdom), 25 mM of the GluK1 receptor antagonist (3S,4aR,6S,8aR)6-[[(2S)-2-carboxy-4,4-difluoro-1-pyrrolidinyl]-methyl]decahydro-3isoquinolinecarboxylic acid (LY466195; kindly provided by Lilly Laboratories, Indianapolis, IN), 10 mM of the nonselective iGluR antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Tocris Cookson Ltd), 200 mM l-glutamate monosodium (Sigma, St. Louis, MO), 25 mM of the GluK1 receptor agonist iodowillardiine (Tocris Cookson Ltd), 25 mM of the AMPA agonist fluorowillardiine (Tocris Cookson Ltd), 50 mM of the GluK1/2 receptor agonist (2S,4R)-4methylglutamic acid (SYM 2081; Tocris Cookson Ltd), 100 mM NMDA (Tocris Cookson Ltd), 200 mM d-serine (Tocris Cookson Ltd), and Pontamine sky blue dye (Gurr 6BX; BDH Laboratory Supplies, Poole, United Kingdom; 2.5% wt/vol in 100 mM sodium acetate; pH 6.5). All compounds were ejected as anions (5-100 nA) and retained with small positive currents. Saline was used as a control and represents the ejection of both Cl2 and OH2 ions, because the pH of the saline was adjusted by the addition of 0.01 M NaOH. Current balance was provided through a barrel containing 200 mM NaCl. Microiontophoretic barrels had resistances of 9 to 100 MV.

2.1. Surgical preparation Male Sprague-Dawley rats (n 5 55; 300-370 g) were anesthetized with 60 mg/kg pentobarbital intraperitoneally (i.p.) and then maintained with pentobarbital infusion (25-35 mg·kg21·h21). The left femoral artery and both femoral veins were cannulated for blood pressure recordings and infusion of anesthetic, neuromuscular blocker, and test compounds, respectively. Animals were positioned on a rat stereotaxic frame and ventilated with oxygenenriched air (Harvard Apparatus, Ltd, Edenbridge, Kent, United Kingdom). Throughout the experiments, the end-tidal CO2 was monitored (Capstar-100; CWE Inc, Ardmore, PA), and blood pressure were monitored and stable. Blood gases were measured at intervals and were within normal limits: arterial blood pH 5 7.37 6 0.06 and pCO2 5 3.70 6 0.42 kPa. The dura mater/middle meningeal artery (MMA) complex was accessed through a craniotomy. A hemi-C1 laminectomy exposed the brain stem at the level of the caudal medulla. Depth of anesthesia was judged by the absence of paw withdrawal and corneal blink reflex and by the lack of fluctuations in blood pressure during muscular paralysis.

2.2. Stimulation of middle meningeal artery and recordings from trigeminocervical complex Stimulation of the MMA was performed by using a bipolar stimulating electrode and the lowest possible stimulus intensity was used (10-16 V, 0.5 Hz, 100-microseconds duration). Extracellular recordings were made from second-order neurons in the region of the TCC, using microiontophoretic electrodes (Kation Scientific, Minneapolis, MN) consisting of a 7-barreled glass pipette and incorporating a carbon-fiber recording electrode (impedance at 1 kHz: 0.4-0.8 MV). The signal from the recording electrode was fed through a head stage amplifier (NL100AK, Neurolog; Digitimer, Herts, United Kingdom) to a series of filters and amplifiers and data were collected using Spike2v5 software (CED, Cambridge, United Kingdom). Poststimulation histograms were constructed as the sum of a total of 25 stimuli to record the response of units to electrical stimulation of the MMA as previously described.6 Background cellular activity was continuously monitored through peristimulus histograms. Neuronal action potential firing in response to microiontophoresis of glutamate receptor agonists was analyzed as cumulative rate histograms. 2.2.1. Receptive fields The cutaneous receptive field was assessed in all 3 dermatomes of the trigeminal innervations, as the recording electrode was advanced in the spinal cord. The receptive field was assessed for both nonnoxious, with gentle brushing, and noxious inputs by pinching with forceps and light brush of the cornea. When a neuron sensitive to noxious stimulation of the ophthalmic dermatome was identified, it was tested for convergent input from the dura mater. All recorded neurons were wide-dynamic-range

2.4. Experimental protocol Trigeminal responses were recorded and challenged by kainate receptor antagonists intravenously (LY466195) or locally by microiontophoresis into the TCC (LY466195, UBP302, CNQX). For each intravenous treatment, MMA stimulation–evoked firing was recorded before (3 baselines) and 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, and 90 minutes after the LY466195 or saline control were injected. For the iontophoretic studies, once neurons were identified by their response to ophthalmic facial receptive field stimulation, they were tested for a stable response to electrical stimulation of the MMA and an increased firing rate to microiontophorized glutamate receptor agonists. Three baseline responses to MMA stimulation were collected 5 minutes apart. The glutamate receptor agonists tested for each experiment were ejected in a random order, and once 5 stable baseline cycles were recorded, then UBP302 (10, 20, 30, 40, 50, and 100 nA) or LY466195 (5, 10, and 20 nA) or their control ions (Cl2 and OH2) were ejected with each current applied during individual ejecting cycles of agonists. CNQX or vehicle control (Cl2 and OH2) was applied at 20 nA over 3 cycles of glutamate receptor agonists. A poststimulus histogram was collected during each ejecting current of the antagonists or their controls and at the end of the recovery period. To assess the effects of the antagonists on noxious and nonnoxious stimulation of the cutaneous facial receptive field, light touch, noxious pinch, and corneal brush were randomly applied during baseline cycles, during each ejecting current of the

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antagonists or their controls, and after the recovery period. At the conclusion of the antagonist or vehicle control ejection, agonists’ cycles were continued for 15 to 30 minutes and the neuronal responses were observed. The location of the recording site was obtained by either direct marking of the site by ejection of charged pontamine sky blue through a BAB-350 pump (Kation Scientific) or by reconstruction from a marked reference point and microdrive readings. On termination of the experiment, the tissue was removed and processed for histologic verification using the brain atlas by Paxinos and Watson.43 2.5. Tissue processing and immunohistochemistry Untreated control male Sprague-Dawley rats (n 5 3) weighing 310 to 330 g were deeply anesthetized with pentobarbital (60 mg/ kg, i.p.) and perfused through the left ventricle with 200 mL of heparinized saline, followed by 500 mL of 4% paraformaldehyde in phosphate buffered saline (PBS 0.1 M, pH 7.4, 4˚C). The trigeminal ganglia and the lower brain stem including the upper cervical segments were removed and cryoprotected at 4˚C to 8˚C in sucrose solution (30% sucrose in 0.1 M PBS with 0.1% sodium azide) until saturated. One TG per animal was cut into 20-mm thick sections and mounted on slides. The brain stem was sectioned into 40-mm thick sagittal sections and processed as free floating in PBS. Sections were first left for 10 minutes in a 3% hydrogen peroxide solution for blocking endogenous peroxidase activity, followed by 1-hour incubation with 10% normal goat serum in PBS with 0.5% Triton X-100 (PBS11; Vector Laboratories, Burlingame, CA) at room temperature. This was followed by a 2-step avidin–biotin blocking solution (Vector Laboratories) for 30 minutes to block nonspecific binding sites and a 48-hour incubation at 4˚C with a solution of rabbit-raised antibody against the amino acids CHQRRTQRKETVA of rat GluK1 subunit located in the intracellular C-terminal (1:250; Millipore, Billerica, MA), made up of 2% normal goat serum in PBS containing 0.5% Triton X-100 (PBS1). This was followed by incubation for 90 minutes at room temperature in goatraised biotinylated anti-rabbit secondary antibody (1:500; Vector Laboratories) in PBS1. After this, the sections were incubated for 30 minutes with an avidin–biotin peroxidase complex (Vectastain ABC kitA; Vector Laboratories) and further incubated with the biotinylated tyramide solution (PerkinElmer, Boston, MA) for 5 minutes, followed by 1-hour incubation in fluoroscein avidin-D (1: 500; Vector Laboratories) made up of PB1 at room temperature. Direct immunohistofluorescence was used for double labeling of calcitonin gene–related peptide (CGRP). After blocking the sections in PB11, sections were incubated overnight at 4˚C in a previously validated mouse anti-CGRP primary antibody (1:2000; the epitope recognized by the antibody resides within the Cterminal 10 amino acids of rat a-CGRP; Sigma),38 and then incubated for 1 hour at room temperature with anti-mouse avidin coupled to Texas Red in PB1 (1:500; Vector Laboratories). Sections were then rinsed, air dried, covered with mounting medium (Vector Laboratories), and examined using a Zeiss Axioplan universal microscope (Carl Zeiss, Thornwood, NY). All steps mentioned above were separated with thorough washes using PBS. Negative control samples were obtained by omitting the primary or secondary antibodies. Sections from the trigeminal ganglia were used as positive controls. 2.6. Data analysis To compare the effect of systemic administration of LY466195, the recorded data after the intravenous injection of LY466195 and

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saline were converted to a percentage of the average of the 3 baselines. The effects of the drugs over time and the interaction of both the drug used and time were evaluated using an analysis of variance with repeated measures followed by Bonferroni tests. The Greenhouse–Geisser correction of the degrees of freedom was applied when the assumption of sphericity was not met. A paired t test (LY466195 vs saline) was used to compare further each time point. For microiontophoresis experiments, the statistical analysis was conducted as described previously.8 Briefly, the response of each cell for each glutamate agonist under test conditions was examined as follows: (1) iGluR agonist (baseline), (2) iGluR coejected with an antagonist or control ions (Cl2 and OH2) at increasing doses, and (3) iGluR agonist (recovery). Five baseline pulses of iGluR agonists were analyzed to avoid variations of the responses of a cell between individual pulses, and the reliability of the measurements was tested using Cronbach alpha (SPSS Inc, Chicago, IL). The baseline response probability to MMA or cutaneous receptive field stimulation was calculated from up to 3 poststimulus histograms and peristimulus histograms, respectively, and repeated during treatment with antagonist or control, separately for each current and following recovery period. Effects of antagonist or vehicle control interventions were analyzed using an analysis of variance for repeated measures followed by paired t test with Bonferroni post hoc correction for multiple comparisons, using the average of the baselines of all tested parameters for comparisons. An independent t test was used to compare the effects of the antagonist UBP302 over 2 different neuronal subpopulations (see Results). Data are expressed as mean percentage of the baseline response 6 SEM, and significance was assessed at the P , 0.05 levels.

3. Results 3.1. Immunohistochemistry The qualitative analysis of immunofluorescent-stained sections revealed GluK1 receptors throughout the TCC (Fig. 1A and B, D–F). Cell bodies and punctate staining were evident in laminae I-III, and cell bodies were mainly seen in deeper laminae. Stained cells were round or pear shaped and ranged in diameter size from 15 to 26 mm. Within lamina I and outer lamina II, where CGRP-positive fibers project, GluK1-like staining was seen in cell bodies (Fig. 1E) and in punctate staining, which might represent proximal processes or GluK1-like primary fibers as some colocalization with CGRP-like fibers was seen (Fig. 1F). The presence of GluK1-like receptors within primary fibers arising from the trigeminal ganglia is very likely, as many trigeminal neurons, mainly of medium and small size, were GluK1 positive (Fig. 1G) as previously reported.47 Some colocalization with CGRP-positive neurons was also seen (Fig. 1H), mainly in small size neurons (;25%-30%). 3.2. Localization and neuronal characteristics A total of 98 WDR neurons were studied from the TCC (Fig. 1I) and each displayed convergent trigeminal viscerosomatic inputs of the ophthalmic dermatome. Only cell bodies identified by the size and shape of the action potential in response to glutamate receptor agonists microiontophoresis were recorded (Fig. 1K). Extracellular recordings were made from neurons responding in a reversible excitatory manner to all iGluR agonists tested and to MMA electrical stimulation with latencies consistent with Ad-fibers (average latency 8-12 milliseconds; Fig. 1J). No unit

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Figure 1. GluK1-like immunofluorescent staining within the trigeminocervical complex (TCC) and trigeminal ganglia and reconstruction of recording sites. (A and B) Photomicrographs of the TCC taken from the C1 level, showing GluK1-like staining (green). Cell bodies and punctate staining were evident in laminae I-III, and cell bodies were mainly seen in deeper laminae. Stained cells were round or pear shaped. (D–F) Within lamina I and outer lamina II, where calcitonin gene–related peptide (CGRP)–like fibers project (red), GluK1-like staining (green) was seen in cell bodies and in punctate staining, which might represent proximal processes or GluK1 primary fibers as some colocalization (yellow) with CGRP was seen (G). (C) A photomicrograph of a negative control section of the TCC. (G) A photomicrograph of GluK1-like cells (green) within the trigeminal ganglion. (H) GluK1-like cells (green) mainly colocalized with small size CGRP-positive cells (red) within the trigeminal ganglion. Arrowheads present examples of GluK1-like positive cells, stars indicate CGRP-like positive cells, and arrows show colocalization of CGRP- and GluK1-like fibers (F) or cells (H). Scale bars 5 100 mm (A, C, D), 50 mm (E, G, H), and 10 mm (B and F). I. Reconstruction of recording sites within the C1 spinal cord level, plotted after Paxinos and Watson,43 identified histologically (solid circles represent pontamine sky blue spots) and by microdrive readings (open circles). A photomicrograph demonstrating an original recording site marked by ejection of pontamine sky blue (arrow) is shown. (J) An original trace showing a cluster of cells in the TCC, firing in response to stimulation of the middle meningeal artery (100 microseconds, 0.5 Hz, 12 V; *indicates stimulus artefact). (K) Original tracing from a neuron in the TCC responding to microiontophoresis of L-glutamate.

was studied with more than 1 antagonist, and between 1 and 3 units were studied per animal.

3.3. Effects of intravenous LY466195 on middle meningeal artery stimulation–evoked activity LY466195 administered intravenously significantly inhibited trigeminocervical activity in response to MMA stimulation at a dose of 100 mg/kg (n 5 7, P , 0.05; Fig. 2A) by a maximum of 33% 6 2% (t12 5 4.48, P , 0.05). LY466195 at 200 mg/kg significantly decreased responses (n 5 5; P , 0.05) at similar levels as the 100 mg/kg dose by a maximum of 32% 6 9% (t6 5 4.20, P , 0.05; Fig. 2A), and there was no significant difference between the effects induced by the 2 doses (P $ 0.40). Cell firing returned to baseline

levels within 90 minutes. At the dose of 50 mg/kg, LY466195 did not display a significant effect on cell firing (n 5 5; P 5 0.13). Intravenous administration of vehicle control had no effect on firing in response to MMA stimulation (n 5 7, P 5 0.18; Fig. 2A). In all animals, neither intravenous administration of LY466195 nor saline control produced any changes on blood pressure. 3.4. Effects of microiontophoretic administration of LY466195 on trigeminal neuronal firing 3.4.1. Effect on middle meningeal artery and receptive field stimulation Ejection of LY466195 significantly inhibited cell firing in response to MMA stimulation (n 5 8, P , 0.005; Fig. 2B

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Figure 2. (A) Effect of intravenously administrated LY466195 on responses of trigeminocervical neurons to middle meningeal artery (MMA) stimulation. LY466195 inhibited firing to dural electrical stimulation at 200 and 100 mg/kg but not at 50 mg/kg. Saline control ejection had no effect on neuronal firing. *P , 0.05. B. Effect of microiontophoresis of LY466195 on responses of trigeminocervical neurons to MMA stimulation and to receptive field characterization. Example of poststimulus histograms from a representative neuron, recorded during baseline conditions and during ejection of LY466195 at currents 5, 10, and 20 nA. (C) Comparison of the MMA stimulation–evoked firing under each condition. Ejection of LY466195 demonstrated a dose-dependent inhibition of the cell firing, maximally at 20 nA. (D) Effects of microiontophoretically delivered LY466195 and its vehicle control (Cl2 and OH–) on the firing rates of second-order neurons in response to receptive field characterization. Microiontophoretic application of LY466195 at 20 nA significantly inhibited firing to both innocuous (brush) and nocuous (corneal brush; pinch) stimulation of the ophthalmic dermatome, whereas a lower ejection current (10 nA) significantly inhibited noxious stimulation of the receptive field. *P , 0.05.

and C) in all neurons tested in a current dose–dependent manner by a maximum of 72% 6 6% (t7 5 4.65, P , 0.05) post-LY466195. LY466195 significantly reduced in a dosedependent manner–evoked firing to both innocuous (brush) and noxious (pinch and corneal brush) mechanical stimulation of the ophthalmic facial cutaneous receptive field in all neurons tested (n 5 6; Fig. 2D).

3.4.2. Effects of microiontophoretic application of LY466195 on postsynaptic firing evoked by glutamate agonists LY466195 was applied by microiontophoresis on cells firing in response to the agonists NMDA (NMDA receptor agonist), fluorowillardiine (AMPA receptor agonist), and iodowillardiine (GluK1 kainate receptor agonist). For all 3 agonists tested, there was no difference across the mean firing of the 5 repeated

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Figure 3. A. Example of the effects of LY466195 on the firing rates to pulsed ejections of fluorowillardiine, iodowillardiine, and N-methyl-d-aspartic acid (NMDA). Cell firing in response to the ejected agonists returned to baseline levels within 2 to 5 minutes after LY466195 microiontophoresis ceased. (B) Comparison of the effect of LY466195 and control ions (Cl2 and OH2) on iodowillardiine-, NMDA-, and fluorowillardiine-evoked responses. Cell firing in response to iodowillardiine and NMDA was significantly inhibited by microiontophoretically administered LY466195. (C) Example of the response of a trigeminocervical neuron to pulsed NMDA. Ejection of serine, LY466195, and control ions are shown with the solid bars. Ejection of LY466195 demonstrated a potent inhibitory effect on NMDA-evoked firing that was not reversed by pretreatment with serine. (D) Comparison of the NMDA-evoked firing during ejection of control ions and LY466195. (E) Poststimulus histograms generated from a representative trigeminocervical neuron after electrical stimulation of the middle meningeal artery (MMA). Microiontophoresis of serine or control ions at the same current had no effect on neuronal firing. Coejection of serine with LY466195 failed to block the inhibitory actions of LY466195. (F) Comparison of the MMA stimulation–evoked firing under the influence of LY466195, serine, and control ions. (G) Example of the effects of CNQX on the firing rates to pulsed ejections of the L-glutamate, iodowillardiine, and SYM 2081. Cell firing in response to the ejected agonists returned to baseline levels within 30 minutes after CNQX microiontophoresis ceased. (B–H) Effects of CNQX on cell firing in response to L-glutamate, SYM 2081, and NMDA, separately for each ejection cycle. CNQX strongly inhibited L-glutamate, SYM 2081, and NMDA-evoked firing. Ejection of control ions had no significant effects on glutamate agonists–evoked firing. (I) Comparison of the MMA stimulation–evoked firing under the influence of CNQX and control ions. CNQX significantly inhibited neuronal firing in response to trigeminovascular stimulation. (J) Poststimulus histograms generated from a representative trigeminocervical neuron after electrical stimulation of the MMA during baseline conditions, microiontophoresis of CNQX, and recovery of neuronal firing. (K) Effects of microiontophoretically delivered CNQX on the firing rates of second-order neurons in response to receptive field characterization. Microiontophoretic application of CNQX at 20 nA significantly inhibited firing to both innocuous (brush) and nocuous (corneal brush; pinch) stimulation of the ophthalmic dermatome, whereas a lower ejection current (10 nA) significantly inhibited noxious stimulation of the receptive field. *P , 0.05 compared with control.

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epochs recorded during baseline (n 5 8), and all responses were reliable (Cronbach a $ 0.94). LY466195 potently inhibited responses to iodowillardiine(P , 0.001) and NMDA- (P , 0.005) evoked firing in a dose–response manner (Fig. 3A and B). Fluorowillardiineevoked firing was not altered by LY466195 across the cohort (P 5 0.09). 3.4.3. Effects of microiontophoretic application of LY466195 and serine on N-methyl-D-aspartate-evoked postsynaptic firing As LY466195 demonstrated a significant action on NMDA receptors, we aimed to investigate whether this effect was because of actions of LY466195 on the glycine/serine binding site of the NMDA receptor. Serine is an endogenous co-agonist of glutamate at the NMDA receptor, increasing the affinity of the receptor for the endogenous agonist glutamate.40 Ejection of serine alone neither affected neuronal firing in response to NMDA ejection (Fig. 3C and D) nor to MMA stimulation (Fig. 3E and F) compared with control ions. Co-ejection of serine with LY466195 failed to reverse the inhibitory actions of LY466195 on NMDAevoked postsynaptic firing (Fig. 3D, F).

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3.5. Effects of microiontophoretic administration of GluK1 receptor antagonist UBP302 on trigeminal neuronal firing 3.5.1. Effects of microiontophoretic application of UBP302 on dural stimulation–evoked activity UBP302 significantly inhibited in a dose-dependent manner– evoked firing to MMA stimulation in 15 of 27 neurons (Fig. 4A). The maximum inhibition of 70% 6 7% was obtained at the ejection current of 100 nA. Evoked responses to MMA stimulation were significantly facilitated by UBP302 in 12 of 27 cells (P , 0.001; Fig. 4B). In some cases, further to the facilitation observed from Ad-fiber input, a decrease in the threshold to C-fiber activation was also observed during application of UBP302. Inhibitory and facilitatory responses during ejection of UBP302 were frequently seen from neurons recorded in the same animal and the firing returned to baseline within 30 minutes in both groups. Ejection of control ions had no effect (n 5 15). Based on the responses of the 2 groups of cells to UBP302 ejection, these units were further analyzed separately and will be referred to as the inhibitory group and facilitatory group. 3.5.2. Effects of microiontophoretic application of UBP302 on receptive field stimulation–evoked activity The effects of UBP302 on cell firing to innocuous (brush) and noxious (pinch and corneal brush) mechanical stimulation of the

Figure 4. Effect of microiontophoresis of UBP302 on responses of trigeminocervical neurons to middle meningeal artery stimulation and to receptive field characterization. Ejection of UBP302 produced 2 opposing responses. (A) In 15 units, ejection of UBP302 demonstrated a dose-dependent inhibition of the cell firing, maximally at 100 nA. (B) In 12 neurons, UBP302 significantly potentiated cell firing. Control vehicle had no effect. Poststimulus histograms from representative cluster of neurons in the inhibitory (A) and facilitatory (B) groups recorded pre-UBP302 ejection during the ejection of UBP302 at 100 nA and 30 minutes after the cessation of ejection. (C) Effects of microiontophoretically delivered UBP302 and its vehicle control (Cl2 and OH2) on the firing rates of secondorder neurons in response to receptive field characterization. Cell firing in response to light brush was not altered by microiontophoretically administered UBP302 neither in the inhibitory group nor in the facilitatory group. Cell firing in response to pinch and to corneal stimulation was significantly inhibited by microiontophoretically administered UBP302 in the inhibitory group. Noxious-evoked responses to both pinch and corneal stimulus were not significantly facilitated across the cohort in the facilitatory group. Control ejection had no significant effect. (D) Example of the effects of UBP302 on the firing rates to pulsed ejections of fluorowillardiine, iodowillardiine, and N-methyl-d-aspartic acid (NMDA). Cell firing in response to the ejected agonists returned to baseline levels within 30 minutes after UBP302 microiontophoresis ceased. (E) Comparison of the current–response curves for UBP302 and control (Cl2 and OH2) on iodowillardiine-, SYM 2081-, L-glutamate-, fluorowillardiine-, and NMDA-evoked responses. Overall, cell firing in response to iodowillardiine (n 5 19), SYM 2081 (n 5 18), and Lglutamate (n 5 18) was significantly inhibited by microiontophoretically administered UBP302, and there was no significant difference between the inhibitory and the facilitatory groups. Cell firing in response to fluorowillardiine (n 5 17) and NMDA (n 5 6) was unaffected by microiontophoretically delivered UBP302. Vehicle control had no significance on any agonist-evoked firing. *P , 0.05.

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ophthalmic facial cutaneous receptive field were tested in 26 WDR neurons (Fig. 4C). Based on the responses of the MMA-evoked activity to UBP302, 14 of the neurons recorded were clustered in the inhibitory group and 12 were clustered in the facilitatory group; neurons were analyzed separately based on their groups. Overall, microriontophoretic ejection of UBP302 had no effect on firing rate in response to nonnoxious stimulation of the ophthalmic cutaneous area (n 5 26; P 5 0.10), and there was no significant difference between the inhibitory and facilitatory groups (P $ 0.07). UBP302 significantly reduced in a dosedependent manner–evoked firing to both pinch and corneal brush stimulation in all neurons clustered in the inhibitory group (pinch: P , 0.001; corneal: P , 0.001), and noxious stimulation–evoked firing returned to baseline within 30 minutes after stopping UBP302 ejection. In the facilitatory group, evoked responses to noxious stimulus were facilitated during UBP302 ejection by a maximum of 23% 6 9%, but overall there was no significant difference across the cohort (pinch: P 5 0.53; corneal: P 5 0.38). For both responses to pinch and corneal stimulation, there was a significant difference between the inhibitory and facilitatory groups (P , 0.05). 3.5.3. Effects of microiontophoretic application of UBP302 on postsynaptic firing evoked by glutamate receptor agonists For all agonists tested, there was no difference across the mean firing of the 5 repeated epochs recorded during baseline (data not shown). UBP302 reversibly and significantly inhibited trigeminal firing in response to microiontophoresed iodowillardiine (kainate receptor agonist) in all 19 neurons tested, demonstrating a dose-dependent effect (n 5 19; Fig. 4D and E). SYM 2081- (kainate receptor agonist) evoked firing was additionally reversibly reduced by UBP302 in a dose-dependent manner (n 5 18; Fig. 4E), in all units. Based on the responses of the MMA-evoked activity to UBP302, iodowillardiine and SYM 2081-evoked firing was inhibited by UBP302 in both the inhibitory (iodowillardiine: n 5 9; SYM 2081: n 5 9) and facilitatory (iodowillardiine: n 5 10; SYM 2081: n 5 9) groups. UBP302 reduced significantly and reversibly the probability of neuronal firing in response to L-glutamate ejection (n 5 18, P , 0.05; Fig. 4E). UBP302 reduced firing in both inhibitory (n 5 9) and facilitatory (n 5 9) grouped cells equally. The effect of UBP302 on fluorowillardiine- (AMPA receptor agonist) and on NMDA-evoked firing was tested in 17 and 9 units, respectively, and UBP302 produce negligible effects on neuronal firing across the cohort for both agonists (Fig. 4E). 3.6. Effects of microiontophoretic administration of the nonselective ionotropic glutamate receptor antagonist CNQX on trigeminal neuronal firing 3.6.1. Effects of microiontophoretic application of CNQX on dural stimulation–evoked activity CNQX at 20 nA inhibited MMA electrical stimulation–evoked cell firing to 43% 6 13% compared with control (P , 0.005), and this effect was ubiquitous among all cells (Fig. 3I and J). 3.6.2. Effects of microiontophoretic application of CNQX on receptive field stimulation–evoked activity CNQX significantly inhibited evoked firing to both nonnoxious stimulation of the V1 receptive field to 24% 6 7% (P , 0.05) and to noxious pinch stimulation to 40% 6 9% (t9 5 5.7; P , 0.005) and corneal mechanical stimulation to 45% 6 17% (n 5 4, P 5

0.05; Fig. 3K). No facilitation was observed on any of the neurons tested with CNQX. 3.6.3. Effects of microiontophoretic application of CNQX on postsynaptic firing evoked by glutamate receptor agonists The baseline firing responses to glutamate receptor agonist application demonstrated good stability, and there was no difference across the mean firing of the 5 repeated epochs. CNQX inhibited L-glutamate–evoked responses to 16% 6 9% (t7 5 5.50, P , 0.005) and abolished SYM 2081-evoked firing (t7 5 4.21, P , 0.005). N-methyl-D-aspartate-evoked firing was significantly reduced to a lesser degree than to L-glutamate and SYM 2081 by a maximum of 67% 6 6% (P , 0.001; Fig. 3G and H).

4. Discussion The data presented demonstrate a robust modulation of trigeminovascular transmission by kainate receptors at TCC neurons receiving nociceptive trigeminovascular inputs. The use of microiontophoretic application of a kainate specific antagonist in our model, with its technical advantage of precise anatomical localization,49 enabled the demonstration of bidirectional regulation of trigeminovascular nociceptive transmission in the TCC mediated by kainate receptors, which at the level of individual neurons in vivo is certainly novel. In this context, it is noteworthy that models of neurogenic dural vasodilation7,13 failed to demonstrate a peripheral action for kainate receptor antagonists, which makes a central action even more attractive. Moreover, the addition of a cohort of animals treated intravenously emphasizes the potentially translational implications of the data. This study further offers a plausible locus of action for the clinically active compound LY466195 in migraine,28 through both kainate and NMDA receptor antagonism in the trigeminovascular pain pathway. This study used electrical stimulation of dural structures to identify nociceptive neurons. The MMA and its surrounding dura mater are densely innervated by A- and C-fibers,10 and there is clear evidence in humans that electrical stimulation of the meninges reproduces at least pain referred to the head.18,44 Middle meningeal artery stimulation has been used extensively as a model for the study of trigeminal nociception and to successfully predict the efficacy of antimigraine drugs in animal models of head pain.9 The use of microiontophoresis in our study enabled us to investigate in isolation the induction of postsynaptic action potentials through exogenous applications of glutamate agonists, thus eliminating the role of presynaptic transmitter release. This is a widely used method that allows one to narrow the field of possible induction mechanisms to those of postsynaptic origin.49 Local ejection of UBP302, a selective GluK1 receptor antagonist, by microiontophoresis blocked in all neurons the postsynaptic activation in response to local application of kainate receptor agonists. This indicates the presence of GluK1 kainate receptors on postsynaptic locations in the TCC, which was further confirmed by means of immunohistochemistry. Interestingly, although postsynaptic firing in response to application of kainate receptor agonists was always inhibited by local application of UBP302, firing evoked by stimulation of the MMA was either inhibited or facilitated by UBP302. This may be because of a different synaptic environment in each neuronal subpopulation where a dominant presence of presynaptic kainate receptors may be responsible for the facilitatory effects (Fig. 5). In this study, we demonstrated the presence of GluK1 receptors in both primary afferents and within neuronal bodies in the TCC. The differential

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Figure 5. Proposed mechanism of action of microiontophoresed UBP302 on trigeminovascular activation. Electrical stimulation of the middle meningeal artery (MMA) will activate fibers arising from the trigeminal ganglion and increase neuronal firing of second-order neurons in the trigeminocervical complex. (A) During baseline recordings, activation of primary afferents innervating the MMA causes glutamate release in the trigeminocervical complex. Glutamate activates postsynaptic kainate receptors in addition to a-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-d-aspartic acid receptors activation, promoting trigeminocervical nociceptive transmission. Presynaptic kainate receptors on primary trigeminal afferents could be activated by glutamate and control glutamate release, whereas activation of kainate receptors on inhibitory interneurons is also possible and will modulate inhibitory synaptic transmission. (B) Local application of UBP302 by microiontophoresis can selectively block postsynaptic kainate receptors on second-order neurons and inhibit nociceptive transmission, most likely in the absence of kainate receptors on the presynaptic trigeminal nerve fiber. Blockade of kainate receptors on inhibitory GABAergic terminals could occur because of UBP302 diffusion, and this could additionally account for the resultant analgesic effect. However, as we did not directly study this, such a blockade of kainate heteroreceptors on GABAergic interneurons is shown with a “?” In such a synaptic environment, postsynaptic kainate receptors play an important role in trigeminovascular nociceptive transmission and their selective blockade could prove a beneficial treatment for migraine. (C) In a different synaptic environment where both pre- and postsynaptic kainate receptors are present on trigeminal fibers and second-order trigeminocervical neurons, respectively, local application of UBP302 by microiontophoresis can selectively block postsynaptic kainate receptors on trigeminocervical neurons. In addition, blockade of presynaptic kainate receptors on primary afferents might inhibit the negative control feedback of glutamate release by kainate receptors. This results in increased glutamate release on stimulation of the trigeminal fibers innervating the MMA. Released glutamate could then act on a bigger scale on a-amino-3-hydroxy-5-methylisoxazole-4propionate and N-methyl-d-aspartic acid receptors and facilitate nociceptive transmission. In these neurons, it is also possible that postsynaptic GluK1 kainate receptors do not mediate sensory transmission.

function and pharmacology of pre- and postsynaptic kainate receptors have been demonstrated before in vitro.30–32 In this study, we further suggest such a differential function in vivo. The inhibition of the MMA stimulation–evoked firing is likely produced by the postsynaptic blockade of GluK1 kainate receptors on these second-order neurons, as seen by the attenuation of the kainate receptor agonist’s postsynaptic activation (Fig. 5A and B). Stimulation of Ad- and C-fibers, but not of nonnociceptive primary fibers, has been previously shown to activate postsynaptic kainate receptors on spinal dorsal horn neurons,35 suggesting that kainate receptors are postsynaptically placed, largely at synapses that receive inputs from nociceptive primary afferent fibers. Interestingly, in this study, in the same neuronal subpopulation, noxious over nonnoxious receptive field responses were preferentially inhibited. Given the data, it seems reasonable to pursue the postsynaptic kainate receptor on second-order neurons in the TCC as a target for the development of novel treatments for migraine. In the spinal cord, it has also been suggested that kainate heteroreceptors on GABAergic terminals36 could be activated by glutamate spillover from neighboring excitatory synapses and occlude GABA/glycine release,27,30 through indirect activation of GABAB autoreceptors,30 thus promoting a possible pronociceptive mechanism. In

this study, diffusion of UBP302 to neighboring GABAergic interneurons may be also a possibility, and therefore blockade of such kainate heteroreceptors would further contribute toward an analgesic effect. However, the direct blockade of kainate receptors that we certainly observed was that of postsynaptic GluK1 kainate receptors on second-order neurons. Interestingly, the GluK1 selective receptor antagonist UBP302, in addition to the inhibition of trigeminovascular activation, also facilitated trigeminovascular transmission in a subset of neurons, despite the selective reduction of postsynaptic firing in responses to kainate receptor agonists. It is thus unlikely that the observed facilitation was because of postsynaptic blockade of kainate receptors or, as discussed above, because of blockade of kainate heteroreceptors on GABAergic terminals, as these would have resulted in inhibition. A nonglutamate receptor action of UBP302 is also rather unlikely, as UBP302 is consistently described as a highly selective and potent GluK1 antagonist. None of the other willardiine derivative antagonists were ever described to have nonglutamate receptor actions.16 We and others have shown that GluK1 subunits are expressed in the TG47 and on primary afferents in the TCC.24 It is believed that presynaptic kainate receptors on primary afferents act as autoreceptors and occlude further glutamate release when

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activated.32 It is likely that strong activation of presynaptic kainate receptors leads to inhibition of glutamate release,25,33 possibly by inactivating calcium and/or sodium channels 29. Thus, the facilitation observed in a subpopulation of trigeminovascular neurons by antagonism of GluK1 receptors could be because of blockade of presynaptic kainate autoreceptors30–32 resulting in enhanced glutamate release in response to trigeminovascular stimulation. The released glutamate could then act on AMPA and NMDA receptors, resulting in enhancement of sensory transmission (Fig. 5A, C). Thus, in a synaptic environment where both presynaptic and postsynaptic GluK1 are present on primary trigeminal fibers and second-order neurons, respectively, a GluK1 antagonist may induce facilitation of trigeminovascular activity, whereas in a synaptic environment where there is a dominant presence of postsynaptic GluK1 receptors, a GluK1 antagonist is more likely to have antinociceptive properties. Although UBP302 demonstrated a trend to also facilitate corneal brush and pinch responses in the facilitatory group where trigeminovascular stimulation was facilitated, this was not significant. Trigeminovascular activation recorded in this study was mainly induced by Ad-fibers, whereas activation induced by corneal brush and pinch is more likely induced by stimulation of C-fibers. It is likely that GluK1 are not located on presynaptic trigeminal C-fibers; however, our immunohistochemistry data suggest that kainate receptors may be found in small size neurons, possibly corresponding to Ad- and C-fibers. The differential effect may be explained because of the modality of stimulation in each case, where in trigeminovascular activation electrical stimulation was used and data were collected over a total of 25 stimuli, whereas during noxious receptive field activation, mechanical stimulation was used and data were collected over a 2-second stimulation of the receptive field. However, it is difficult to safely reach a conclusion for these effects. In contrast to the dual effects of UBP302, application of CNQX resulted only in inhibition of nociceptive transmission in the TCC, further suggesting that the facilitation seen is a specific kainate receptor–mediated effect. Microiontophoretic application of the clinically active compound LY466195 also failed to demonstrate any bidirectional regulation of excitatory synaptic transmission in the TCC. This was probably because of the fact that although LY466195 demonstrated a potent antagonist action on GluK1 receptor–evoked firing in accordance with its pharmacologic characterization,50 to our surprise, a significant action on NMDA receptors was additionally recorded. With most kainate receptor antagonists described to date, it is more common to have some action on AMPA than on NMDA receptors.26 In vitro data of LY466195 demonstrated some competitive binding affinity on NMDA receptors, with a 55-fold selectivity for GluK1 receptors over NMDA.50 Other kainate or AMPA/kainate receptor antagonists, such as kynurenic acid21 and CNQX,34 have an antagonist action on the glycine/serine site of the NMDA receptor. Our data suggest that LY466195 has no actions on the glycine/serine binding site of the NMDA receptors, as serine failed to prevent its inhibitory actions over NMDA-evoked firing. In a randomized double-blind study, LY466195 was effective in relieving acute migraine,28 although patients reported visual side effects, that may be attributed to blockade of both receptors. Although a global blockade of glutamate receptors may indeed seem to have stronger analgesic effects, the widespread distribution of AMPA and NMDA receptors in the CNS, as well as the essence of their physiological activity for normal neuronal function, will very likely induce unacceptable clinical side effects.5 Kainate

receptors, however, are not as widely distributed within the CNS; but they are certainly found in areas of interest in terms of the pain pathways involved in migraine, such as in the trigeminal ganglia, the TCC, and the thalamus. In conclusion, our data provide evidence for the regulation of trigeminovascular transmission by both postsynaptic and presynaptic kainate receptors, indicating a modulatory role of kainate receptors in trigeminovascular nociceptive processing at the level of TCC. Differential activation of kainate receptors has revealed novel roles of this receptor in pro- and antinociceptive mechanisms in the TCC, and its differential targeting in terms of synaptic localization might provide new options in migraine therapeutics.

Conflict of interest statement P. J. Goadsby has consulted for Eli-Lilly on migraine therapeutics. Eli-Lilly had no involvement of any type in the work or manuscript. The remaining authors have no conflicts of interest to declare. The work has been funded by the Migraine Trust, EUROHEADPAIN European Union FP7, and the Sandler Family Foundation. Article history: Received 11 July 2014 Received in revised form 14 November 2014 Accepted 2 December 2014 Available online 30 December 2014

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