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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Pain. Author manuscript; available in PMC Jan 1, 2012.
Published in final edited form as:
PMCID: PMC3005011
NIHMSID: NIHMS244370

Dural afferents express acid-sensing ion channels: a role for decreased meningeal pH in migraine headache

Abstract

Migraine headache is one of the most common neurological disorders. The pathological conditions that directly initiate afferent pain signaling are poorly understood. In trigeminal neurons retrogradely labeled from the cranial meninges, we have recorded pH-evoked currents using whole-cell patch-clamp electrophysiology. Approximately 80% of dural afferent neurons responded to a pH 6.0 application with a rapidly activating and rapidly desensitizing ASIC-like current that often exceeded 20 nA in amplitude. Inward currents were observed in response to a wide range of pH values and 30% of the neurons exhibited inward currents at pH 7.1. These currents led to action potentials in 53%, 30% and 7% of the dural afferents at pH 6.8, 6.9 and 7.0, respectively. Small decreases in extracellular pH were also able to generate sustained window currents and sustained membrane depolarizations. Amiloride, a non specific blocker of ASIC channels, inhibited the peak currents evoked upon application of decreased pH while no inhibition was observed upon application of TRPV1 antagonists. The desensitization time constants of pH 6.0-evoked currents in the majority of dural afferents was less than 500 ms which is consistent with that reported for ASIC3 homomeric or heteromeric channels. Finally, application of pH 5.0 synthetic-interstitial fluid to the dura produced significant decreases in facial and hind-paw withdrawal threshold, an effect blocked by amiloride but not TRPV1 antagonists, suggesting that ASIC activation produces migraine-related behavior in vivo. These data provide a cellular mechanism by which decreased pH in the meninges following ischemic or inflammatory events directly excites afferent pain-sensing neurons potentially contributing to migraine headache.

Keywords: pain, migraine, ASIC, dural afferent, headache, meninges

Introduction

Migraine is a multifaceted disorder consisting of unilateral throbbing headache, aura, nausea, vomiting, photophobia and phonophobia [13]. While multiple hypotheses regarding the pathophysiology of migraine headache have been proposed, the exact nature of the triggering process itself is largely unknown [19]. Activation of trigeminal neurons innervating the intracranial meninges and their related large blood vessels is likely required to generate the headache experienced during a migraine attack. Prior work has found that meningeal nociceptors can be sensitized by inflammatory mediators [27; 25; 14] and mediators released from degranulating meningeal mast cells [20; 32]. However, the cellular mechanisms that initiate pain signaling in these neurons are unknown as most of the mediators do not directly open ion channels to produce firing of action potentials.

Acid sensing ion channels (ASICs) belong to the ENaC/DEG (epithelial amiloride-sensitive Na+ channel and degenerin) family of ion channels. ASICs are neuronal voltage-insensitive cationic channels activated by increases in the concentration of extracellular protons [30]. ASICs are expressed throughout the nervous system, including primary sensory neurons, while ASIC3 is largely restricted to the periphery [21]. Peripheral sensory neurons expressing ASIC3 innervate visceral organs including the colon, heart as well as skeletal muscles [29; 16; 22; 23] and this channel has been proposed to participate in pain originating from these organs. With respect to migraine, ASICs on dural afferents have been proposed as a sensor of decreased extracellular pH within the dura [4]. However, this has never been determined experimentally. The aim of this study was to investigate the effects of decreased extracellular pH on dural afferents, including the mechanism by which these neurons may respond to decreased pH within the dura. Additionally, these studies examined the ability of decreased meningeal pH to produce migraine – related behavior in awake animals.

Materials and Methods

Animals

Adult male Sprague Dawley rats (175–200g) were maintained in a climate-controlled room on a 12 hr light/dark cycle with food and water ad libitum. All procedures were performed in accordance with the policies and recommendations of the International Association for the Study of Pain, the National Institutes of Health guidelines for the handling and use of laboratory animals, and by the Institutional Animal Care and Use Committee of the University of Arizona.

Surgical Preparation

I. Tracer injection

Dural afferents were identified as previously described [7] with several modifications. Seven days prior to the sacrifice, animals were anesthetized with a combination of ketamine and xylazine (80 mg/kg and 12 mg/kg; Sigma-Aldrich). Under a dissecting microscope, two holes (3 mm in diameter) were made in the skull using a Dremel Multipro 395 fitted with a dental drill bit (Stoelting) leaving a thin layer of bone at the bottom of the hole. Fine forceps were used to carefully remove the remaining bone and expose but not damage the dura. Fluorogold (5 μl/hole; 4% in synthetic interstitial fluid) was then applied onto the dura. A small piece of gelfoam was retained in the hole to increase the absorption of the dye and prevent spread of the tracer outside of the hole. Holes were covered with bone wax to prevent tracer spread and the incision was closed with sutures. Immediately postoperatively, animals received a single subcutaneous injection of gentamicin (8mg/kg) to minimize infection. Undamaged dura at the injection sites was evaluated at the time the animals were sacrificed and only animals with intact dura and no signs of damage were used for further experiments.

I. Dura Cannulation

Dura cannulae were implanted as previously described [11]. Animals were anesthetized with a combination of ketamine and xylazine (80 mg/kg and 12 mg/kg; Sigma-Aldrich). A 2cm incision was made to expose the skull. A 1mm hole (1mm left of midline, 1mm anterior to bregma) was made with a hand drill (DH-0 Pin Vise; Plastics One, Roanoke, VA) to carefully expose the dura. A guide cannula (22 GA, #C313G; Plastics One), designed to extend 0.5 mm from the pedestal to avoid irritation of the dural tissue, was inserted into the hole and sealed into place with glue. Two additional 1mm holes were made caudal to the cannula to receive stainless-steel screws (#MPX-080-3F-1M; Small Parts, Miami Lakes, FL), and dental acrylic was used to fix the cannula to the screws. A dummy cannula (#C313DC; Plastics One) was inserted to ensure patency of the guide cannula. Immediately postoperatively, animals received a single subcutaneous injection of gentamicin (8mg/kg) to minimize infection. Rats were housed separately and allowed 6 to 8 days of recovery.

Cell culture

Seven days following fluorogold application, trigeminal ganglia were removed, enzymatically treated, and mechanically dissociated as previously described for mouse ganglia [10]. Rats were anesthetized with isoflurane (Phoenix Pharmaceuticals) and sacrificed by decapitation. The trigeminal ganglion (TG) were removed and placed in ice-cold Hanks balanced-salt solution (divalent free). Ganglia were cut into small pieces and incubated for 25 mins in 20 U/ml Papain (Worthington) followed by 25 mins in 3 mg/ml Collagenase TypeII (Worthington). Ganglia were then triturated through fire-polished pasteur pipettes and plated on poly-D-lysine (Becton Dickinson) and laminin (Sigma)-coated plates. After several hours at room temperature to allow adhesion, cells were cultured in a room-temperature, humidified chamber in Liebovitz L-15 medium supplemented with 10% FBS, 10 mM glucose, 10 mM HEPES and 50 U/ml penicillin/streptomycin. Cells were used within 24 h post plating.

Electrophysiology

Whole cell patch-clamp experiments were performed on isolated rat TG using a MultiClamp 700B (Axon Instruments) patch-clamp amplifier and pClamp 10 acquisition software (Axon Instruments). Recordings were sampled at 5 kHz and filtered at 1 kHz (Digidata 1322A, Axon Instruments). Pipettes (OD: 1.5 mm, ID: 0.86 mm, Sutter Instrument) were pulled using a P-97 puller (Sutter Instrument) and heat polished to 2.5 – 4 MΩ resistance using a microforge (MF-83, Narishige). Series resistance was typically <7 MΩ and was compensated 60–80%. All recordings were performed at room temperature. A Nikon TE2000-S Microscope equipped with a mercury arc lamp (X-Cite® 120) was used to identify FG-labeled dural afferents. Data were analyzed using Clampfit 10 (Molecular Devices) and Origin 8 (OriginLab). Pipette solution contained (in mM) 140 KCl, 11 EGTA, 2 MgCl2, 10 NaCl, 10 HEPES, 2 MgATP, and 0.3 Na2GTP, 1CaCl2 pH 7.3 (adjusted with N-methyl glucamine), and was ~ 320 mosM. External solution contained (in mM) 135 NaCl, 2 CaCl2, 1 MgCl2, 5 KCl, 10 Glucose, 5 HEPES, and 5 MES, pH 7.4 (adjusted with N-methyl glucamine), and was ~ 320 mosM. 5 HEPES/5 MES (4-morpholineethanesulfonic acid) buffer was used to prepare extracellular solutions with pH ranging from 6.0 to 7.4. Solutions were rapidly changed during recordings using gravity-fed flow pipes positioned near the cell and controlled by computer driven solenoid valves. The solution exchange time was ~ 20 ms. No currents were observed when solutions were switched from pH 7.4 to pH 7.4 using our drug application system. A cutoff of 20 pA was selected as a minimum amplitude for response for all the experiments based on preliminary experiments measuring currents in response to pH 7.1 application. Current amplitudes were slightly larger than 20 pA at pH 7.1, and this value was selected to capture an accurate representation of pH 7.1-responsive neurons.

Behavior test

Rats were acclimated to suspended Plexiglas chambers (30cm long × 15cm wide × 20 cm high) with a wire mesh bottom (1 cm2). Ten μl of vehicle or testing solution was injected through an injection cannula (28GA, #C313I; Plastics One) cut to fit the guide cannula. Withdrawal thresholds to probing the face and hind-paws were determined at 1-hour intervals after administration. A behavioral response to calibrated von Frey filaments applied to the midline of the forehead, at the level of the eyes, was indicated by a sharp withdrawal of the head. Paw withdrawal (PW) thresholds were determined by applying von Frey filaments to the plantar aspect of the hind-paws, and a response was indicated by a withdrawal of the paw. The withdrawal thresholds were determined by the Dixon up-down method. Maximum filament strengths were 8 and 15 gm for the face and hind-paws, respectively.

Data analysis

All data are presented as means ± SEM unless otherwise noted. Behavioral studies among groups and across time were analyzed by two-factor ANOVA. Data were converted to area over the time-effect curve and normalized as a percentage of the pH 5 – treated group to allow for multiple comparisons. Significant differences between groups were assessed by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison post hoc.

Drugs

Fluorogold was purchased from Fluorochrome, LLC. and dissolved in synthetic interstitial fluid (pH 7.4, 310 Osm) to 4%. Amiloride was purchased from Sigma. Amiloride was dissolved in DMSO to 1M as a stock solution and diluted to designated concentration. Final DMSO concentration never exceeded 0.1% for patch clamp experiments. Capsazepine was from Ascent Scientific and AMG-9810 was from Tocris. For the behavioral experiments, stock amiloride solutions (1 M in DMSO) and stock AMG-9810 solutions (100 mM in DMSO) were prepared and diluted in pH 5 synthetic interstitial fluid (SIF) to the final concentration of 10 mM and 1 mM, respectively. Vehicle control was pH 5.0 SIF with 1% DMSO.

Results

Extreme pH sensitivity of dural afferents

Patch clamp electrophysiology was performed on trigeminal ganglion neurons (TG) in culture from rats in which fluorogold was previously applied onto the dura. Retrogradely-labeled cells were selected for recording. Several important controls were performed for this technique to confirm that recorded cells were dural afferents. 1) Holes were made in the skull where a thin layer of bone was left intact at the bottom such that fluorogold did not penetrate the skull and contact the dura. Fluorogold thus remained in the hole and could label tissues other than dura. The hole was sealed with gel foam and bone wax. Fluorogold-positive cells were rarely observed in trigeminal cultures from these animals; 2) since UV light is required to excite fluorogold and this may damage cells, we studied cells from naïve animals and labeled cells from fluorogold-labeled animals that had ASIC-like currents before and after 10 min of UV light exposure (passed through the fluorogold filter cube) and have seen no significant differences in the properties of these cells following UV light exposure. Importantly, under typical experimental conditions the normal length of time cells were exposed to UV light during the process of locating fluorogold-positive neurons was less than 1 min; 3) we compared our preliminary patch-clamp results to data we obtained using Dil as a retrograde tracer (this tracer was used in [14]) and have observed no significant differences; and 4) we observed identical properties, including rapidly activating and rapidly desensitizing ASIC-like currents with amplitudes above 20 nA as well as action potential firing upon decreased pH application, in a fraction of randomly-selected trigeminal neurons taken from unlabeled animals (the innervation target of these neurons is unknown), indicating that the properties that we observed in retrogradely-labeled cells did not occur due to the labeling process. Thus, fluorogold-positive neurons were used for this study since we believe that these cells are dural afferents and that they are not significantly altered by the retrograde labeling.

Examples of currents evoked by a 5s step from pH 7.4 to the indicated pH from a representative dural afferent are shown in Figure 1A. Among 160 dural afferents from 11 rats, 80% exhibited ASIC-like pH 6-evoked currents, ranging from 0.07 to 63 nA (Figure 1B). Approximately 50% of the dural afferents generated currents with amplitudes well above 20 nA. Most of the dural afferents that exhibited pH 6.0-evoked currents also exhibit pH 6.8-evoked currents, ranging from 0.03 to 6.6 nA (Figure 1B). To calculate the percentage of neurons responding with inward currents to pH 6.9, 7.0 and 7.1, the minimum amplitude for response was set at 20 pA and neurons generating ASIC-like currents larger than 20 pA were counted as positive. In response to pH 6.9, 7.0 and 7.1, 73%, 56% and 30% of dural afferents exhibited currents, respectively (Figure 1B, n = 30). Densities of pH 6.0-evoked currents in dural afferents were determined with some cells demonstrating up to 1058 pA/pF (Figure 1C) suggesting a high density of channel expression.

Figure 1
The action of modest pH stimuli on dural afferents. A) Recordings from a dural afferent in response to a 5s pH step from 7.4 to 6.0, 6.8, 6.9, 7.0 and 7.1, respectively. B) Percentage of dural afferents responding to step from pH 7.4 to pH 6.0, 6.8, 6.9, ...

pH-evoked currents in dural afferents exhibit variable kinetics

The pH-evoked currents in dural afferents were fast inactivating suggesting the presence of ASIC1 or ASIC3-containing subtypes. To attempt to determine which subtype mediates these pH-evoked currents, the time constant for recovery from desensitization at pH 6.0 was measured. The decay time constant varied in dural afferents (Figure 2A) and single exponentials fit to the data showed time constants ranging from 142 to 3102 ms (Figure 2B). However, 75% of the dural afferents (n = 128) had decay time constant of 0 – 500 ms (Figure 2B). Further analysis of the currents with decay time constant less than 500 ms showed that 18.75 % and 44.53% of the dural afferent displayed decay time constant between 100 – 200 ms and 200 – 300 ms, respectively (Figure 2B). Prior work has shown that homomeric ASIC3 decay time constants average 320 ± 70 ms, which suggests the presence of ASIC3 homomers in dural afferents. Several lines of evidence indicate that pH evoked currents with decay time constants less than 300 ms are mediated by ASIC3 heteromers. The decay time constants for ASIC1a/ASIC3, ASIC1b/ASIC3, ASIC2a/ASIC3 and ASIC2b/ASIC3 average 160 ± 30, 230 ± 10, 190 ± 20 and 230 ± 20 ms, respectively [15]. The mean decay time constant of wild type DRG pH-evoked current was shorter than that of homomeric ASIC3 transfected in COS-7 cells and was mimicked by coexpression of ASIC3 with other ASIC subunits [1]. Additionally, 25% of dural afferents had time constants that suggest the presence of other ASIC subunits as they were longer than those shown previously for channels containing ASIC3. Taken together, the extreme sensitivity of dural afferents to changes in pH along with the desensitization time constants suggests that pH-evoked currents in dural afferents are most likely mediated by ASICs but the exact makeup of these subtypes is yet to be determined.

Figure 2
Dural afferents pH evoked currents exhibit variable kinetics. A) Examples of acid evoked currents from two representative dural afferents in response to a 5s pH step from 7.4 to 6.0. ASIC3 like (left) and ASIC1a like (right) B) Histograms showing the ...

pH-evoked currents in dural afferents are blocked by the ASIC antagonist amiloride

In order to further determine whether dural afferent pH-evoked currents were mediated by ASICs, the effects of amiloride, a non-specific blocker of the ENaC/DEG channels [17], were determined. Current amplitude in the presence of the antagonists was normalized to the average current amplitude in response to the preceding control pH applications. As shown in Figure 3, ,11 mM amiloride reversibly blocked the pH 6.0-evoked current (Figure 3A and C; average block 92% at 1 mM and 58% at 10 μM), while 10 μM capsazepin or 10 μM AMG9810 produced virtually no effect (Figure 3B, C; average block 3.05% and 1.26%, respectively). Previous studies have shown that 10 μM AMG 9810 fully blocks proton-induced TRPV1 activation [12] thus the lack of effect of either capsazepine or AMG-9810 indicates that TRPV1 channel do not contribute to the pH-evoked currents in dural afferents at these proton concentrations.

Figure 3
Amiloride blockade of pH 6.0 evoked currents in dural afferents. A–C) pH was stepped from 7.4 to 6.0 for 1s or 5s every 20s. pH 6.0 evoked current in a representative dural afferent is reversibly blocked by 1 mM amiloride (A) but not 10 μM ...

Amiloride block of ASIC currents is less evident at higher pH [31]. The mechanism by which this occurs is not clear but this demonstrates that there is unusual pharmacology between amiloride and ASICs that appears to depend on the pH used as a stimulus i.e. amiloride concentrations that block ASIC currents at pH 6.0 are not necessarily the same as those that block currents at pH 7.0. Current evoked by pH 6.9 application was partially blocked by 1 mM amiloride (53% block compared to 92% block at pH 6.0 shown above), but not by 10 μM capsazepine or 10 μM AMG-9810 (Figures 4A and 4B). These data indicate that pH-evoked currents in dural afferents at these higher pH values were also generated by ASICs and not TRPV1. In transfected cells, ASIC3 is known to generate a “window current” due to a window of overly between the activation and inactivation curves around neutral pH. This property allows the channel to generate sustained depolarizing currents for at least 20 mins. Previously, amiloride has been shown to enhance the window current in cells transfected specifically with ASIC3 subunits [31]. Similar effects were observed here (Figure 4C) since the pH 7.0-evoked sustained currents were increased by 1 mM amiloride. Since the decay time constant of pH 6.0-evoked currents in dural afferents were mostly within the range of ASIC3-containing subtypes and amiloride could increase dural afferent pH 7.0-evoked window current, the subtypes responsible for these currents in dural afferents most likely contain ASIC3.

Figure 4
Amiloride exhibits paradoxical effect on higher pH. A) pH 6.9 evoked current in a representative dural afferent is blocked by 1 mM amiloride. B) The percentage of pH 6.9 evoked peak current amplitude blocked by 1 mM amiloride (n = 9), 10 μM capsazepine ...

pH-evoked firing of action potentials in dural afferents

Current clamp recordings were performed to determine the effects of decreased pH on membrane excitability. Responses of cells to application of solutions of different pH were initially performed under voltage-clamp conditions to determine the current amplitudes (Figure 1A). Subsequently, responses to different pH applications were recorded in the current-clamp configuration. The percentage of dural afferents, firing action potentials at different pH values was plotted in Figure 5B. In 53% of the dural afferents, a short burst of action potentials was rapidly evoked by a 5s application of pH 6.8 (Figure 5A). In 30% of the dural afferents, pH 6.9 evoked either a single action potential or a burst of action potentials. Remarkably, pH 7.0 also evoked action potentials in 7% of the dural afferents. No action potentials were observed in response to application of pH 7.1 solutions. However, this pH was often able to evoke small membrane depolarizations in dural afferents (Figure 5B).

Figure 5
pH evoked depolarization and firing of action potentials in dural afferents. A) pH evoked depolarization and firing of action potential recorded in a representative dural afferent by a 5s step from pH 7.4 to indicated pH. The 4 traces are on same vertical ...

pH evoked sustained current in dual afferents

In retrogradely-labeled dural afferents which exhibited pH 7.0-evoked currents, 80% (n = 32) exhibited sustained currents during prolonged application with no sign of development of complete desensitization by the end of application (a sustained pH 7.0-evoked current is shown in a representative dural afferent in Figure 6). This 60 sec sustained current (Figure 6A) was able to evoke a sustained 60 sec membrane depolarization (Figure 6B), which was consistent among the cells tested in both voltage and current clamp with prolonged application of pH 7.0 solutions. Given that these window currents are able to produce sustained membrane depolarization, and window currents can presumably last for the duration of exposure of the cell to moderate pH values, these properties may be important in prolonged afferent signaling during migraine headache in the presence of decreased dural pH.

Figure 6
Small pH changes evoke sustained current in dural afferents. A) The sustained current is undiminished throughout a 60s stimulus to pH 7.0 (beginning and ending pH is 7.4) in a representative cell. B) In turn, 60s stimulus to pH 7.0 evoked an undiminished ...

Cutaneous allodynia following acidic stimulation of the dura

Application of pH 5 SIF solution to the dura produced significant (p < 0.0001) time dependent and reversible reductions in withdrawal thresholds to tactile stimuli applied to the face or the hind-paws (Figs 7A, B) compared with pH 7.4 SIF application. Maximal effects occurred 2 hours after pH 5 application, and facial and hind-paw responses approached baseline by 5 hrs after pH 5 application (Figure 7A, B). Coapplication of amiloride (100 nmol) with pH 5 prevented facial and hind-paw cutaneous allodia (Figure 7C). In contrast, AMG-9810 (10 nmol) application did not prevent pH 5 induced facial and hind-paw allodynia (Figure 7C). To test that the AMG-9810 dose used here was sufficient to block TRPV1 activity, we examined facial and hind-paw allodynia produced following application of 0.01 nmol capsaicin to the dura. AMG-9810 significantly blocked the capsaicin – induced decrease in facial and hind-paw withdrawal threshold (p < 0.01, See Supplementary Figure 1.) indicating that this dose is sufficient to block any TRPV1-mediated contribution to the behavior shown in Figure 7.

Figure 7
Application of pH 5.0 SIF solution to the dura elicited cutaneous allodynia via activation of ASICs. Withdrawal thresholds to tactile stimuli applied to the face A) and the hind-paws B) were measured in rats before and immediately after dural application ...

Discussion

Understanding the mechanisms that directly activate primary afferent neurons innervating the cranial meninges is important in understanding the events that initiate migraine headache. The studies described here demonstrate that even small decreases in extracellular pH are able to directly excite primary dural-afferent neurons via the opening of ASICs. This is the first study providing experimental evidence that ASICs are important in dural afferent signaling and these findings further suggest that decreased pH within the dura is an initiating factor in the pathophysiology of migraine headache.

The expression of ASIC channels on dural afferents would allow these neurons to immediately respond to changes in pH within the dura thus initiating afferent signaling. However, the source of a change in pH within the dura is not known. Cortical-spreading depression (CSD), defined as a spreading wave of cortical excitation followed by depression of neuronal activity, has been linked to migraine, especially to migraine aura [2]. Cortical spreading depression has been shown to be accompanied by dural ischemia [18] which could produce a drop in dural pH. Given the fact that dural afferents express a high density of ASIC channels and ASICs are extremely sensitive to pH change, even small decreases in pH resulting from dural ischemia can activate ASIC channels and initiate signaling.

Alternatively, recent studies support the hypothesis that episodes of local sterile meningeal inflammation contribute to migraine headache pathogenesis [19]. Mast cells are known to reside mostly within the dura compared to other meningial layers [9; 28] and they have been demonstrated to be in direct contact with afferent endings within the dura [24]. Mast cell degranulation is hypothesized to be an early event leading to the activation and sensitization of dural afferents. Mast cell degranulation is known to occur following stress (via the release of corticotrophin releasing factor or CRF), CGRP release, nitroglycerin infusion, and increased estrogen levels, all factors associated with migraine in human [19]. Prior work has shown that the intragranular pH of isolated mast cells was 5.55 ± 0.06 [8], which makes it possible that mast cell degranulation could acidify the environment surrounding sensory nerve endings. The studies described here demonstrate that changes in pH from 7.4 to pH 6.8, 6.9 or 7.0 alone were sufficient to directly excite many dural afferents and to produce sustained membrane depolarization in others. Thus, even small changes in pH due to the release of the acidic mast-cell granular contents could lead to activation of dural afferents via opening of ASICs. Further acidification as well as sensitization of dural afferents by other mast-cell derived substances [20] could enhance this activation leading to increased pH-induced excitation of dural afferents. Given the clear temporal dissociation between the short duration of action potential firing shown here and the time course of the behavioral response (as well as the time course of migraine headache), sensitization of these responses leading to prolonged excitation may better explain how ASIC-dependent signaling could mediate many hours of pain.

Protons can activate both ASICs and TRPV1. TRPV1 is presumably expressed on dural afferents since these neurons have been shown to respond to capsaicin [3]. However, several lines of evidence support a role for ASICs at the pH values used in this study. The decay time constants of dural afferent pH evoked current were within the range of ASICs. The pH-evoked currents were not blocked by capsazepine or AMG-9810 but were blocked by amiloride. Additionally, amiloride enhanced dural afferent pH 7.0-evoked sustained current. While the mechanism by which amiloride increases sustained currents at this pH is unknown, this effect was observed in ASIC3-containing channels previously [31]. Taken together, these findings further support the conclusion that pH-evoked currents in dural afferents are mediated by ASICs.

The studies described here also show that dural afferents are able to generate window currents at pH 7.0. These window currents have been shown to last for at least 20 min at pH 7.0 [31] and would presumably last as long as the pH stimulus is present. Although the pH that might be achieved within the dura prior to or during migraine headache is not known, pH 7.0 is not far from normal physiological pH. Further, sustained currents and membrane depolarizations were observed at pH 7.1. Although these currents/depolarizations were not able to evoke action potentials under the normal, non-sensitized recording conditions used here, other pathological events may also occur during migraine attack (e.g. mast-cell degranulation). This may convert sustained pH-induced depolarization to sustained firing of action potentials in sensitized neurons. Thus, persistent activity through ASICs may contribute to the sustained activation of dural afferents leading to the development of migraine headache.

The pH of different cellular compartments, bodily fluids, and organs is usually tightly regulated. Consequently, noxious afferent signaling is a strategy utilized by many systems to signal changes in pH to avoid tissue damage. Thus, it is not surprising that the expression of ASICs and the ability to generate large pH-induced inward currents is not unique to neurons innervating the dura. For example, a recent study of trigeminal ganglion neurons innervating masseter muscle showed that 64% of these neurons displayed robust ASIC-like current at pH 6.8 (average amplitude 4.9 ± 0.5 nA) and the average amplitude of pH 6.0-evoked currents in these neurons was almost 12 nA. [6]. Although our study is the first demonstration of ASIC-dependent signaling from the dura, our findings are in line with pH-induced activation of afferents innervating other tissues. These studies highlight the importance of signaling changes in pH throughout the body but unlike other tissues, the mechanisms leading to pH changes within the meninges have yet to be fully determined.

These studies also demonstrate both facial and hind-paw allodynia following application of pH 5.0 solution to the dura. The majority of migraine patients experience cutaneous allodynia during the headache phase [5]. Thus, this behavioral response represents migraine-related behavior in rats following exposure of the dura to a decrease in pH. Although the pH used in the behavioral experiments described here (pH 5.0) was significantly lower than those used in the in vitro experiments, it is not clear how quickly this solution is buffered and what pH is ultimately present at the nerve endings that are embedded within the dura. Prior studies using pH 4 solutions injected into the muscle have produced allodynia that is absent in ASIC3 knockout mice [26]. The pH stimulus in this prior study was likely rapidly buffered also but it nonetheless indicates that ASICs can mediate responses even at pH values lower than that used here The response to pH 5.0 application in the present study was blocked by the amiloride and not by AMG-9810 indicating that ASICs mediate these effects regardless of the final pH at the nerve endings. Further, these results show that despite the transient ASIC-mediated activity observed in vitro, a single application of a decreased pH solution to the dura is sufficient to produce prolonged allodynia. The exact mechanisms leading to prolonged ASIC-mediated behavior are not yet clear but will be examined in future studies.

In conclusion, the studies described here have elucidated the role of ASIC channels in mediating dural afferent activity following changes in pH. These findings provide direct evidence that decrease in extracellular pH can initiate afferent signaling from the dura and produce migraine – related pain behavior, suggesting that inhibitors of ASICs may represent new candidates for migraine therapy.

Supplementary Material

01

Supplementary Figure 1:

AMG-9810 blocked capsaicin – induced cutaneous allodynia. Withdrawal thresholds to tactile stimuli applied to the face A) and the hind-paws B) were measured in rats before and immediately after dural application of pH 7.4 SIF containing 0.01 nmol capsaicin (n = 8), pH 7.4 SIF (n = 8) or pH 7.4 SIF containing 0.01 nmol capsaicin + 10 nmol AMG9810 (n = 9). For both facial and hind-paw responses, two-factor analysis of variance indicated that response thresholds of capsaicin-treated rats were significantly (p < 0.0001) less than those of SIF-treated rats. C) Data were converted to area over the time-effect curve and normalized as a percentage of the capsaicin – treated group to allow for multiple comparisons. Significant (p < 0.05) differences among means for each group were determined by student’s t-test. Coapplication of AMG-9810 (black bars) significantly abolished behavioral signs of tactile allodynia of the face and hind-paw evoked by capsaicin (white bars) (p < 0.01).

Acknowledgments

The authors wish to acknowledge the technical support of Ning Qu. This work was supported by funding from The University of Arizona (GD), The University of Arizona Foundation (GD), and The American Pain Society (GD).

Footnotes

Conflict of interest

The authors declare that they have no conflicts of interest.

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