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 20nA 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 constant of pH 6.0-evoked currents in the majority of dural afferents was less than 500ms 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.

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, 7.0 and 7.1, respectively (n = 30). Cutoff was set at 20 pA. C) Current density (pA/pF) of pH 6.0 evoked currents in dural afferents (n = 30).

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 distribution of desensitization time constants (s) (n = 128).

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 capsazepine (B) or 10 μM AMG-9810 (C). D) The current amplitude after drug treatment was normalized to the average current amplitude in response to the preceding control pH applications. Blockade of pH 6 evoked peak current amplitude by amiloride, capsazepine or AMG-9810 was averaged (Mean ± SEM). pH 6.0 evoked current was blocked by 10 μM and 1 mM amiloride (n = 9 and 10, respectively), but not 10 μM capsazepine (n = 10) or AMG-9810 (n = 13).

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 (n = 5) and 10 μM AMG-9810 (n = 14), respectively. C) 1 mM Amiloride enhanced sustained current evoked by a pH step from 7.4 to pH 7.0.

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 scales. B) Percentage of dural afferents exhibiting firing of action potentials in response to indicated 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 depolarization in the same cell.

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 of pH 5 SIF (n = 18) or pH 7.4 SIF (n = 8). For both facial and hind-paw responses, two-factor analysis of variance indicated that response thresholds of pH 5 SIF-treated rats were significantly (p < 0.0001) less than those of pH 7.4 SIF-treated rats. C) Application of pH 5 SIF was given alone or with either amiloride (gray bars, 100 nmol, n = 10) or AMG-9810 (black bars, 10 nmol, n = 9). Vehicle control was pH 5 SIF containing 1% DMSO (white bars). Significant (p < 0.05) differences among means for each group were determined by analysis of variance followed by Dunnett’s post hoc test. Coapplication of amiloride significantly abolished behavioral signs of tactile allodynia of the face and hind-paw (p < 0.05). Cotreatment with AMG-9810 failed to prevent development of behavioral signs of tactile allodynia of the face or hind-paw.