All animal experiments were performed in accordance with the European Community Council Directive of September 22, 2010 (2010/63/EEC) and approved by the Animal Care and Use Committee of the University of Eastern Finland and the Ethics Committee of Kazan Federal University. Electrical activity of TG nerve was recorded using isolated hemiskull preparations obtained from adult (P35–36) rats as described previously (Shatillo et al., 2013). Firing activity was recorded from the nervus spinosus (V3 branch of the TG nerve) which was isolated and cleaned from the dura mater. This nerve innervates a region of the medial meningeal artery, supposed to initiate migraine pain and this model is widely used to investigate molecular mechanisms of migraine pain. The isolated preparation was washed for 20 min with Krebs solution containing (in mM): 120 NaCl; 2.5 KCl; 2 CaCl₂; 1 MgCl₂; 11 glucose; 1 NaHPO₄; 24 NaHCO₃ constantly gassed with 95% O₂/5% CO₂ and the pH kept at 7.2–7.4.

TG neurons were isolated from P9–P12 rats. Animals were anesthetized and decapitated. TG ganglia were excised and enzymatically dissociated in F12 medium containing 0.25 mg/ml trypsin, 1 mg/ml collagenase, and 0.2 mg/ml DNAase (Sigma) at 37°C. Cells were plated on poly-l-lysine-coated glasses in F12 medium with 10% fetal bovine serum and cultured for 1–2 days at 37°C in an atmosphere containing 5% CO₂. During experiments cells were continuously perfused (at 2 ml/min) with a solution containing (in mM): 148 NaCl; 5 KCl; 1 MgCl₂; 2 CaCl₂; 10 HEPES; 10 D-Glucose; pH adjusted to 7.2 with NaOH. The intracellular solution for patch clamp experiments contained (in mM): 145 KCl; 2 MgCl₂; 10 HEPES; 5 EGTA; 0.5 CaCl₂; 2 Mg-ATP; 0.5 Na-GTP; 5 KCl; pH adjusted to 7.2 with KOH.

For Ca²⁺ imaging experiments cells were incubated for 40 min at 37°C in F12 medium supplemented with FBS 10% (Gibco Invitrogen, Carlsbad, CA, USA) containing fluo-3-AM (5 μM, Life Technologies, Foster City, CA, USA) followed by a 10–15 min washout period.

Capsaicin, HC 030031 and capsazepine were dissolved in dimethyl sulfoxide (DMSO), dithiothreitol (DTT)—in external solution. DMSO in used concentration did not change the nociceptive activity (Zakharov et al., 2015). All substances were purchased from Sigma-Aldrich (St. Louis, MO, USA). NaHS (Sigma-Aldrich, St. Louis, MO, USA) was used as a source of H₂S. In solution NaHS dissociates to give HS⁻ which associates with H⁺ to produce H₂S. At 20°C—22.3% of total sulfide is present as H₂S (Sitdikova et al., 2014). The real-time measurements of H₂S in the chamber using amperometric sensors indicate a rapid loss of sulfide via H₂S volatilization by bubbling with about 50% H₂S loss within 3 min (Deleon et al., 2012; Sitdikova et al., 2014). In our experiments NaHS was used in a concentration of 100 μM which yields about 11 μM H₂S in the perfusion system which constantly flows to the recording chamber. Stock solutions of NaHS were prepared immediately before each experiment and kept hermetically sealed in a dark place.

TG nerve firing was recorded using a DAM 80 amplifier (band pass 0.001–3 kHz, gain 1000; World Precision Instruments, Sarasota, FL, USA). The nervus spinosus was placed inside the fire-polished glass recording microelectrode with a tip diameter of ~150 μm, filled with Krebs solution. A recovery period of at least 15 min was used to obtain stable baseline conditions. Control recordings of meningeal spikes were performed for 10 min previous to drug application. Signals were digitized at 125 kHz using a data acquisition board NI PCI6221 (National Instruments, Austin, TX, USA), and WinEDR software (Strathclyde University, Glasgow, UK). Five standard deviations (SD) were used to set the threshold for spike detection.

TRPV1 receptors are predominantly expressed in small- and medium-diameter neurons, which were used in our patch clamp experiments. TRPV1 currents were recorded at a holding potential of −70 mV using the whole-cell configuration of the patch clamp technique. TRPV1 currents were evoked by local application of capsaicin in a concentration of 1 μM for 2 s using a fast perfusion system (Rapid Solution Changer, RSC-200, BioLogic Science Instruments, Grenoble, France), with a solution exchange rate of ~20 ms. To prevent the desensitization of TRPV1 receptors, agonist was applied at intervals of 5 min. Responses to capsaicin were measured using a HEKA-10 amplifier and HEKA Patch Master Software (HEKA Electronic, Germany).

Fluorescence signals of neurons were recorded by microscope imaging setup (TILL Photonics GmbH, Munich, Germany) with a light excitation wavelength of 488 nm using respective filters. Images were collected in a time-lapse mode (500 ms exposure time) with a 12-bit CCD camera (SensiCam, Kelheim, Germany). Fluorescent signals from single cells were quantified as ΔF/F0, where F0 is the background subtracted baseline fluorescence and ΔF is the increment over baseline. The inhibitor of TRPV1—capsazepine (10 μM), the inhibitor of TRPA1—HC 030031 (50 μM), NaHS (100 μM, 2 s) and capsaicin (1 μM, 2 s) were applied via a fast perfusion system as indicated above, followed by application of a 50 mM KCl-containing solution to differentiate neurons. Data were analyzed off-line using Origin Pro 2015 (MicroCal, Northampton, MA, USA) software.

For each experiment we used at least three independent replicates (animals) and n means the number of cells. Normality of sample data was evaluated with Shapiro-Wilk test and for equal variances using F-test Origin Pro 2015 (OriginLab Corp., Northampton, MA, USA). Differences were considered as statistically significant at p < 0.05. All values are given as mean ± SEM. Statistical significance was determined by paired Student’s t-test and Mann-Whitney test.

First, we tested if H₂S can induce the nociceptive effect in meningeal nerves using a technique of suction electrode recording from meningeal TG nerve fibers (Zakharov et al., 2015). The peripheral part of the TG nerve (nervus spinosus) was placed inside the microelectrode and the orthodromic spontaneously generated action potentials (AP; spikes) generated at the periphery were recorded (Figure 1). Bath application of 100 μM NaHS induced a significant increase in the frequency of nociceptive spikes during the first 5 min to 320 ± 88% of control (control 0.53 ± 0.08 s⁻¹, vs. 1.35 ± 0.13 s⁻¹ in the presence of NaHS; n = 10, p < 0.0001; Figures 1A,D). During the next 5 min of application, the frequency of spikes did not differ significantly from control (0.95 ± 0.30 s⁻¹, p = 0.2) and it returned to control level after 15 min of drug application (0.40 ± 0.01 s⁻¹; p = 0.3; Figure 1A).

As previous studies suggested the action of NaHS in different tissues could be mediated either by TRPV1 or TRPA1 receptors (Trevisani et al., 2005; Andersson et al., 2012; Okubo et al., 2012; Lu et al., 2014; Hajna et al., 2016), we next examined the pro-nociceptive effect of NaHS in the presence of the specific inhibitors of these receptors. Inhibition of TRPV1 receptors by capsazepine (25 μM) did not significantly change TG nerve firing (0.40 ± 0.11 s⁻¹ in control, vs. 0.32 ± 0.13 s⁻¹ in capsazepine; n = 6, p = 0.14). Remarkably, the subsequent application of NaHS in the presence of capsazepine did not affect the frequency of nociceptive firing. The frequency of AP was 0.56 ± 0.30 s⁻¹ (p = 0.5) during first 5 min of NaHS application, 0.44 ± 0.27 s⁻¹ (p = 0.85) and 0.27 ± 0.15 s⁻¹ during 10 and 15 min of application, respectively (p = 0.25; Figures 1B,D). The inhibition of TRPA1 by HC 030031 (50 μM) did not change firing itself (0.61 ± 0.11 s⁻¹ in control, vs. 0.55 ± 0.09 s⁻¹ in HC 030031; n = 7, p = 0.51), however, partially prevented the action of NaHS (increase to 161 ± 13%; 0.85 ± 0.13 s⁻¹; n = 7, p = 0.007; Figures 1C,D). The NaHS effect after inhibition of TRPA1 was significantly lower than in control conditions. During 10 and 15 min of NaHS application the frequency of AP returned to the control level (0.70 ± 0.12 s⁻¹, p = 0.13 and 0.82 ± 0.19 s⁻¹, p = 0.27, respectively; Figure 1C). These results suggested the involvement of TRPV1 and TRPA1 receptors in the pro-nociceptive firing induced by NaHS in TG nerve fibers in meninges.

Next, we studied the effect of NaHS on TRPV1 receptors in single TG neurons using patch clamp recordings. The application of the TRPV1 receptor agonist capsaicin (1 μM, 2 s) induced inward currents in 22 out of 31 neurons with an average amplitude of 1345 ± 330 pA (n = 22; Figures 2A,B). In control, the repetitive application of capsaicin (interval 5 min) revealed two fractions of neurons: in one fraction, the amplitude of TRPV1 currents did not significantly change (n = 14, Figure 2C), whereas in another fraction rundown of currents was observed (n = 8, Figure 2D). These findings are consistent with an intrinsic heterogeneity of TRPV1 receptors with different rates of desensitization which was found in isolated sensory neurons previously (Akopian et al., 2007; Storti et al., 2015). We did not reveal differences in the membrane capacitance between two groups of TG neurons (36.9 ± 4.7 pF, n = 14 vs. 33.4 ± 7.7 pF, n = 8, p = 0.67). Superfusion with NaHS (100 μM) for 5 min revealed bidirectional effects on the fractions of cells with a different rate of desensitization. In a fraction of 14 cells with a low rate of desensitization, NaHS transiently increased currents from 1578 ± 501 pA to 2357 ± 715 pA (n = 14, p = 0.0065) followed by rundown during 10 and 15 min of NaHS application (Figures 2A,C). In other eight cells, we only observed a reduction of capsaicin-induced currents after 5 min exposure to NaHS from control value of 768 ± 168 pA to 414 ± 141 pA in the presence of NaHS (n = 8, p = 0.016; Figures 2B,D). In both cases, the effect of NaHS was not washable. The activating NaHS effects on TRPV1 currents could be explained by its reducing action on disulfide bonds of the TRPV1 channel protein (Susankova et al., 2006). Indeed, pre-application of 1 mM DTT prevented the increase of the current amplitude by NaHS. Thus, in the presence of DTT, NaHS decreased the amplitude of TRPV1 currents from 1360 ± 330 pA to 1025 ± 371 pA (n = 11, p = 0.0033) by 5 min of application with further reduction to 622 ± 171 (p < 0.0002) and then to 532 ± 167 pA (p < 0.0001), by 10 and 15 min, respectively (data not shown).

To investigate the direct effect of NaHS on TRPV1 currents, NaHS (100 μM) was applied through the fast perfusion system for 2 s. In cells responding to capsaicin, NaHS induced an inward current with an average amplitude of 413 ± 114 pA (n = 12; Figure 2E). Application of the TRPV1 receptor antagonist capsazepine (10 μM) completely blocked currents induced by NaHS and capsaicin (n = 5, Figure 2E). NaHS and capsaicin-induced currents were washable from the capsazepine inhibition. Subsequent application of the TRPA1 receptor selective antagonist HC 030031 (50 μM) on the same cell did not affect currents induced by NaHS and capsaicin (n = 5, Figure 2E). These data suggest that NaHS can directly activate TRPV1 receptors in rat TG neurons.

In order to explore the action of the H₂S donor on intracellular Ca²⁺ level in a large population of isolated neurons, we applied capsaicin (1 μM) and NaHS (100 μM) for 2 s before and after inhibition of TRPV1 or TRPA1 receptors by capsazepine (10 μM) or HC 030031 (50 μM), respectively. The subsequent application of KCl (50 mM) was used to distinguish neurons from glial cells (Kilic et al., 2017). As shown in Figures 3A,B NaHS induced Ca²⁺ transients in 41% neurons (104 of 251 cells); 43% of neurons tested were also sensitive to capsaicin (107 cells; Figure 3B). In 59% of cells responding to capsaicin, NaHS also induced an increase of intracellular Ca²⁺ (63 of 107 cells). Inhibition of TRPV1 receptors by capsazepine abolished Ca²⁺ responses evoked by NaHS in 37% of cells, which responded to NaHS and in 63% of cells the increase of [Ca²⁺]_in evoked by NaHS was still observed (Figures 3A,C). After inhibition of TRPA1 receptors by HC 030031, 80% of cells still showed an increase of [Ca²⁺]_in by NaHS application and in 20% of cells the NaHS response was eliminated (Figures 3A,C). In summary, in this approach the action of NaHS was partially sensitive to TRPV1 and TRPA1 antagonists.

The TG system is directly involved in sensory and nociceptive conductance and TG nerve firing is involved in pain initiation during migraine. TRPA1 and TRPV1 receptors are widely expressed in capsaicin-sensitive sensory nerves (Huang et al., 2012). Activation of TRPV1 and TRPA1 on meningeal nerve endings induces the release of vasoactive neuropeptides CGRP and substance P and contributes to different forms of headache including migraine (Giniatullin et al., 2008; Benemei et al., 2013). Our results demonstrate that the donor of H₂S—NaHS directly increases firing in TG nerve and this effect is mainly mediated by activation of TRPV1 as capsazepine, TRPV1 antagonist, completely abolished the effect of NaHS. At the same time the TRPA1 antagonist HC 030031 partially prevented the increase of TG nerve firing indicating the involvement of TRPA1 receptors in the H₂S effect.

A number of indirect studies indicate the activation of TRPV1 and TRPA1 in afferent endings by H₂S. It was shown that NaHS similar to capsaicin induced the release of CGRP and substance P from sensory nerves in the airways of guinea pig which causes bronchoconstriction in vivo (Patacchini et al., 2004; Trevisani et al., 2005). NaHS evoked contractions of the urinary bladder by activation of capsaicin-sensitive afferents and release of sensory neuropeptides (Patacchini et al., 2004). In the guinea-pig and human colon H₂S caused mucosal Cl⁻ secretion (Schicho et al., 2006) and increased afferent firing in rat intestinal mesenteric nerves by activation of TRPV1 in afferent endings (Lu et al., 2014). On the other hand activation of TRPA1 by NaHS in afferent nerve fibers was reported to mediate an increased cutaneous blood flow by the release of CGRP and substance P in the mouse ear model (Hajna et al., 2016), whereas vasodilatory effects of H₂S were reduced in mice lacking TRPA1 receptors (Pozsgai et al., 2012).

The focal application of NaHS on TG neurons induced inward currents similar to capsaicin which were inhibited by the TRPV1 antagonist capsazepine but were not sensitive to the inhibitor of TRPA1 antagonist HC 030031, which indicates a direct activation of TRPV1 receptors by H₂S. At the same time superfusion of TG neurons with NaHS induced a bidirectional effect on capsaicin induced currents. In 63% of neurons NaHS induced an increase of the current amplitude during first minutes with subsequent desensitization, which can be explained by the reduction of disulfide bonds by H₂S. Indeed, the sulfhydryl redox agent DTT in our experiments prevented the facilitating effect of NaHS. The redox modulation of TRPV1 receptors is well-known and it was shown than DTT greatly potentiated both native and recombinant rat TRPV1 channels at extracellularly located sites (Susankova et al., 2006). H₂S is known for its reducing action which is responsible for its effects on Ca²⁺-activated K⁺ channels and NMDA-receptors (Abe and Kimura, 1996; Sitdikova et al., 2010; Kimura, 2016). In 36% of cells a constant decrease of TRPV1 currents was observed during NaHS superfusion, which may be explained by the rapid desensitization of TRPV1 receptors. This suggestion is supported by the variability in desensitization of capsaicin responses between different cell fractions. Indeed, the existence of several pools of TRPV1 receptors with slow and fast kinetics and with distinct rates of desensitization have been reported in TG neurons (Akopian et al., 2007; Storti et al., 2015; Zakharov et al., 2015) which may be determined by their lipid environment, coupling to caveolin, functional states, including redox state or phosphorylation (Storti et al., 2015). Moreover, the kinetics of TRPV1-mediated current depends on the co-expression of TRPA1 channels, with likely involvement of intracellular Ca²⁺ or other intracellular messengers affecting TRPV1 receptor desensitization (Masuoka et al., 2017). Similarly to our findings, in DRG neurons NaHS directly induced inward currents which were inhibited by capsazepine and A784168 (Lu et al., 2014). Our results suggest that H₂S is a putative agonist of TRPV1 receptors in somas of TG neurons.

Fluorescence studies demonstrate that H₂S increased the intracellular Ca²⁺ level in 41% of TG neurons cells, however, only 59% of H₂S-sensitive cells responded to capsaicin, which reflects variability of H₂S molecular targets and co-expression of other types of calcium-permeable receptors in TG neurons. Thus, in DRG neurons, the TRPV1 current densities were significantly smaller in allyl isothiocyanate (AITC)-sensitive DRG neurons than in AITC-insensitive cells and spontaneous TRPA1 channel activity inhibited the TRPV1 channels via Ca²⁺ elevation (Masuoka et al., 2017). Indeed 37% of H₂S induced Ca²⁺ transients were inhibited by capsazepine which indicates mainly activation of TRPV1 receptors. However, 20% of H₂S induced Ca²⁺ transients were also abolished by HC 030031 indicating the activation of TRPA1 receptors in a small fraction of neurons, which was supported by a number of other studies. It was shown that in TG neurons application of NaHS increased [Ca²⁺]_in in 20–42% of capsaicin-sensitive neurons with close correspondence between neurons that responded to NaHS and to AITC (Hajna et al., 2016). NaHS also evoked inward currents in DRG neurons and in CHO cells expressing TRPA1 receptors, which were inhibited by a TRPA1 antagonist (Miyamoto et al., 2011; Andersson et al., 2012; Ogawa et al., 2012).

However, it should be noted that high concentrations of NaHS (1–10 mM) were used in those studies which may activate TRPA1 indirectly by formation of reactive oxygen species (Andersson et al., 2008). Moreover, the main mechanism of TRPA1 activation appears to be oxidation of reactive cysteine residues whereas reducing agents induce an inhibition of TRPA1 (Macpherson et al., 2007). H₂S being a reducing agent cannot induce direct oxidation of the thiol groups of proteins (Greiner et al., 2013). Recent studies report that polysulfides generated in NaHS and Na₂S solutions or by the chemical interaction of H₂S and NO are able to activate TRPA1 receptors (Hatakeyama et al., 2015; Kimura, 2016; Miyamoto et al., 2017). It appears possible therefore that the effective molecules which activate TRPA1 receptors in our and previous studies were polysulfides. However, in our experiments relatively low concentration of NaHS (100 μM, effectively 11 μM, see “Materials and Methods” Section) were used which probably is insufficient to generate sufficient amounts of polysulfides to activate TRPA1. Moreover, TRPV1 stimulation by H₂S can cause Ca²⁺ dependent desensitization of TRPA1 receptor as TRPA1 is highly co-expressed with TRPV1 (Akopian et al., 2007; Palazzo et al., 2013).

The gating machinery of the TRPV1 receptor is complicated as different ligands are acting at distinct extra- and intracellular sites. The sites responsible for the reducing action of DTT are located at the extracellular part of the TRPV1 protein (Susankova et al., 2006) whereas capsaicin acts from the intracellular side (Gavva et al., 2004). Thus, the S512Y and Y511A point mutations at the intracellular part of the S3 segment were able to eliminate capsaicin sensitivity (Jordt and Julius, 2002). As the effect of NaHS was inhibited by capsazepine, a competitive antagonist of the TRPV1 receptor with structural similarities to capsaicin (Pingle et al., 2007), we propose that H₂S activates the gating of the TRPV1 receptor at the same locus as capsaicin. However, the exact mechanisms of H₂S action on the TRPV1 receptor have to be determined in future experiments.

In summary, our data suggest that H₂S induces pro-nociceptive firing in the peripheral part of the TG nerve through activation of TRPV1 and TRPA1 receptors. This is consistent with the ability of H₂S to induce membrane currents and Ca²⁺ transients in cell bodies of TG neurons, which were mediated by TRPV1 and TRPA1 receptors. The endogenous H₂S producing enzyme CBS is abundantly expressed in rat TG neurons (Feng et al., 2013) and it is known that inflammation upregulates CBS expression in TG neurons at both protein and mRNA levels (Miao et al., 2014). A similar increase of CBS expression was observed also in rat DRG neurons after streptozotocin (Zhang et al., 2013) and complete Freund adjuvant treatments (Qi et al., 2013). We suggest that up-regulation of CSE or CBS during migraine related neuro-inflammation in meninges generates H₂S resulting in activation of pro-nociceptive TRPV1 and TRPA1 receptors. Associated release of neuropeptide CGRP from TRPV1/TRPA1 positive peptidergic nerve fibers should further support both neuronal sensitization (Giniatullin et al., 2008) and the long-lasting nociceptive firing underlying migraine pain. Therefore, targeting the CBS-H₂S-TRP signaling in the trigemino-vascular system might represent a novel therapeutic strategy for alleviation of TG pain.