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Keywords:

  • glycinergic mIPSCs;
  • icilin;
  • pre-synaptic facilitation;
  • trigeminal subnucleus caudalis;
  • TRPA1

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Statement of conflicts of interest
  8. References

J. Neurochem. (2012) 122, 691–701.

Abstract

The effect of icilin, a potent agonist of transient receptor potential ankyrin 1 (TRPA1) and TRPM8, on glycinergic transmission was examined in mechanically isolated rat medullary dorsal horn neurons by use of the conventional whole-cell patch-clamp technique. Icilin increased the frequency of glycinergic spontaneous miniature inhibitory post-synaptic currents (mIPSCs) in a dose-dependent manner. Either allyl isothiocyanate(AITC) or cinnamaldehyde, other TRPA1 agonists, also increased mIPSC frequency, but the extent of facilitation induced by AITC or cinnamaldehyde was less than that induced by icilin. However, menthol, a TRPM8 agonist, had no facilitatory effect on glycinergic mIPSCs. The icilin-induced increase in mIPSC frequency was significantly inhibited by either HC030031, a selective TRPA1 antagonist, or ruthenium red, a non-selective transient receptor potential channel blocker. Icilin failed to increase glycinergic mIPSC frequency in the absence of extracellular Ca2+, suggesting that the icilin-induced increase in mIPSC frequency is mediated by the Ca2+ influx from the extracellular space. In contrast, icilin still increased mIPSC frequency either in the Na+-free external solution or in the presence of Cd2+, a general voltage-dependent Ca2+ channel blocker. The present results suggest that icilin acts on pre-synaptic TRPA1-like ion channels, which are permeable to Ca2+, to enhance glycinergic transmission onto medullary dorsal horn neurons. The TRPA1-like channel-mediated enhancement of glycinergic transmission in medullary dorsal horn neurons would contribute to the regulation of pain information from the peripheral tissues.

Abbreviations used
[Ca2+]terminal

intraterminal Ca2+concentration

15d-PGJ2

15-deoxy-Δ12,14-prostaglandin J2

AITC

allyl isothiocyanate

APV

DL-2-amino-5-phosphonovaleric acid

CNQX

6-cyano-7-nitroquinoxaline-2,3-dione

DRG

dorsal root ganglia

ECl

Cl equilibrium potential

Iicilin

icilin-induced membrane currents

I-V

current-voltage

K-S

Kolmogorov-Smirnov

RR

ruthenium red

SG

substantia gelatinosa

TG

trigeminal ganglia

TRP

transient receptor potential

TRPA1

transient receptor potential ankyrin 1

TTX

tetrodotoxin

Vc

trigeminal subnucleus caudalis

VDCC

voltage-dependent Ca2+channel

VH

holding potential

Trigeminal sensory nuclei of the brainstem are divided into main sensory, oral, interpolar, and caudal nuclei (Olszewski 1950). Among them, the trigeminal subnucleus caudalis (Vc) extensively receives primary afferents including Aδ- and C-fibers from orofacial tissues, and orofacial nociceptive transmission mediated by the excitatory neurotransmitter glutamate is mainly processed in neurons within the Vc (Jacquin et al. 1986; Ambalavanar and Morris 1992; Crissman et al. 1996). Neurons in the substantia gelatinosa (SG) region of the Vc are mainly interneurons that also receive primary afferents, and project their axon terminals to the SG and adjacent laminae (Li et al. 1999). Although the reason why the Na+-free external solution increases the basal frequency of mIPSCs should be further elucidated, changes in ionic gradient or pH by removing extracellular Na+ might affect the probability of spontaneous glycine release (see also Doi et al. 2002; Jang et al. 2006; Sinning et al. 2011). Therefore, changes in the excitability of medullary dorsal horn neurons via a network of local interneurons would play an important role in the processing of nociceptive transmission (Furue et al. 2004). In a line with this idea, the application of glycine and GABAA receptor antagonists results in the enhancement of the excitatory response elicited by primary afferent stimulation in medullary dorsal horn neurons (Grudt and Williams 1994). In addition, the dysfunction in glycinergic inhibitory transmission within the medullary dorsal horn is known to induce mechanical allodynia (Miraucourt et al. 2007, 2008). These results suggest that glycinergic, in addition to GABAergic, inhibitory transmission is involved in the SG neuronal network within the Vc.

Transient receptor potential (TRP) channels are non-selective cation channels, and multiple types of TRP channels have been identified in the central and peripheral nervous system (Clapham et al. 2005; Minke 2006). They are classified in six main subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPML (mucolipin), TRPP (polycistin), and TRPA (ankyrin) (Clapham et al. 2005; Minke 2006). Of them, TRP ankyrin 1 (TRPA1), which is predominantly expressed on sensory neurons such as dorsal root ganglia (DRG) and trigeminal ganglia (TG) (Story et al. 2003; Kim et al. 2010), is initially known to be activated by noxious cold (Story et al. 2003). However, TRPA1 is also activated by a number of chemical compounds, such as mustard oil, ginger, garlic, cinnamon oil, and icilin (Bandell et al. 2004; Jordt et al. 2004; Bautista et al. 2006). As TRPA1 is a non-selective cation channel permeable to Na+ and Ca2+ (Wang et al. 2008; Karashima et al. 2010), its activation leads to a membrane depolarization. For example, the activation of TRPA1 depolarizes central terminals of primary afferent fibers to enhance glutamate release onto spinal dorsal horn neurons (Kosugi et al. 2007; Jiang et al. 2009). On the other hand, TRPA1 is not found in central neurons including the spinal cord as well as brainstem (Patapoutian et al. 2003; Story et al. 2003; Kobayashi et al. 2005), although TRPA1 has been recently observed in the post-synaptic dendrites of SG neurons in the medullary and spinal dorsal horn (Kim et al. 2010). In this study, therefore we have examined the effect of icilin, a TRPA1 agonist on spontaneous glycine release in medullary dorsal horn neurons. The present results suggest that icilin acts on pre-synaptic TRPA1-like ion channels, which are insensitive to some TRPA1 agonists, but blocked by general TRPA1 antagonists, to enhance glycinergic transmission onto medullary dorsal horn neurons.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Statement of conflicts of interest
  8. References

Preparation

All experiments complied with the guiding principles for the care and use of animals approved by the Council of the Physiological Society of Korea and the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and every effort was made to minimize both the number of animals used and their suffering.

Sprague Dawley rats (12–16 days old, either sex, Samtako Bio Korea, Osan, Korea) were decapitated under ketamine anesthesia (100 mg/kg, i. p.). The brainstem was carefully removed and placed into an ice-cold incubation medium (see Solutions) saturated with 95% O2 and 5% CO2. Transverse slices (400 μm) containing the Vc were made from the brainstem using a microslicer (VT1000S; Leica, Nussloch, Germany), and they were kept in an incubation medium (see Solutions) saturated with 95% O2 and 5% CO2 at (22–24°C) for at least 1 h before the mechanical dissociation. Details of mechanical dissociation have been described previously (Rhee et al. 1999; Akaike and Moorhouse 2003). Briefly, slices were transferred into a culture dish (Primaria 3801; Becton Dickinson, Rutherford, NJ, USA) containing a standard external solution (see Solutions), and the SG region of the Vc was identified under a binocular microscope (SMZ-1; Nikon, Tokyo, Japan). Mechanical dissociation was accomplished using a custom-built vibration device and a fire-polished glass pipette oscillating at 50–60 Hz (0.3–0.5 mm) on the surface of the SG region. The slices were removed and the mechanically dissociated neurons were left for 15 min to allow the neurons to adhere to the bottom of the culture dish. These dissociated medullary dorsal horn neurons retained a short portion (∼ 50 μm in length) of their proximal dendrites.

Electrical measurements

All electrical measurements were performed using conventional whole-cell patch recordings and a patch-clamp amplifier (Axopatch 200B; Molecular Devices, Union City, CA, USA), as described previously (Choi et al. 2007; Bae et al. 2010). Neurons were voltage clamped at a holding potential (VH) of 0 mV, except where indicated. Patch pipettes were made from borosilicate capillary glass (G-1.5; Narishige, Tokyo, Japan) using a pipette puller (P-97; Sutter Instrument Co., Novato, CA, USA). The resistance of the recording pipettes filled with internal solution was 4–6 MΩ. The liquid junction potential and pipette capacitance were compensated for. Neurons were viewed under phase contrast on an inverted microscope (TE2000; Nikon). Membrane currents were filtered at 1 kHz, digitized at 4 kHz, and stored on a computer equipped with pCLAMP 10 (Molecular Devices). During the recordings, 10 mV hyperpolarizing step pulses (30 ms in duration) were periodically applied to monitor the access resistance. All experiments were performed at (22–25°C).

Data analysis

Glycinergic spontaneous miniature IPSCs (mIPSCs) were counted and analyzed using the MiniAnalysis program (Synaptosoft, Inc., Decatur, GA, USA) as described previously (Jang et al. 2002; Choi et al. 2007; Bae et al. 2010). Briefly, mIPSCs were detected automatically using an amplitude threshold of 10 pA, and then visually accepted or rejected based upon the rise and decay times. Basal noise levels during voltage-clamp recording were less than 10 pA. The average values of both the frequency and amplitude of mIPSCs during the control period (5–10 min) were calculated for each recording, and the frequency and amplitude of all the events during the icilin application (1–2 min) were normalized to these values. The inter-event intervals and amplitudes of a large number of synaptic events obtained from the same neuron were examined by constructing cumulative probability distributions and then compared using the Kolmogorov-Smirnov (K-S) test with Stat View software (SAS Institute, Inc., Cary, NC, USA). Numerical values are provided as the mean ± SEM using values normalized to the control. Significant differences in the mean amplitude and frequency were tested using Student’s paired two-tailed t-test using absolute values rather than normalized ones. Values of p < 0.05 were considered significantly different.

RT-PCR

Total RNA was extracted from the TG and Vc of Sprague Dawley rats (12–16 days old) using QIAzol solution (QIAGEN, Valencia, CA, USA). Isolated RNA (2 μg each) was reverse transcribed using the Omniscript reverse transcription kit (QIAGEN). The resultant cDNA was amplified using gene-specific primer pairs with GoTaq Flexi DNA polymerase (Promega, Madison, WI, USA) as described by the manufacturer. The primer sequences were 5′-CCC CAC TAC ATT GGG CTG CA-3′ and 5′-CCG CTG TCC AGG CAC ATC TT-3′ for TRPA1, 5′-AAG TCC CTC ACC CTC CCA AAA G-3′ and 5′-AAG CAA TGC TGT CAC CTT CCC-3′ for β-actin, The reaction was performed as described previously (Dong et al. 2010).

Solutions

The ionic composition of the incubation (or bath) solution consisted of (in mM) 124 NaCl, 3 KCl, 1.5 KH2PO4, 24 NaHCO3, 2 CaCl2, 1.3 MgSO4, and 10 glucose saturated with 95% O2 and 5% CO2. The pH was about 7.45. The standard external solution was (in mM) 152 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 Hepes. The Ca2+-free external solution was (in mM) 150 NaCl, 3 KCl, 2 EGTA, 3 MgCl2, 10 glucose, and 10 Hepes. The Na+-free external solution was (in mM) 150 N-methyl-D-glucamine-Cl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 Hepes. All these external solutions were adjusted to a pH of 7.4 with Tris-base. For recording glycinergic mIPSCs, these external solutions routinely contained 300 nM tetrodotoxin (TTX), 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 50 μM DL-2-amino-5-phosphonovaleric acid (APV), 10 μM SR95531, and 30 μM picrotoxin, except where indicated. GABAergic mIPSCs were recorded in the presence of 300 nM TTX, 1 μM strychnine, 10 μM CNQX and 50 μM APV, and glutamatergic mEPSCs were recorded in the presence of 300 nM TTX, 1 μM strychnine, 10 μM SR95531 and 50 μM APV. The ionic composition of the internal (pipette) solution for voltage-clamp studies consisted of (in mM) 135 Cs-methanesulfonate, 5 tetraethylammonium-Cl, 5 CsCl, 2 EGTA, 2 ATP-Mg, and 10 Hepes with a pH adjusted to 7.2 with Tris-base.

Drugs

The drugs used in this study were glycine, picrotoxin, (+)−bicuculline, strychnine, menthol, AITC, cinnamaldehyde, ruthenium red (RR), TTX, CNQX, APV, CdCl2, N-methyl-D-glucamine, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), bicuculline, ATP-Mg (from Sigma, St. Louis, MO), and icilin, HC030031, SR95531 (from Tocris, Bristol, UK). All solutions containing drugs were applied using the ‘Y–tube system’ for rapid solution exchange (Murase et al. 1989).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Statement of conflicts of interest
  8. References

Glycinergic mIPSCs in mechanically dissociated SG neurons

After the brief mechanical dissociation of the SG region of the Vc, small neurons (10–15 μm in somatic diameter) with variously shaped somata, including fusiform, triangular, and multipolar forms were found. When these neurons were held at a VH of 0 mV using a whole-cell patch-clamp technique, the spontaneous outward synaptic currents were recorded in the presence of 300 nM TTX (voltage-dependent Na+ channel blocker), 10 μM CNQX (AMPA/KA receptor blocker), 50 μM APV (NMDA receptor blocker), and 10 μM SR95531 (GABAA receptor blocker). These spontaneous currents were reversibly blocked by 1 μM strychnine (n = 4), a glycine receptor antagonist (Fig. 1a). Figure 1(b) shows typical synaptic events at various VH conditions and their current-voltage (I-V) relationship. The reversal potential of spontaneous synaptic currents estimated from the I-V relationship was −70.3 mV (n = 5). This value was similar to the theoretical Cl equilibrium potential of −78.7 mV, which was calculated from the Nernst equation using extracellular and intracellular Cl concentrations of 159 mM and 7 mM, respectively. These results indicate that the spontaneous outward synaptic currents are glycinergic mIPSCs mediated by synaptic glycine receptors.

image

Figure 1.  Glycinergic mIPSCs recorded from mechanically dissociated SG neurons. (a) A typical trace of mIPSCs observed before, during, and after the application of 1 μM strychnine at a VH of 0 mV. The standard extracellular solution contained 300 nM tetrodotoxin, 10 μM CNQX, 50 μM APV, and 10 μM SR95531. Insets: typical traces of mIPSCs in the absence (control) and the presence of strychnine with an expanded time scale. (b) i: Typical traces of glycinergic mIPSCs at various VHs. ii: A plot of the mean amplitude of mIPSCs at various VH values. The calculated reversal potential of mIPSCs was −70.3 mV, which is close to the theoretical Cl equilibrium potential (−78.8 mV). Each point was the mean and SEM from the five neurons. (c) Representative traces of current response induced by 100 μM icilin in the standard extracellular solution, in the presence of 1 μM strychnine, and in the presence of 30 μM picrotoxin. Icilin was applied at 4 min intervals, and either strychnine or picrotoxin was pre-incubated 1 min before the icilin application. (d) Current-voltage relationship of the Iicilin. A linear voltage-ramp command (−100 to 0 mV, 1 s) was applied to the same SG neuron in the absence (a) and presence (b) of 100 μM icilin. Inset represents a typical trace of Iicilin (b-a) with an expanded amplitude scale. The reversal potential of Iicilin was −78.1 mV.

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In these conditions, we examined the effect of icilin, a cooling agent that activates both TRPA1 and TRPM8 (Baraldi et al. 2010), on glycinergic mIPSCs. The application of icilin (100 μM) immediately increased the frequency of glycinergic mIPSCs and this effect rapidly disappeared after the icilin washout (Fig. 1c). Icilin (100 μM) also induced small outward membrane currents (32.7 ± 6.6 pA, n = 12), which were completely blocked by 30 μM picrotoxin, a GABAA and glycine receptor blocker (n = 6, Fig. 1c), or 30 μM bicuculline, another GABAA receptor antagonist (n = 10, data not shown). However, the icilin-induced membrane currents (Iicilin) were not affected by 1 μM strychnine (n = 6, Fig. 1c). The reversal potential of Iicilin determined by voltage-ramp experiments was similar to the theoretical ECl (Fig. 1d). On the other hand, picrotoxin (30 μM) had little effect on the frequency and amplitude of glycinergic mIPSCs, suggesting that synaptic glycine receptors are likely to be αβ heteromeric receptors (see also Pribilla et al. 1992; Rajendra et al. 1997; Jeong et al. 2003). In all subsequent experiments, picrotoxin (30 μM) was added to the standard external solution to block the Iicilin.

Icilin acts pre-synaptically to increase spontaneous glycine release

Icilin (100 μM) greatly increased the mean frequency of glycinergic mIPSCs to 1425 ± 240% of the control (p < 0.01, n = 21), but it also slightly decreased the mean amplitude of mIPSCs to 84 ± 5% of the control (p < 0.01, n = 21, Fig. 2a and b). In addition, icilin (100 μM) shifted the cumulative distribution of inter-event interval to the left (p < 0.01, K-S test, Fig. 2b), consistent with an increase in mIPSC frequency. However, icilin (100 μM) also shifted the cumulative distribution of the current amplitude to the left (p < 0.05, K-S test, Fig. 2b), consistent with a decrease in mIPSC amplitude. A decrease in mIPSC amplitude might result from the icilin-mediated post-synaptic effects, such as the direct inhibition of post-synaptic glycine receptors. As expected, icilin (100 μM) slightly but significantly decreased 30 μM glycine-induced currents (91 ± 2% of the control, n = 8, p < 0.05, Fig. 2c). On the other hand, icilin (100 μM) had no effect on the rise time (2.3 ± 0.15 ms for the control and 2.3 ± 0.14 ms for icilin, n = 15, p = 0.67) and decay time constant of glycinergic mIPSCs (8.4 ± 0.5 ms for the control and 7.7 ± 0.4 ms for icilin, n = 15, p = 0.17, data not shown). In addition, the VH did not affect the extent of icilin-induced increase in mIPSC frequency (Fig. 2d). These results suggest that icilin mainly acts pre-synaptically to increase spontaneous glycine release onto medullary dorsal horn neurons. The icilin-induced increase in mIPSC frequency was well reproducible during the repeated applications with a time interval of 10 min (first; 1708 ± 243%, second; 1651 ± 399%, third; 1508 ± 206% of the control, n = 10, data not shown). Icilin increased mIPSC frequency in a concentration-dependent manner, where icilin even at a 10 μM concentration increased glycinergic mIPSC frequency to 206 ± 43% of the control (n = 19, p < 0.05, Fig. 2e). In another set of experiments, we observed the effects of icilin on glutamatergic and GABAergic transmission in medullary dorsal horn neurons. While icilin (100 μM) had no significant effect on the frequency of GABAergic mIPSCs (Fig. 2f(i)), it greatly increased glutamatergic mEPSC frequency to 1181 ± 302% of the control (n = 12, p < 0.01, Fig. 2f(ii)).

image

Figure 2.  Effect of icilin on glycinergic mIPSCs. (a) A typical trace of glycinergic mIPSCs before, during, and after the application of 100 μM icilin. Insets: typical traces of mIPSCs in the absence (control) and the presence of icilin with an expanded time scale. (b) Cumulative probability distributions for the inter-event interval (i, < 0.01, K-S test) and current amplitude (ii, < 0.05, Kolmogorov-Smirnov test) of glycinergic mIPSCs shown in (a). Three hundred fifty events for the control and 590 events for icilin were plotted. Insets: 100 μM icilin-induced changes in mIPSC frequency (i) and amplitude (ii). Each column was the mean and SEM from 21 experiments. **< 0.01. (c) i: Typical traces of 30 μM glycine-induced currents, the absence and the presenceof 100 μM icilin. ii: The icilin-induced changes in glycine-induced peak currents. A column represents the mean and SEM from eight experiments. *< 0.05. (d) Changes of the icilin-induced increase in mIPSC frequency at VHs of 0 1mV (= 21) or -60 mV (= 7). At a VH of -60 mV, inwardly directed glycinergic mIPSCs were recorded using the high Cl ([Cl]= 70 mM)-based pipette solution. *< 0.05, **< 0.01, NS; not significant. (e) Concentration-response relationship of mIPSC frequency facilitation against icilin concentration. Each point represents the mean and SEM from 6 to 19 experiments. (f), Typical traces of GABAergic mIPSCs (i) or glutamatergic mEPSCs (ii) before, during, and after the application of 100 μM icilin. GABAergic mIPSCs were recorded at a VH of 0 mV in the presence of 300 nM tetrodotoxin, 10 μM CNQX, 50 μM APV and 1 μM strychnine. Glutamatergic mEPSCs were recorded at a VH of −60 mV in the presence of 300 nM tetrodotoxin, 50 μM APV, 10 μM SR95531 and 1 μM strychnine.

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Pharmacological properties of icilin-induced increase in glycinergic mIPSCs

Icilin is a potent agonist of TRPM8, and it can activate TRPM8 in addition to TRPA1 (Baraldi et al. 2010). Therefore, we examined the effects of various agonists on glycinergic mIPSCs. In SG neurons responding to icilin, menthol (300 μM), a specific TRPM8 agonist (Baraldi et al. 2010), had no facilitatory effect on glycinergic mIPSCs (146 ± 20% of the control, n = 6, p = 0.43, Fig. 3a and b). In addition, menthol at 30 μM to 1 mM concentrations had no facilitatory effect on glycinergic mIPSCs (data not shown). We also examined the effect of AITC, a specific TRPA1 agonist (Baraldi et al. 2010), on glycinergic mIPSCs. However, the pattern of AITC-induced increase in mIPSC frequency was distinct from that of icilin, as AITC (300 μM) elicited a long lasting increase in glycinergic mIPSC frequency (Fig. 3a). AITC increased glycinergic mIPSC frequency to 382 ± 58% of the control during the application (n = 11, p < 0.01, Fig. 3a and b). However, the AITC-induced facilitation of mIPSC frequency lasted for over 10 min after the washout of AITC (Fig. 3a). The ability of AITC to form the disulfide conjugation with cysteine residues of proteins including TRPA1 might be related to such differential facilitatory actions on spontaneous glycine release (Macpherson et al. 2007). We also examined the effect of cinnamaldehyde, which is a potent agonist of TRPA1 (Baraldi et al. 2010), on glycinergic mIPSCs. While cinnamaldehyde at 100 μM and 300 μM concentrations had no facilitatory effect on glycinergic mIPSCs (data not shown), it at a 1 mM concentration significantly increased glycinergic mIPSC frequency to 341 ± 113% of the control (n = 5, p < 0.05, Fig. 3b). However, cinnamaldehyde (1 mM) also decreased the mean mIPSC amplitude to 62 ± 5% of the control (n = 5, p < 0.05), although cinnamaldehyde had no inhibitory effect on the glycine-induced currents (data not shown).

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Figure 3.  Effects of menthol and allyl isothicocyanate (AITC) on glycinergic mIPSCs. (a) A typical trace of glycinergic mIPSCs before, during, and after the application of 100 μM icilin (upper), 300 μM menthol (middle), and 300 μM AITC. (b) Icilin (100 μM, = 11)-, menthol (300 μM, = 6)-, AITC (300 μM, = 11)-, and cinnamaldehyde (Cinna; 1 mM, = 5)-induced changes in mIPSC frequency. Each column and error bar indicates the mean and SEM. All frequencies were normalized to the control value (dotted line). *< 0.05, **< 0.01, NS; not significant.

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As icilin and AITC are known to be TRPA1 agonists, the above results strongly suggest that the icilin-induced increase in spontaneous glycine release might be mediated by putative pre-synaptic TRPA1. To verify this, we examined the effect of HC030031, a selective TRPA1 antagonist (McNamara et al. 2007; Baraldi et al. 2010), on the icilin-induced increase in spontaneous glycine release. HC030031 (100 μM) by itself slightly increased the basal frequency of glycinergic mIPSCs (215 ± 43% of the control, n = 9, p < 0.05). The extent of icilin-induced increase in mIPSC frequency (1551 ± 277% of the control, n = 9) was greatly reduced in the presence of 100 μM HC030031 (317 ± 57% of the HC030031 condition, n = 9, p < 0.01, Fig. 4a(ii) and b(i)). We also examined the effect of RR, a non-selective TRP channel blocker (Minke 2006), on the icilin-induced increase in mIPSC frequency. RR at a 3 μM concentration significantly increased the basal frequency of glycinergic IPSCs (530 ± 143% of the control, n = 9, p < 0.01). Although the reason why RR increased the basal frequency of mIPSCs should be further elucidated, changes in mitochondrial Ca2+ regulation might be related to this phenomenon as RR is known to alter the homeostasis of mitochondrial Ca2+ (Hajnóczky et al. 2006). In the presence of 3 μM RR, however, the icilin-induced increase in mIPSC frequency (897 ± 145% of the control, n = 9) was greatly reduced (125 ± 7% of the RR condition, n = 9, p < 0.01, Fig. 4a(iii) and b(ii)). On the other hand, the Iicilin was not blocked by either 100 μM HC030031 or 3 μM RR (Fig. 4c), suggesting that the Iicilin is not mediated by TRPA1.

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Figure 4.  Effects of HC030031 and ruthenium red (RR) on the icilin-induced increase in mIPSC frequency. (a) Typical traces of glycinergic mIPSCs recorded before (left) and during (right) the application of 100 μM icilin in the control external solution (i), in the presence of 100 μM HC030031 (ii), and in the presence of 3 μM RR (iii). (b) Changes of the icilin-induced increase in mIPSC frequency in the absence and presence of 100 μM HC030031 (= 9; i) and 3 μM RR (= 9; ii). Open circles and connected lines represent the individual results, whereas closed circles and error bars indicate the mean and SEM. **< 0.01. (c) Representative traces of current response induced by 100 μM icilin in the presence of 100 μM HC030031 (i, = 5) or 3 μM RR (ii, = 5). Icilin was applied at 4 min intervals, and either HC030031 or RR was pre-incubated 1 min before the icilin application. These experiments were performed in the presence of 1 μM strychnine.

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It has been well established that peripheral TRPA1 is activated by noxious cold (< 17°C) (Story et al. 2003). Therefore, we examined the effect of cold extracellular solution on glycinergic mIPSCs. However, the application of cold (15°C) extracellular solution had no facilitatory effect on mIPSC frequency (132 ± 17% of the control, n = 14, p = 0.67, Fig. 5a(i) and b(i)). Another potential endogenous candidate to activate peripheral TRPA1 might be prostaglandins, such as 15d-PGJ2, which has been recently suggested to activate TRPA1 in DRG neurons (Cruz-Orengo et al. 2008). However, 15d-PGJ2 at a 20 μM concentration had no facilitatory effect on mIPSC frequency (113 ± 12% of the control, n = 12, p = 0.15, Fig. 5a(ii) and b(ii)). In another set of experiments, we directly observed whether medullary dorsal horn neurons indeed express TRPA1 using a RT-PCR method. However, transcript for TRPA1 was not found at the Vc (Fig. 5c). The lack of detection of TRPA1 transcript was not as a result of an inadequate RNA primer used, because TRPA1 transcript was clearly detected from the TG (Fig. 5c). Taken together, TRPA1 is unlikely to be expressed in medullary dorsal horn neurons.

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Figure 5.  Effects of low temperature and 15d-PGJ2 on GABAergic mIPSCs. (a) Typical traces of glycinergic mIPSCs recorded before (left) and during (right) the application of cold (15°C) external solution (i) and 20 μM 15d-PGJ2 (ii). (b) Changes in mIPSC frequency in the absence and presence of cold (15°C) external solution (= 14; i) and 20 μM 15d-PGJ2 (= 12; ii). Open circles and connected lines represent the individual results, whereas closed circles and error bars indicate the mean and SEM. NS; not significant. (c) Total RNA was extracted from the TG and Vc and RT-PCR was performed with these RNA samples. The clear expression of TRPA1 transcript was detected at cDNA samples from the TG but not Vc.

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Mechanisms underlying the icilin-induced increase in glycinergic mIPSCs

We next examined the mechanisms underlying the icilin-induced increase in spontaneous glycine release. As an increase in the intraterminal Ca2+ concentration ([Ca2+]terminal) promotes the probability of neurotransmitter release (Wu and Saggau 1997), we first observed the effect of Ca2+-free (plus 2 mM EGTA) external solution on the icilin-induced increase in mIPSC frequency. In the Ca2+-free external solution, glycinergic mIPSC frequency slightly decreased, but the effect was not significant statistically (73 ± 20% of the control, n = 5, p = 0.24, Fig. 6a). However, the icilin-induced increase in mIPSC frequency (1547 ± 266% of the control, n = 5) was completely occluded in the Ca2+-free external solution (109 ± 6% of the Ca2+-free condition, n = 5, p < 0.01, Fig. 6a and b(i)). In addition, the extent of icilin-induced increase in mIPSC frequency was highly dependent on the extracellular Ca2+ concentration (Fig. 6b(ii)). The results suggest that the icilin-induced facilitation of spontaneous glycine release is mediated by the Ca2+ influx from the extracellular space.

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Figure 6.  Effects of extracellular Ca2+on the icilin-induced increase in mIPSC frequency. (a) Typical traces of glycinergic mIPSCs recorded before (left) and during (right) the application of 100 μM icilin in the control external solution (i) and in the Ca2+-free external solution (ii). (b) i: Changes of the icilin-induced increase in mIPSC frequency in the absence and presence of extracellular Ca2+. Open circles and connected lines represent the individual results from five experiments, whereas closed circles and error bars indicate the mean and SEM. **< 0.01. ii: Changes of the icilin-induced increase in mIPSC frequency in the presence of various concentrations of extracellular Ca2+([Ca2+]o). Each column was normalized to the respective control (dotted line) and represents the mean and SEM from 5 to 12 experiments. NS; not significant, *< 0.05, **< 0.01.

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We further examined the effect of extracellular Na+ on icilin-induced increase in mIPSC frequency. The Na+-free external solution slightly decreased the basal mIPSC frequency (84 ± 5% of the control, n = 7, p < 0.05, Fig. 7a). Although the reason why the Na+-free external solution increases the basal frequency of mIPSCs should be further elucidated, changes in ionic gradient or pH by removing extracellular Na+ might affect the probability of spontaneous glycine release (see also Jang et al. 2006; Sinning et al. 2011). However, the extent of icilin-induced increase in mIPSC frequency (1619 ± 172% of the control, n = 7) was not affected even in the Na+-free external solution (1447 ± 170% of the Na+-free condition, n = 7, p = 0.19, Fig. 7a(ii) and b(i)). Finally, we observed the effect of Cd2+, a general voltage-dependent Ca2+ channel (VDCC) blocker, on the icilin-induced increase in spontaneous glycine release. The application of 200 μM Cd2+ to SG neurons had no effect on the basal frequency of mIPSCs (89 ± 7% of the control, n = 6, p = 0.16, Fig. 7a). However, the extent of icilin-induced increase in mIPSC frequency (1363 ± 288% of the control, n = 6) was not affected even in the presence of in the 200 μM Cd2+ (1465 ± 329% of the Cd2+ condition, n = 6, p = 0.26, Fig. 7a(iii) and b(ii)). The results suggest that the icilin-induced facilitation of spontaneous glycine release is not mediated by a pre-synaptic depolarization and/or the activation of VDCCs.

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Figure 7.  Effects of Na+-free external solution and Cd2+on the icilin-induced increase in mIPSC frequency. (a) Typical traces of glycinergic mIPSCs recorded before (left) and during (right) the application of 100 μM icilin in the control external solution (i), in the Na+-free external solution (ii), and in the presence of 200 μM Cd2+(iii). (b) Changes of the icilin-induced increase in mIPSC frequency in the absence and presence of extracellular Na+ (= 7; i) and 200 μM Cd2+(= 6; ii). Open circles and connected lines represent the individual results, whereas closed circles and error bars indicate the mean and SEM. NS; not significant.

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Icilin increases action potential-dependent glycine release

Finally, to elucidate the physiological significance of the present finding, we observed the effect of icilin on action potential-dependent glycine release in the slice preparation. In the presence of 10 μM SR95531, 10 μM CNQX and 50 μM APV, glycinergic eIPSCs were recorded from SG neurons at a VH of 0 mV by the paired-pulse stimulation through a glass pipette placed near to the SG area. These eIPSCs were completely blocked by 1 μM strychnine (data not shown, see also Choi et al. 2010). In nine of 14 neurons tested, bath applied icilin (100 μM) slightly, but significantly increased the amplitude of the first eIPSCs (eIPSC1; 116 ± 4% of the control, n = 6, p < 0.05, Fig. 8a and b) and decreased the paired-pulse ratio (PPR) from 1.14 ± 0.04 to 1.08 ± 0.03 (95 ± 1% of the control, n = 6, p < 0.05, Fig. 8a and b). The results suggest that icilin acts pre-synaptically to increase the probability of glycine release in more physiological condition.

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Figure 8.  Effect of icilin on action potential-dependent glycinergic eIPSCs. (a) A typical time course of the first eIPSC (eIPSC1) amplitude (i) and PPR (eIPSC2/eIPSC1; ii) before, during, and after the application of 100 μM icilin. Insets represent typical traces of the numbered region. (b) Icilin-induced changes in the eIPSC1 amplitude (i) and PPR (ii). Each column was the mean and SEM from nine experiments. *< 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Statement of conflicts of interest
  8. References

Previous reports have shown that TRPA1 is expressed on pre-synaptic nerve terminals as well as axons in a subset of primary sensory neurons (Story et al. 2003; Kim et al. 2010). Although the activation of pre-synaptic TRPA1 enhances spontaneous glutamate release from central terminals of primary afferents onto spinal dorsal horn neurons (Kosugi et al. 2007; Jiang et al. 2009), functional roles of TRPA1 in neurotransmitter release within central neurons are still unknown. In this study, therefore we investigated the effect of icilin on spontaneous glycine release onto acutely isolated medullary dorsal horn neurons. To our knowledge, there is little information about the unspecific effects of icilin except its agonistic action on TRPA1 and TRPM8. We found that icilin significantly increased glycinergic mIPSC frequency in a concentration-dependent manner. Although icilin slightly decreased the mean amplitude of glycinergic mIPSCs, this effect would be because of the direct inhibitory action of icilin on post-synaptic glycine receptors, as icilin slightly inhibited the IGly. Considering that the reduction of amplitude below the threshold could cause a decrease in the detectable synaptic currents, the icilin-induced increase in mIPSC frequency should reflect an increase in release probability at pre-synaptic sites. Furthermore, the preparation used in this study further supports the pre-synaptic action of icilin because mechanically dissociated neurons retain cell-free pre-synaptic nerve terminals, and thus should exclude any non-presynaptic effects, such as those associated with changes in soma excitability (for review, see Akaike and Moorhouse 2003). Together, icilin is likely to act pre-synaptically to increase spontaneous glycine release onto medullary dorsal horn neurons.

On the other hand, icilin induced outward membrane currents at a VH of 0 mV in medullary dorsal horn neurons in the presence of SR95531, a competitive GABAA receptor antagonist. The voltage-ramp experiments suggest that icilin elicited the Cl currents. The Iicilin is unlikely to be mediated by glycine receptors, because strychnine had no inhibitory effect on the Iicilin. In addition, the Iicilin might be not mediated by TRPA1, as the Iicilin was not blocked by either HC030031 or RR. As the Iicilin was clearly inhibited by picrotoxin and bicuculline, GABAA receptor blockers, icilin seems to activate GABAA receptors, which are insensitive to SR95531. These results are consistent with previous studies showing that the tonic Cl currents mediated by GABAA receptors are not affected by SR95531, but blocked by picrotoxin or bicuculline (Semyanov et al. 2003; Gao and Smith 2010), suggesting that icilin activates high-affinity extra-synaptic GABAA receptors, which may be composed of α4/6 and/or δ-subunits. Further studies should be needed to reveal the detailed properties of Iicilin.

Icilin is also known to be 400–600 times more potent than menthol, a specific TRPM8 agonist (Baraldi et al. 2010), at TRP channels, and thus it can activate TRPM8 rather than TRPA1. In this study, we found that AITC, a potent TRPA1 agonist (Baraldi et al. 2010), also significantly increased glycinergic mIPSC frequency. However, menthol even at higher concentrations had no effect on glycinergic mIPSCs. Given that menthol at 10 μM–1 mM concentrations can activate TRPM8 to elicit the cation currents in DRG neurons (Okazawa et al. 2000; Peier et al. 2002; Reid et al. 2002), icilin is likely to act on pre-synaptic TRPA1 to enhance spontaneous glycine release onto medullary dorsal horn neurons. Furthermore, we found that the icilin-induced increase in mIPSC frequency was greatly reduced by the specific TRPA1 antagonist HC030031 as well as the non-selective TRP antagonist RR. Similarly, a previous study has shown that the TRPA1-mediated Ca2+ influx is blocked by either HC030031 or RR in a concentration-dependent manner (McNamara et al. 2007). In this stage, however, it remains unclear whether icilin acts on pre-synaptic TRPA1 to increase spontaneous glycine release, because the mRNA of TRPA1 is not found in central neurons (Patapoutian et al. 2003; Story et al. 2003; Kobayashi et al. 2005). Although TRPA1-like protein has been occasionally detected on dendrites of central trigeminal neurons, the cell types to which these dendrites belong are not identified (Kim et al. 2010). Similarly, a previous study has shown that AITC had no direct facilitatory effect on glycine release in spinal dorsal horn neurons (Kosugi et al. 2007). In consistent with this speculation, we found that either low temperature (15°C) or 15d-PGJ2, which are known to activate peripheral TRPA1 (Story et al. 2003; Cruz-Orengo et al. 2008), had no facilitatory effect on glycine release. Furthermore, we found that the facilitatory effects of AITC and cinnamaldehyde, much better characterized TRPA1 agonists, were modest in comparison to that of icilin. These pharmacological results suggest that icilin might act on pre-synaptic TRPA1-like other ion channels rather than TRPA1 to increase spontaneous glycine release onto medullary dorsal horn neurons. Further studies should be needed to reveal the exact expression pattern, cellular localization, and endogenous ligands of functional TRPA1 and/or TRPA1-like channels in the medullary dorsal horn region.

In general, an increase in the [Ca2+]terminal would be mediated by the influx of Ca2+ from the extracellular space via pre-synaptic VDCCs, Ca2+-permeable receptors, or ion channels. Alternatively, an increase in the [Ca2+]terminal could be accomplished by the release of Ca2+ from pre-synaptic Ca2+ stores. In mechanically isolated medullary dorsal horn neurons, the icilin-induced increase in spontaneous glycine release was highly dependent on the extracellular Ca2+ concentration as icilin had no facilitatory effect on mIPSC frequency in the absence of extracellular Ca2+. This suggests that the influx of Ca2+ from the extracellular space, rather than the Ca2+ release from pre-synaptic Ca2+ stores, plays a pivotal role in the icilin-induced increase in spontaneous glycine release. In addition, as the icilin-induced increase in mIPSC frequency was not affected either in the absence of extracellular Na+ or in the presence of Cd2+, the contribution of Ca2+ influx passing through pre-synaptic VDCCs following the icilin-induced pre-synaptic depolarization might not be involved in the facilitatory action of icilin on spontaneous glycine release. Another potential route for the entry of Ca2+ from the extracellular spaces might be other TRP channel subtypes, as TRPC channels are involved in the store-operated as well as receptor-operated Ca2+ entry (for review, Clapham et al. 2005; Minke 2006). Although it is still unknown whether icilin acts on such Ca2+ entry channels because RR is often used as a general TRP channel blocker, our present results showing the selective blockade of HC030031 suggest that such Ca2+ entry channels are not involved in the icilin-induced increase in spontaneous glycine release. Taken together, the results suggest that the icilin-induced increase in mIPSC frequency is likely to be mediated by the Ca2+ influx passing through pre-synaptic TRPA1-like channels. Although the Ca2+ permeability of pre-synaptic TRPA1-like channels is unknown in this stage, the permeability of Ca2+ to monovalent cations, e.g., PCa/PNa, of TRPA1 in nociceptive sensory neurons is known to be 0.84–5.7 (Story et al. 2003; Wang et al. 2008; Karashima et al. 2010).

In the DRG and TG, TRPA1 is predominantly expressed on a subset of small- and middle-sized neurons (Story et al. 2003; Kobayashi et al. 2005; Bautista et al. 2006; Kim et al. 2010), which are parent neurons of C- and Aδ-fibers, indicating that TRPA1 mediates not only cold sensation but also nociception from the peripheral tissues. In fact, previous studies have shown that TRPA1 is involved in inflammatory hyperalgesia and neuropathic pain (Obata et al. 2005; Eid et al. 2008; Ji et al. 2008), and that the formalin-induced pain is partially mediated by TRPA1 (McNamara et al. 2007). Furthermore, the activation of TRPA1 has been shown to enhance spontaneous glutamate release from central terminals of primary afferents (Kosugi et al. 2007; Jiang et al. 2009), although it should be revealed how pre-synaptic TRPA1 expressed on central terminals of primary afferents is activated in physiological conditions. These results suggest that the activation of TRPA1 expressed on peripheral tissues induces pain. In this study, we found that icilin acts on pre-synaptic TRPA1-like channels to increase the frequency of glycinergic mIPSCs in medullary dorsal horn neurons. Considering that the dysfunction in glycinergic inhibitory transmission induces mechanical allodynia in the medullary dorsal horn (Miraucourt et al. 2007, 2008), our present results suggest that TRPA1-like channels expressed on central inhibitory nerve terminals might reduce pain by enhancing glycinergic transmission. Although icilin also enhanced spontaneous glutamate release onto medullary dorsal horn neurons, it is still unknown whether icilin acts on peripheral TRPA1 or central TRPA1-like channels to enhance spontaneous glutamate release because excitatory nerve terminals originate from primary afferents as well as local excitatory interneurons. Further studies should be needed to verify whether central TRPA1-like channels are involved in the regulation of pain information from the peripheral tissues.

In conclusion, our present results suggest that icilin acts on pre-synaptic TRPA1-like channels, which are permeable to Ca2+, to enhance spontaneous glycine release onto medullary dorsal horn neurons. The TRPA1-like channel-mediated enhancement of glycinergic transmission in medullary dorsal horn neurons would contribute to the regulation of pain information from the orofacial tissues.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Statement of conflicts of interest
  8. References

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0028239).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Statement of conflicts of interest
  8. References