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.
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- Materials and methods
- Statement of conflicts of interest
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.