Sensory neurons display long-term hyperexcitability after crush or transection of their peripheral axons [7, 9, 10, 41–44]. During the healing process, injury-induced hyperexcitability of primary afferent neurons is present until recovery of sensory axons and reinnervation of the peripheral target is achieved . A variety of factors maintain this hyperexcitable state, which can become persistent and induce neuropathic pain in a proportion of patients [1, 45]. Changes in the expression of several voltage-dependent channels contribute to the generation of hyperexcitability in sensory neurons, and particularly in nociceptors [6, 46–53]. In contrast, how background conductances tune the excitability of sensory neurons is largely unknown. DRG and trigeminal neurons express several members of the K2P family of background K+ channels [16, 17, 20], with TREK-1 being involved in polymodal pain perception , and the TREK/TRAAK family in heat and cold pain perception . In small and medium-sized DRGs, many of which are nociceptors, TREK-2 and TRESK channels have a major contribution to total background current, while TREK-1 and TRAAK carry a smaller fraction of the current [18, 19].
We have found that among those channels, only TRESK channels are down-regulated in response to injury both after in vivo or in vitro axotomy upon cell dissociation. This decrease in channel expression is well correlated with an increase in the injury marker ATF3 [33–37] and the α2δ1 subunit of the L-type Ca2+ channel . This is similar to what had been previously described in Aplysia nociceptors, where peripheral axon injury produces persistent hyperexcitability of nociceptive neurons and a reduction of the background S-type K+ current [8, 9]. Interestingly, this current, which shares similar pharmacological and electrophysiological properties with TREK-1 channels in mammals , contributes to sensory neuron hyperexcitability by increasing Rin and decreasing rheobase current to AP firing . Also, a decrease in the S-type K+ current, which is persistently activated upon membrane depolarization (like TRESK), will favor repetitive firing of the neuron [8, 9, 18]. In contrast to the studies in Aplysia where recordings were performed in a semi-intact ganglia preparation, in the present study it was not possible to compare changes in background currents between neurons after axotomy due to the fact that acute dissociation also induces hyperexcitability in nociceptive neurons. Although we have not studied the time-dependence development of hyperexcitability in vitro after cell dissociation, this seems to appear quite early, since we could record hyperexcitable neurons as short as 3-4 h after plating. This observation has also been reported by others [11, 31] and shows a good correlation with the expression of injury markers (ATF3 and Cacna2d1), changes in TRESK expression, or the lack of difference between background currents recorded in dissociated neurons (Figure 2) in conditions where most of the background current should be carried by TRESK (at room temperature other background channels are mostly inactivated; [19, 32]). Interestingly, another study has reported up-regulation of TRESK expression in DRG neurons after several days in culture (Suppl. Fig S6 ), opening the possibility to down-regulation of TRESK channels after acute dissociation followed by recovery of their expression levels after several days in culture together with regenerative outgrowth of neurites.
Despite the proposed role of leak K+ channels in setting membrane potential, we did not found differences in resting membrane potential after axotomy, which is in agreement with the lack of difference found in the resting membrane potential of DRG neurons from wild-type or TRESK[G339R] functional knockout mice . This suggests that some compensation by other channels may be present in the knockout mouse or that TRESK has not a prominent role in setting resting membrane potential but on neuronal excitability. In fact, we observed an increase in TREK-1 expression in injured neurons compared with sham surgery (Figure 1B), which might compensate for TRESK reduction to maintain resting membrane potential. As mentioned, our electrophysiological recordings were done at room temperature, were some K2P channels appear to have a very low open probability . If some compensation by other K2P channels was present, we might have underestimated their contribution when recording membrane potential or current, since it is possible that these channels were not active. On the other hand, it is also possible that low levels of TRESK expression after axotomy may be sufficient to maintain resting membrane potential but make the neuron more easily activated in response to depolarizing stimuli.
In this study we have used the sanshool derivative IBA which has been shown to elicit pungent burning, cooling and tingling sensations in humans . IBA produces a transient depolarization of the resting membrane potential that is sufficient to activate the DRG neuron and induce Ca2+ entry (Figure 5A), as proposed for hydroxy-α-sanshool  and IBA . It is possible that the depolarization found in vitro after IBA application (~40 mV) may be larger than in physiological conditions due to the downregulation of TRESK after neuronal dissociation. Nevertheless, the effects found in vivo (Figure 6, 7) as well as recently reported data , suggested that even if TRESK is normally expressed, the block elicited by IBA is sufficient to depolarize the neuron and induce neuronal firing.
Despite not being completely selective for TRESK channels (Figure 5B), it seems that the major action of IBA is due to the blocking effect on this channel since an overall block of the K+ current can be seen in native DRGs (Figure 4E, F). It has been suggested that hydroxy-α-sanshool and, by extension IBA, could activate other channels such as TRPV1 or TRPA1 [54, 56]. In contrast, others have discarded this effect from studies on knockout mice . Our study and a recent characterization of IBA effects on DRG neurons  show that this compound activates different subsets of neurons, some of them expressing TRPV1, TRPA1 or TRPM8, but also some neurons not responding to well-known agonists of these TRP channels. Therefore, it seems that effects of IBA are mainly mediated by inhibition of K2P channels although it can't be completely ruled out that IBA does some unidentified effect on intracellular calcium signaling or on TRPs. A detailed study on IBA selectivity remains to be performed.
In this study, most IBA-sensitive neurons were in the small- and medium-size range and about 70% of them responded to capsaicin. Therefore, it is likely that most of those neurons were unmyelinated nociceptors. The other 30% only responded to IBA, but not to capsaicin, probably representing either the fraction of neurons with slowly conducting C-fibers insensitive to capsaicin or D-hair fibers (Aδ). Large DRGs activated by IBA probably correspond to large myelinated sensory afferents with Aβ axons [23, 24]. In agreement to these observations, the important biological role of TRESK is further demonstrated by the potent activation of peripheral C-nociceptor units in vivo after IBA injection in the rat paw. This is consistent with the effects of hydroxy-α-sanshool on the skin-nerve preparation  or peripherally applied IBA on low threshold mechanosensitive neurons and in wide dynamic range type neurons, that receive input from mechanoreceptors and nociceptors . IBA-induced activation seemed to occur in a particular class of peripheral C-nociceptors, namely the mechano-insenstive ones, but not in the mechano-sensitive ones. The majority of mechano-insensitive C-nociceptors are peptidergic, NGF-dependent, IB4-negative peripheral nociceptors, which have been recently shown to have an important role in neuropathic pain conditions [57, 58]. Our findings suggest that background currents mediated by TRESK may be important in this specific class of peripheral nociceptors. Blockage of TRESK channels in vivo not only induced spontaneous activity in C-nociceptors, but also resulted in a behavioral sensitization to mechanical stimuli. The decrease in the threshold for evoked mechanical pain after IBA injection or TRESK knockdown, opens the possibility that C-fibers that are mechanically insensitive in normal conditions, became sensitive after decreasing the total amount of background current. Despite the apparent paradox that pain and hyperalgesia to mechanical stimulation are encoded by mechano-insensitive nociceptors, mechanical sensitivity of previously mechanically-insensitive C-fibers have been already reported due to sensitization by capsaicin  or tonic pressure . Although the cellular mechanisms underling these changes are still unknown, different possibilities exist, like unmasking of stretch-activated membrane channels, release of chemical mediators generated by mechanical stimulation or a decrease/block of a K+ conductance (e.g. TRESK), which will make mechanical stimulation more effective to activate the fiber.
Injection of IBA in the rat hindpaw produced a dose-dependent nocifensive behavior that shows a good correlation with the effects of this compound in cultured sensory neurons, in the activation of sciatic nerve C-fibers and with recently reported results . Consistent with these effects, sanshool-containing water produced aversion in mice  and burning sensation in humans . In contrast, another study failed to demonstrate any nocifensive behavior after topical application of sanshool to the rat hindpaw . Skin penetration of sanshool after topical application may not be sufficient to reach and activate nociceptor terminals, but direct drug injection in the paw is able to activate them, like in reports by Sawyer et al. , Klein et al.  and in the present study. Our finding that knocking down TRESK expression decreases the threshold to mechanical painful stimuli is also consistent with the effects found on animal behavior and to the suggested involvement of TRESK in mediating tingling paresthesia [24, 38], therefore implicating TRESK channels in pain sensation. The apparent selectivity of TRESK silencing on mechanical but not heat thresholds is difficult to rationalize with the present findings, but could be due to an incomplete knock down of TRESK expression. We cannot rule out that effects on thermal painful perception will appear with higher levels of silencing or by completely knocking out the channel expression. Similarly, a decrease in mechanical withdrawal threshold but not in heat withdrawal latency after IBA injection has been reported . In addition, a recent report shows only a slight increase in thermal nociceptive sensitivity (20% decrease in latency in the hot plate test) in TRESK knockout mice . A detailed study on the role of this ion channel in different sensory modalities should come from further analysis of this TRESK-deficient mouse.
The regulation of TRESK currents after injury shown here suggests a possible role of this channel in the generation of allodynia and/or hyperalgesia caused by nerve injury. Blocking or silencing the channel we also show that TRESK participates in nociceptor excitability and behavioral responsiveness in normally behaving animals, but the role of TRESK in pathological conditions (after injury or in different pain models) remains to be further investigated. TRESK is particularly interesting since it is the only background channel activated by an increase in intracellular Ca2+ [15, 62], a common signaling mechanism found after activation of nociceptors by many compounds. Between resting membrane potential and spike threshold, a decrease in TRESK currents may be critical for opposing depolarizing inputs, as other major outward currents are inactivated (except background currents), outside the voltage range for effective activation, or relatively inactive in the absence of Ca2+ influx that occurs during action potentials. This is in general agreement with the results from the TRESK[G339R] functional knockout mice  or the recently reported association of a dominant-negative mutation in the human channel in certain cases of familial migraine with aura . A decrease in TRESK functionality may also underlie the appearance of CIPS (Cyclosporine-Induced Pain Syndrome) due to the use of calcineurin inhibitors (cyclosporine; FK506) [63, 64] or the increase in the anesthetic isoflurane (a TRESK activator) requirement after cyclosporine treatment . Because inhibiting calcineurin will impair TRESK activation in response to stimuli-induced Ca2+ increase, a higher requirement of this volatile anesthetic will be needed to achieve anesthesia, as recently shown in the knockout mice .