Here, we sought to identify the subtypes of sensory neurons that underlie the tingling paresthesia elicited by hydroxy-α-sanshool. Our findings show that sanshool is the first pharmacological agent identified that can discriminate between distinct subsets of mechanosensory neurons (). Among Aδ fibers, virtually all D-hair afferents were vigorously excited by sanshool, whereas AM nociceptors were completely unresponsive. D-hair afferents are the most sensitive of all mechanoreceptors, with mechanical thresholds below the measurable limit (Brown and Iggo, 1967
; Burgess et al., 1968
; Koltzenburg et al., 1997
). They are a key fiber type required for normal tactile acuity and movement detection (Brown and Iggo, 1967
; Wetzel et al., 2007
). In addition, D-hairs have also been implicated in diabetic peripheral neuropathy (Jagodic et al., 2007
; Shin et al., 2003
), which often leads to tingling paresthesia in patients.
Sanshool also activated rapidly adapting Aβ mechanoreceptors that encode the movement of guard hair follicles. Similar to rapidly adapting Aδ D-hair receptors, sanshool was more potent in activating Aβ fibers with rapidly adapting properties to mechanical force than those with slowly adapting properties. Thus, among all myelinated fibers, sanshool activates rapidly adapting fibers far more extensively than slowly adapting fibers. Spontaneous activity in rapidly adapting myelinated fibers has been implicated in both injury- and disease-evoked paresthesia, as well as in post-ischemic paresthesia; however, the exact neuronal subtypes that mediate tingling paresthesia have not been characterized (Nordin et al., 1984
; Ochoa and Torebjork, 1980
A subset of slowly-adapting Aβ fibers also responded to sanshool, albeit with lower firing intensities. Most slowly adapting Aβ fibers are thought to be “SA-I” that innervate Merkel cells and encode sustained pressure to skin, but some slowly adapting Aβ fibers are “SA-II” and sense skin stretch (Srinivasan et al., 1990
; Lamotte et al., 1998
). Interestingly, in rat 35% of slowly adapting Aβ fibers in the sural nerve and in mice 52% of slowly adapting Aβ fibers in the saphenous nerve are reported to be SA-II stretch sensors (Leem et al., 1993
; Maricich et al., 2009
). Furthermore, a distinguishing feature of SA-II receptors is that they are approximately six times less sensitive to skin indentation than SA-I receptors (Johansson and Vallbo, 1979
). Two findings support the idea that the sanshool-sensitive slowly adapting Aβ fibers are SA-II type skin stretch sensors. First, the proportion of sanshool-sensitive slowly adapting Aβ fibers (36%) is consistent with the proportion of SA-II type skin stretch sensors. Second, the sanshool-sensitive SA-Aβ fibers were ~5 fold less sensitive to sustained force than the sanshool-insensitive population.
Sanshool activated a unique subset of C-fibers that has an intrinsically slower conduction velocity than other C-fibers. Conduction velocity is largely dependent on fiber diameter and myelination which influence the internal resistance and membrane capacitance of nerve axons. Thus, we may observe this difference because sanshool-sensitive channels are expressed on the smallest diameter C-fibers. However, conduction velocity also correlates with the length constant of a nerve fiber, which is directly proportional to membrane resistance (Koester and Siegelbaum, 2000; Hodgkin and Rushton, 1946). An intriguing possibility is that sanshool-sensitive channels, potentially KCNK18 channels, decrease membrane resistance and thereby, directly slow the conduction velocity. Previous studies of tingling paresthesia in humans have failed to report aberrant activity of Aδ or C-fibers (Nordin et al., 1984
; Ochoa and Torebjork, 1980
). However, this may be due to technical difficulties in recording from patients experiencing tingling paresthesia. Our data implicate both D-hair afferents and the unique population of slowly conducting C-fibers in tingling paresthesia.
Our data lend support to the hypothesis that sanshool elicits tingling paresthesia through selective activation of mechanosensitive somatosensory neurons (). Human psychophysical testing shows that sanshool exhibits its sensory effects ~60 seconds after application (Bryant and Mezine, 1999
). Our behavioral data show that mice respond to the effects of sanshool with a characteristic latency of 50 seconds, which is strikingly similar to that observed in humans. The sanshool response latency is significantly slower than latencies to capsaicin or mustard oil. In addition, sanshool consumption fails to elicit the nocifensive responses of nose rubbing and wiping that are commonly observed following consumption of capsaicin or mustard oil (unpublished observations). Moreover, no differences were observed in the sanshool response latency between wild type and TRPA1-/-
animals. Thus, sanshool-evoked behaviors more likely result from tingling paresthesia, rather than painful irritation. This is consistent with the activation pattern of Aδ and Aβ fibers by sanshool, as well as with results from human psychophysical studies demonstrating that sanshool does not elicit pain sensations (Bryant and Mezine, 1999
; Sugai et al., 2005
). Although sanshool also activates a subset of C-fibers, it is unclear whether these C-fibers actually transmit pain signals. Several studies have demonstrated the existence of C-fibers that transmit information other than pain. For example, a recent study demonstrated the existence of unmyelinated C-fibers that code for pleasant touch sensations in humans (Loken et al., 2009
). In addition, C-fibers that transmit sensations of brushing and itch have also been reported (Zotterman, 1939
). Finally, specific labeling of neurons that express a Mas-related G protein-coupled receptor, MrgprB4
, revealed a unique subpopulation of C-fibers that specifically innervate the skin, but not the viscera; these fibers are hypothesized to function as touch receptors, rather than nociceptors (Liu et al., 2007
). Further analysis at the molecular and behavioral levels is required to elucidate the exact role of this new class of sanshool-sensitive C-fibers.
Common among all sanshool-sensitive fibers is the presence of action potential bursting, which we observed in 29% of fibers. Bursting is exhibited by many neurons within the central nervous system, as well as some peripheral neurons. A short burst of action potentials may temporally summate to provide high-fidelity neuronal transmission (Williams and Stuart, 1999
) or foster long term potentiation to strengthen neuronal synapses (Liu et al., 2008
). In the peripheral nervous system, bursting has been described in trigeminal afferents in the brainstem that are thought to play a key role in the central pattern generator circuit regulating mastication in rodents (Brocard et al., 2006
; Hsiao et al., 2009
). Bursting is also associated with tingling paresthesia. Microelectrode recordings show robust bursting of sensory afferents in normal human subjects experiencing tingling paresthesia (Ochoa and Torebjork, 1980
). In addition, neuronal recordings from patients suffering from activity-dependent tingling paresthesia showed robust bursting of myelinated, rapidly-adapting mechanoreceptors that increased with the degree of paresthesia. Finally, in rat models of diabetic neuropathy, robust bursting of medium diameter fibers increased in diabetic neurons as compared to wild type neurons (Jagodic et al., 2007
). Indeed, tingling paresthesia is a common complaint of diabetic patients with neuropathy. We speculate that the bursting pattern may underlie the tingling sensation commonly associated with chewing Szechuan peppers.
Activation of TRPA1 and TRPV1, and inhibition of the two-pore potassium channels KCNK3 (TASK-1), 9 (TASK-3) and 18 (TRESK) have been proposed as mechanisms by which sanshool activates neurons (Koo et al., 2007
; Riera et al., 2009
; Menozzi-Smarrito et al., 2009
) (Bautista et al., 2008
). However, we demonstrate that sanshool-evoked fiber responses are of similar prevalence and amplitude in the presence or absence of TRPA1 and TRPV1 selective antagonists. Likewise, sanshool-evoked behaviors were similar between wild type and TRPA1-/-
animals. These data suggest that neither TRPA1 nor TRPV1 mediate the excitatory effects of sanshool. In somatosensory neurons, expression and electrophysiological studies show the presence of KCNK18 channels (Dobler et al., 2007
; Kang et al., 2008
), but expression of KCNK3 and 9 have not been demonstrated; however, KCNK3 and 9 are expressed by keratinocytes in the skin (Kang and Kim, 2006
). Thus, sanshool may act directly on KCNK channels in sensory neurons as well as in keratinocytes, which are known to modulate sensory neuron function (Koizumi et al., 2004
; Lumpkin and Caterina, 2007
) to induce tingling paresthesia. The bursting behavior observed in response to sanshool application is also consistent with a model of potassium channel blockade. Bursting in trigeminal neurons has been linked to the activity of Kv1 channels (Hsiao et al., 2009
), and TEA-insensitive potassium channel(s) may contribute to burst firing (Brocard et al., 2006
). However, analysis of KCNK-deficient mice is required to test this hypothesis. Recently, two other members of the KCNK channel family, KCNK2 (TREK-1) and KCNK4 (TRAAK), have been shown to regulate responses to thermal and mechanical stimuli in nociceptors (Maingret et al., 1999
; Noel et al., 2009
). Thus the KCNK family of channels may play key roles in a variety of mechanosensitive sensory fibers. Again, the analysis of mice lacking KCNK channels will be required to test this hypothesis.
Our finding that sanshool robustly activates a distinct subset of D-hair, ultra-sensitive light touch receptors in the skin and targets novel, uncharacterized populations of Aβ and C-fiber nerve afferents shows that sanshool is an innovative tool for physiological and molecular studies. In addition, characterization of sanshool-sensitive mechanoreceptors represents an essential first step in identifying the cellular and molecular mechanisms underlying tingling paresthesia that accompanies peripheral neuropathy and injury.