D , von Frey response thresholds for individual animals before and after topical application of mustard oil MO. After the baseline measurement, MO was applied topically to the hindpaw, and after 30 min, von Frey thresholds were measured. Animals were then either treated with vehicle or sanshool solution. E , response latency to radiant heat stimuli applied to the hindpaw in mice before and after MO treatment.
MO was applied topically to the hindpaw, and after 30 min, animals were either treated with vehicle or sanshool solution. Error bars are s. Because sanshool is used as a traditional medicine to treat inflammatory pain, we next measured the effect of sanshool on mechanical and heat sensitivity in a model of neurogenic inflammation. Following induction of hypersensitivity by mustard oil, topical application of sanshool greatly attenuated the decrease in mechanical threshold while vehicle had no effect Fig.
Thus, sanshool selectively attenuates mechanical pain hypersensitivity. We next used the ex-vivo skin—nerve preparation to determine if sanshool alters sensory transduction in the peripheral nervous system, and if so, whether sanshool targets specific subsets of somatosensory fibre types.
Nerve fibres were mechanically stimulated while the receptive fields were exposed to either vehicle 0. Sanshool inhibition was observed at a wide range of applied mechanical forces Fig. These data demonstrate that sanshool strongly inhibits mechanical responses in slowly-adapting, myelinated fibres, especially the AM fibres, which mediate sharp acute mechanical pain as well as the hypersensitivity to mechanical pain after neurogenic inflammation Lin et al.
These findings are consistent with the use of toothache tree extracts to treat inflammatory hypersensitivity in areas densely innervated by myelinated mechanosensitive pain fibres e. Fibres were stimulated with either a mechanical or an electrical stimulus. Data are from this study and Lennertz et al. B , percentage of nerve fibres that exhibit mechanical inhibition in sanshool. C , number of action potentials fired at baseline and after 3 min in sanshool for each fibre tested. Solid bar represents the mean. D , sanshool concentration—inhibition curve for AM fibres using a mN force stimulus.
Data were fitted by a non-linear four-parameter regression model. E , dose-dependent inhibition of AM fibres by sanshool in response to increasing mechanical force. Sanshool may attenuate mechanically evoked AP firing by either inhibiting mechanosensitive channels that trigger excitability, or by directly inhibiting voltage-gated channels.
Thus, we questioned whether sanshool inhibits AP firing using an electrical stimulus, which would bypass mechanical transduction. The time course of recovery of electrical excitability correlated well with the recovery of mechanical excitability data not shown. This experiment suggests that sanshool inhibits the initiation or propagation of APs in slowly adapting, myelinated fibres, independently of its effect on mechanical transduction. A , example recordings demonstrate the inhibition of electrically evoked action potentials in AM fibres but not in C fibres.
B , percentage of nerve fibres that exhibit electrical inhibition in the presence of sanshool. Some neurons failed to generate an AP top while others remained excitable bottom in the presence of sanshool. Neurons that fired spontaneous APs in the presence of sanshool were not included in the analysis. E , thresholds for AP firing in neurons resistant or sensitive to the inhibitory effect of sanshool. Consistent with its effect on sensory fibres, sanshool also inhibited electrically evoked AP firing in a subset of cultured DRG neurons Fig. Sanshool-sensitive neurons had distinct physical and physiological characteristics compared to sanshool-resistant neurons.
This result is consistent with the inhibition of myelinated fibres in our teased fibre recordings Fig.
Zanthoxylum clava-herculis is a spiny tree or shrub native to the southeastern United States. Along with the related Zanthoxylum americanum, it is sometimes called "toothache tree" or "tingle tongue" because chewing on the leaves, bark. Toothache tree may refer to one of several American trees: Aralia spinosa Zanthoxylum clava-herculis or Zanthoxylum americanum (Northern prickly ash).
Small diameter neurons tend to express higher levels of Na v channels that are resistant to the specific sodium channel blocker TTX, such as Na v 1. Second, neurons inhibited by sanshool had a more hyperpolarized threshold for AP firing at baseline Fig. Na v channels expressed in sensory neurons are activated at different membrane potentials depending on the channel subtype Rush et al.
Therefore, sanshool-sensitive neurons may express Na v channels that are activated at a more hyperpolarized membrane potential and are preferentially inhibited by sanshool.
We next used whole-cell voltage clamp recording to examine the effects of sanshool on endogenous Na v channels in sensory neurons. The application of sanshool caused a rapid and reversible reduction in voltage-gated sodium current amplitude Fig. Classification of neurons based on their sensitivity to TTX showed that TTX sensitivity is weakly correlated with sanshool sensitivity Fig. Inset shows the voltage protocol for the traces. B , representative time course of sanshool-induced voltage-gated sodium current inhibition. Veh, vehicle; San, sanshool; Wash, washout.
C , whole-cell voltage-gated sodium conductance of a representative DRG neuron shown in A. Data points were fit with a Boltzmann function. Neurons were classified according to sensitivity to TTX. Data points were fit with a double Boltzmann function see Methods.
G , average shift in the midpoints of the steady-state inactivation curves derived from double Boltzmann fits. Many Na v channel inhibitors act by preferentially binding to the inactivated state of the channel, which promotes a hyperpolarizing shift in the steady-state inactivation curve Hille, ; Bean et al. We thus measured voltage-dependent steady-state inactivation of Na v channels in the absence and presence of sanshool Fig.
Sensory neurons express multiple sodium channel subtypes that differ in sensitivity to voltage-dependent inactivation. As such, our data were best fit with either a single or a double Boltzmann function to determine a single or two midpoint s for inactivation V h , respectively see Materials and methods for fit assumptions Rush et al. Sanshool caused a significant hyperpolarizing shift in both V h1 and V h2 Fig. If sanshool dampens sensory neuron excitability by inhibiting Na v channels, sanshool sensitivity may correlate with the expression of sanshool-sensitive Na v channel subtypes.
Our initial characterization showed that sensory neurons expressing TTX-sensitive Na v channels tend to be more strongly inhibited, while those expressing TTX-resistant Na v channels are less suppressed by sanshool Fig.
However, TTX sensitivity of AP firing did not correlate strictly with sanshool sensitivity, as many TTX-sensitive large diameter neurons were still resistant to the inhibitory effect of sanshool. We thus sought to determine the specific Na v channel subtypes expressed in neurons inhibited by sanshool.
We used calcium imaging to functionally classify neurons and then performed qPCR on subgroups of neurons to identify Na v channel subtype expression. Neurons were classified into three groups based on soma size and calcium response to sanshool Fig. Group I neurons are larger in diameter and activated by sanshool, as previously described Lennertz et al. Group III small diameter neurons are activated by capsaicin and thus include a population of C fibre nociceptors. A , schematic for classifying cultured sensory neurons based on calcium response properties to sanshool and capsaicin.
Circle diameter represents relative cell body size, which in turn correlate with myelination. B , distribution of Na v channel subtype expression in groups of dissociated somatosensory neurons classified as in A. C , conductance—voltage relationship of human Na v 1. Solid bar represents the mean percentage of inhibition. E , dose—response of sanshool-induced current inhibition in hNa v 1.
Data points for hNa v 1. F , relationship between mean percentage of sanshool-induced hNa v inhibition and average midpoint for steady-state inactivation at baseline. Percentage current inhibition for each cell expressing hNa v subtype was derived as in D. Data points were fit with a Boltzmann function to obtain the inactivation midpoint potential, V h see Methods. G , steady-state inactivation curves of hNa v 1.
Solid bar represents the mean shift. The three groups of neurons displayed unique combinations of Na v channel subtype expression Fig. Group I neurons expressed all subtypes tested except for Na v 1. Similarly, group II neurons expressed most Na v channel subtypes, including Na v 1. Group III neurons only expressed Na v 1. These results are consistent with the previously described distribution of Na v channel subtypes based on relative cell size Black et al. Notably, larger diameter neurons that express both Na v 1. These data suggest that Na v 1.
To test this, we characterized the degree of current block at 0 mV and the shift in steady-state inactivation induced by sanshool in Na v channel subtypes expressed heterologously. All Na v channels tested displayed voltage-gated currents that were reduced, to varying degrees, by sanshool Fig.
Notably, Na v 1. There are several classes of Na v channel inhibitors that have different mechanisms of action. Local anaesthetics, such as lidocaine, act by binding to the open or inactivated state of Na v channels Hille, ; Ragsdale et al. Plotting the strength of sanshool-induced current inhibition against the mid-point for steady-state inactivation showed a clear correlation.
The inhibition by sanshool tended to be stronger as the fraction of inactivated channels at resting potential increased Fig. In DRG neurons, sanshool caused a hyperpolarizing shift in the steady-state inactivation curve of endogenous sodium channels Fig. Interestingly, even though sanshool shifted the steady-state inactivation of all heterologously expressed Na v channels, sanshool caused the biggest shift in Na v 1.
These results suggest that at resting potential, sanshool is likely to have the most profound inhibitory effect on the activity of Na v 1. Overall, sanshool inhibits Na v channel subtypes to varying degrees, and differences in the relative contribution of each channel subtype to excitability may dictate sanshool sensitivity among the different groups of neurons.
Extracts from the toothache tree have been used for decades to treat inflammatory pain. Yet, the molecular mechanisms that mediate the analgesia remained unknown. Here we demonstrate that topical application of sanshool to the skin blocks mechanical but not heat hypersensitivity following inflammation. Renae rated it really liked it Aug 22, Deb rated it really liked it Sep 02, Barrie Fearn rated it really liked it Apr 14, Edward Sargisson rated it did not like it Sep 20, Carole rated it really liked it Aug 15, Heidi Wouters rated it really liked it Oct 04, Cierra rated it it was amazing Nov 28, Dina rated it it was amazing Jan 24, Martha rated it it was ok Oct 24, Enrique rated it it was amazing Dec 27, Bonnie rated it really liked it Jan 04, Barb rated it liked it Jan 19, Mary Baskin added it Aug 10, Sigmund marked it as to-read Aug 05, Mikaela Roque added it Sep 17, Mona Hobby added it Aug 17, Katie marked it as to-read Oct 06, Frida marked it as to-read Jun 13, Joshua Sirias is currently reading it Sep 10, The genus name is sometimes spelled Xanthoxylum.
Along with the related Zanthoxylum americanum , it is sometimes called " toothache tree " [3] [4] or " tingle tongue " because chewing on the leaves, bark, or twigs causes a tingling numbness of the mouth, tongue, teeth and gums.
It was used for such medicinal purposes by both Native Americans and early settlers to treat toothache because of this. The tree has a rounded crown and requires plentiful water and sunlight. Its leaves are browsed by deer and its fruit is eaten by birds. The fruit passes through birds, which helps the seeds to germinate. The name Hercules' club is also applied to Aralia spinosa , also native to eastern North America. Unlike Zanthoxylum, Aralia has large twice-compound leaves and very large leaf scars, so the trees are easily distinguished. The benzophenanthridine alkaloid chelerythrine is the major active natural product found in Z.