Response of A) rapidly adapting Aβ and B) slowly adapting Aβ fibers to sanshool. Left: Representative response to a sustained (suprathreshold) mechanical force. Middle: Response to 200 μM hydroxy-α-sanshool. Right: Maximal firing rate in response to sanshool or mechanical stimuli (100 mN force; 10 sec). C) Number of mechanically-evoked action potentials in sanshool-sensitive and sanshool-insensitive slowly adapting Aβ fibers. Sustained force stimuli (5-100 mN; 10 sec each) were applied to each receptive field before the receptive field was exposed to sanshool. *** indicates a significant difference between the two cohorts (2-way ANOVA followed by Tukey's multiple comparison test; p < 0.001) D) Representative examples of sanshool-sensitive and sanshool-insensitive slowly adapting Aβ fibers in response to mechanical stimuli.

A) Latency to arrest drinking behavior after water-deprived mice were given water containing the indicated compounds. Sanshool (1 mM), capsaicin (1 μM) and mustard oil (100 μM) were added to a 0.5 % saccharine solution. ** and *** identify significant differences between drinking latencies (n=8-11 mice per agonist; Student's t-test; p < 0.01 and p < 0.001 respectively). B) Latency to the first action potential in different fiber types evoked by sanshool (200 μM), capsaicin (1 μM) or mustard-oil (1 mM). Latencies to sanshool were compared across fiber type and the C-fiber latencies to different agonists were compared. *, ** and *** indicate a significant difference between groups (one-way ANOVA followed by a Tukey-Kramer post-hoc test; p < 0.05, p < 0.01 and p< 0.001 respectively).

The putative cutaneous innervation pattern of sanshool-sensitive (left) and sanshool-insensitive (right) mechanosensory afferents examined in this study are shown (Brown and Iggo 1967; Burgess et al., 1968). The dermal and epidermal layers of the skin are represented in yellow and brown, respectively. Thickness of sensory fibers represents degree of myelination. Application of sanshool to the skin induces tonic or bursting activation of subsets of fibers, leading to sensation of tingling paresthesia (bottom).

A) Classification of cultured sensory neurons based on chemical sensitivity (n=1187). B) Examples of calcium responses to chemical stimuli and staining with neurofilament 200 antibody in cultured sensory neurons. A presumptive D-hair (arrow head) and nociceptor (arrow) are indicated. 74% of sanshool-sensitive and GABA-sensitive but capsaicin-insensitive neurons stained for NF200, n = 23; compared to 49% of all neurons tested, n = 669; p < 0.05, Fisher's exact test). C) The proportion of sanshool-sensitive and GABA-sensitive (San+, GABA+) but capsaicin-insensitive (Cap-) sensory neurons in cultures with or without neurotrophin 4 (NT4), a growth factor which promotes D-hair development. n = 1187 and 1293, in the absence and presence of NT4, respectively, from three independent cultures. ** p < 0.01, two proportion z-test. D) Example of a D-hair fiber response to sustained (suprathreshold) mechanical force and subsequently, exposed to 200 μM hydroxy-α-sanshool, and percentage of responsive fibers. E) Maximal response of D-hair fibers to sanshool and sustained mechanical stimuli. F) Example of an AM fiber response to sustained (suprathreshold) mechanical force and subsequently, exposed to 200 μM hydroxy-α-sanshool, and percentage of responsive fibers.

A) Representative C-fiber responses. Top trace: A C-fiber that was sensitive to hydroxy-α-sanshool (200 μM) but not to mustard oil (1 mM) or capsaicin (1 μM). Bottom trace: A C-fiber insensitive to sanshool and mustard oil but sensitive to capsaicin. B) Proportion of C-fibers that respond to sanshool (San), mustard oil (MO) or capsaicin (Cap), in the presence or absence of TRPA1 and TRPV1 inhibitors (HC-030031, 100μM and A-425619, 3μM, respectively). Fisher's exact test; p = 0.42 for sanshool-sensitive fibers. C) Fraction of C-fibers responsive to sanshool and their sensitivity to capsaicin and mustard oil (“+” indicates sensitivity to the chemical agonist whereas “-” indicates insensitivity; also, 5/32 fibers responded to none of the chemical agonists). D) Action potentials fired per min in response to sanshool, capsaicin or mustard oil, in the presence or absence of TRPA1 and TRPV1 inhibitors. “n.s.” indicates no significant difference between groups (Student's t-test; p=0.40). ** and *** identify a significant difference between groups (one-way ANOVA followed by a Tukey-Kramer post-hoc test; p < 0.01 and p < 0.001 respectively). E) Conduction velocities of all C-fibers that were sanshool-sensitive (grey) or insensitive (white). Conduction velocities were measured prior to sanshool stimulation and are therefore an inherent property of the C-fiber unaffected by sanshool exposure. *** identifies a significant difference between sanshool-sensitive and insensitive fibers (Student's t-test; p < 0.01).

A) Representative traces of D-hair, RA-Aβ and C-fibers that exhibited burst pattern action potential firing, and the number of such fibers found in each class. The box in each example at the left indicates a region of interest that is expanded to the right of each trace. Note that the time scale is identical for each fiber, thereby reflecting differences in their pattern of burst firing. No SA-Aβ fibers displayed burst firing. B) Comparison of burst firing properties across fiber types. Left: Average number of action potentials fired per burst. Right: Average duration of individual burst and length of interval between bursts for each fiber type. *indicates a significant difference among groups (one-way ANOVA; p < 0.01).