J Neurophysiol 105: 1701–1710, 2011.
First published January 27, 2011; doi:10.1152/jn.00922.2010.
A tingling sanshool derivative excites primary sensory neurons and elicits
nocifensive behavior in rats
Amanda H. Klein,1 Carolyn M. Sawyer,1 Karen L. Zanotto,1 Margaret A. Ivanov,1 Susan Cheung,1
Mirela Iodi Carstens,1 Stephan Furrer,2 Christopher T. Simons,2 Jay P. Slack,2 and E. Carstens1
1
Department of Neurobiology, Physiology and Behavior, University of California, Davis, California; and 2Givaudan Flavors,
Cincinnati, Ohio
Submitted 26 October 2010; accepted in final form 25 January 2011
calcium imaging; dorsal root ganglion cell; transient receptor potential
channels; capsaicin
SZECHUAN (OR SICHUAN) PEPPERS from the plants of genus Xanthoxylum are commonly used in Eastern Asian cuisine. These
peppers are unique due to the numbing and tingling sensations
they impart to the oral cavity. The tingling and “buzzing”
sensation caused by the Szechuan peppers has been linked to a
family of alkylamides collectively called “sanshools” (Yasuda
et al. 1982; Kashiwada et al. 1997). Of the many sanshools
found in the Szechuan pepper, the active isomer responsible for
the tingling sensation has been narrowed to hydroxy-␣-sanshool (Crombie and Tayler 1957) in human psychophysical
studies on the tongue (Bryant and Mezine 1999; Sugai et al.
2005). ␣-Hydroxy sanshool has been reported to excite cells
expressing TRPV1 and TRPA1 (Koo et al. 2007; Riera et al.
Address for reprint requests and other correspondence: E. Carstens, Univ. of
California, Davis, Dept. of Neurobiology, Physiology and Behavior, 1 Shields
Ave., Davis, CA 95616 (e-mail: [email protected]).
www.jn.org
2009) or alternatively to act via inhibition of the two-pore
potassium channel (KCNK) subfamilies KCNK3, KCNK9, and
KCNK18 (Bautista et al. 2008).
Currently, there are sanshool-derived alkylamides that can
by synthesized with properties almost identical to hydroxy-␣sanshool (Menozzi-Smarrito et al. 2009). The hydroxy-␣-sanshool analog used in this study, (2E,4E,8Z)-N-isobutylundeca2,4,8-trienamide (IBA), was derived from unsaturated amides
and the pungency was associated with two main structural
requirements: a (CH⫽Z⫽CH-CH2-CH2-CH⫽E⫽CH) motif and a N-isobutylcarboamide group (Galopin et al. 2004)
(Fig. 1A).
IBA has been reported to elicit tingling, cooling, and pungent burning sensations in humans (Albin and Simons 2010).
We hypothesized that IBA will directly excite primary sensory
dorsal root ganglion (DRG) neurons cells similar to the effects
of hydroxy-␣-sanshool and that some of these neurons will also
be activated by agonists of transient receptor potential (TRP)
channel TRPV1 that responds to noxious heat and capsaicin
(Caterina et al. 1997; Jung et al. 1999), TRPA1 that responds
to a variety of irritant chemicals including cinnamic aldehyde
(CA) (Bandell et al. 2004), and TRPM8 that responds to
cooling and menthol (Peier et al. 2002; Bautista et al. 2007;
Colburn et al. 2007). Given contrasting reports that hydroxy␣-sanshool acts largely via TRPV1 and TRPA1 (Koo et al.
2007; Riera et al. 2009) or by inhibiting KCNK channels
(Bautista et al. 2008), we tested whether IBA activation of
DRG cells is affected or not by a TRP channel antagonist. We
further hypothesized that the sensation elicited by IBA would
be aversive and hence elicit nocifensive behavioral responses
in rats. Finally, if IBA acts at least partly via TRP channels, we
hypothesized that it would enhance heat and mechanical sensitivity as has been shown previously for the TRPV1 agonist
capsaicin (Gilchrist et al. 1996) and the TRPA1 agonist CA
(Tsagareli et al. 2010). An abstract of this study has appeared
previously (Sawyer et al. 2009b).
MATERIALS AND METHODS
Calcium Imaging
Cell culture. Under a protocol approved by the University of
California Davis Institutional Animal Care and Use Committee, juvenile (⬃3 wk, ⬃100 g) male Sprague-Dawley rats were euthanized
under isoflurane anesthesia and lumbar dorsal root ganglia extracted
and placed into Petri dishes containing HBSS (GIBCO, Invitrogen
Life Sciences). Ganglia were minced with fine spring scissors and
incubated in 40 ␮l papain (no. 3126; Worthington Biochemical,
Lakewood, NJ) with 1 mg L-cysteine (Sigma) in 1.5 ml HBSS for 5
0022-3077/11 Copyright © 2011 the American Physiological Society
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Klein AH, Sawyer CM, Zanotto KL, Ivanov MA, Cheung S,
Carstens MI, Furrer S, Simons CT, Slack JP, Carstens E. A tingling
sanshool derivative excites primary sensory neurons and elicits nocifensive behavior in rats. J Neurophysiol 105: 1701–1710, 2011. First
published January 27, 2011; doi:10.1152/jn.00922.2010.—Szechuan
peppers contain hydroxy-␣-sanshool that imparts desirable tingling, cooling, and numbing sensations. Hydroxy-␣-sanshool activates a subset of
sensory dorsal root ganglion (DRG) neurons by inhibiting two-pore
potassium channels. We presently investigated if a tingle-evoking sanshool analog, isobutylalkenyl amide (IBA), excites rat DRG neurons and,
if so, if these neurons are also activated by agonists of TRPM8, TRPA1,
and/or TRPV1. Thirty-four percent of DRG neurons tested responded to
IBA, with 29% of them also responding to menthol, 29% to cinnamic
aldehyde, 66% to capsaicin, and subsets responding to two or more
transient receptor potential (TRP) agonists. IBA-responsive cells had
similar size distributions regardless of whether they responded to capsaicin or not; cells only responsive to IBA were larger. Responses to
repeated application of IBA at a 5-min interstimulus interval exhibited
self-desensitization (tachyphylaxis). Capsaicin did not cross-desensitize
responses to IBA to any greater extent than the tachyphylaxis observed
with repeated IBA applications. These findings are consistent with psychophysical observations that IBA elicits tingle sensation accompanied
by pungency and cooling, with self-desensitization but little cross-desensitization by capsaicin. Intraplantar injection of IBA elicited nocifensive
responses (paw licking, shaking-flinching, and guarding) in a dose-related
manner similar to the effects of intraplantar capsaicin and serotonin. IBA
had no effect on thermal sensitivity but enhanced mechanical sensitivity
at the highest dose tested. These observations suggest that IBA elicits an
unfamiliar aversive sensation that is expressed behaviorally by the limited
response repertoire available to the animal.
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SANSHOOL EXCITES DRG CELLS AND EVOKES NOCIFENSIVE BEHAVIOR
min in a 37°C rocking water bath. Cells were centrifuged at 200 g for
2 min, and media were suctioned away. The ganglia were then incubated
in 2 mg/ml collagenase type II (CLS2; Worthington Biochemical) in
HBSS for 5 min in a 37°C rocking water bath. Cells were centrifuged
again at 200 g for 1 min and gently triturated using fire-polished glass
pipettes with completed media consisting of MEM media with Earle’s
salts and glutamine (GIBCO, Invitrogen Life Sciences) and 10% donor
horse serum (Quad Five, Ryegate, NJ) with 1% ⫻100 MEM vitamin
solution and penicillin-streptomycin (GIBCO). The isolated DRG cells
were plated in 40-␮l aliquots on 25-mm round glass coverslips (Bellco,
Vineland, NJ) coated with 1 mg/ml poly-D-lysine (Sigma) for 1 h. Cells
were given 2 ml of complete media after 1 h postplating and kept in a
37°C water-jacketed CO2-injected incubator under 5% CO2 in air, with
fresh media given after 24 h.
Imaging and chemical stimulation. Following the manufacturer’s
protocol, DRG cells were incubated for 1 h in 1 mM fura-2 AM
(Molecular Probes) dissolved in dimethyl sulfoxide (F1221; Invitrogen Life Sciences) to a final concentration of 10 ␮M in 5 mM
glucose-supplemented Ringer solution (140 mM NaCl, 4 mM KCl, 2
mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, with pH adjusted to
7.4 using NaOH) containing 0.1% Pluronic (F127; Invitrogen Life
Sciences). Cells were rinsed with Ringer solution and incubated for
another 10 min before being placed on a custom-made aluminum
perfusion block designed to fit a thermal stage (HCC100A; Dagan,
Minneapolis, MN) and viewed through a Nikon inverted microscope
(Eclipse TS100; Melville, NY). Fluorescence images obtained at
340/380 nm wavelengths were viewed through a CoolSnap camera
(Technical Instruments, San Francisco CA) attached to a Lambda LS
lamp and a Lambda 10 –3 optical filter changer (Sutter Instrument,
Novato, CA). Ratiometric measurements were made using Simple
PCI software (Compix, Cranberry Township, PA) with an intermittent
pause of 3 s between measurements.
Solutions were administered to one end of the perfusion block by
a gravity-fed eight-channel solenoid-operated perfusion system
(ValveLink 8.2; AutoMate Scientific, San Francisco, CA) and removed via a vacuum line at the other end. The chemicals used were
250 ␮M L-menthol (Givaudan Flavors, Cincinnati OH), 200 ␮M CA,
200 ␮M allyl isothiocynate (AITC; Sigma Chemical, St. Louis MO),
1 ␮M capsaicin (Sigma; all dissolved in 0.015% EtOH in Ringer
J Neurophysiol • VOL
solution), and 50 ␮M IBA (Givaudan; dissolved in 0.25% propylene
glycol). Concentrations for capsaicin, menthol, and CA were chosen
to allow comparison with previous calcium imaging studies that used
these chemicals at the same or similar concentrations (Peier et al.
2002; Behrendt et al. 2004; Bautista et al. 2007; Hjerling-Leffler et al.
2007). The 50-␮M IBA concentration was determined to excite DRG
cells in pilot studies and is similar to the concentration of hydroxy␣-sanshool (100 ␮M) used in a prior study imaging sensory neurons
(Bautista et al. 2008). Each chemical was superfused for a period of
30 s, except capsaicin, which was delivered for 10 s. Vehicles were
applied separately as controls and had no effect on the baseline ratio
of any cells (data not shown).
In the first experiment, IBA was applied first followed 5 min later
by reapplication of IBA to test for self-desensitization (tachyphylaxis). Cells were subsequently tested for responses to menthol, CA,
and occasionally AITC, delivered in a randomized order to avoid
order effects, followed by capsaicin. Capsaicin was tested after the
other TRP agonists to avoid cross-desensitization. Lastly, Ringer
solution containing 144 mM K⫹ was applied. Only those DRG cells
that responded to high-K⫹ Ringer solution were included for data
analysis.
A second set of experiments investigated capsaicin cross-desensitization of IBA-evoked responses. Capsaicin was applied first, followed by IBA 5 min later. Cells that were unresponsive to capsaicin
but that responded to subsequent application of IBA were of particular
interest as potential mechanoreceptors involved in tingle.
A third experiment tested the effects of general TRP channel
blocker ruthenium red (RR; Sigma; 10 ␮M). The purpose of this
experiment was to determine if activation of TRP channels mediates
IBA-evoked responses of DRG cells. Naïve DRG cells were perfused
with RR, beginning at least 10 s before, and continuously during,
chemical application of menthol, cinnamaldehyde, capsaicin, or IBA
in various sequences.
Data analysis. Each DRG cell’s response was calculated as the
highest (maximum) ratio change during the 3 min poststimulus period,
relative to the baseline level averaged over a 1-min period before
chemical application. Peak responses were corrected by subtracting
the prestimulus baseline ratio. A positive response to a chemical was
defined as at least a 20% change from baseline. A pivot table was
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Fig. 1. Isobutylalkenyl amide (IBA) and dorsal root ganglion (DRG) cells. A: chemical structures of hydroxy-␣-sanshool (left) and IBA (right). B: fluorescence
microscopic images of 3 DRG cells responding to IBA. Black and red arrows point to 2 small DRG cells, and green circle shows a larger cell. Left shows cells
before, and right shows cells after first (IBA#1) and second (IBA#2) applications of IBA (50 ␮M) at a 5-min interstimulus interval, followed lastly by high-K⫹.
Note increased 340/380 nm ratio (i.e., redder colors) after each stimulus. Scale (25 ␮m) applies to all micrographs. C: graph plots 340/380 nm ratios vs. time
for 3 DRG cells shown in B. Cell 1 (green) only responded to IBA and high K⫹ while cells 2 and 3 (black and red) responded to all chemicals tested: IBA (50
␮M), cinnamic aldehyde (CA; 200 ␮M), menthol (250 ␮M), allyl isothiocynate (AITC; 200 ␮M), capsaicin (1 ␮M), and high-K⫹. D: size distribution of DRG
neurons according to IBA- and TRP channel agonist sensitivity. Black bars: all cells responding to high-K⫹. Blue bars: cells responsive to IBA but not capsaicin,
menthol, or CA. Red bars: IBA- and capsaicin-sensitive cells. Green bars: IBA- and menthol-sensitive cells. Distribution of IBA- and CA-sensitive cells was
similar to that of IBA- and menthol-sensitive cells and is omitted for clarity. Bin width ⫽ 0.5 ␮M. Size distribution of IBA-only responsive cells was significantly
different compared with IBA- and capsaicin-sensitive cells (P ⬍ 0.01, unpaired t-test) and to IBA- and menthol-sensitive cells (P ⬍ 0.001).
SANSHOOL EXCITES DRG CELLS AND EVOKES NOCIFENSIVE BEHAVIOR
Table 1. Coincidence of responses of IBA-sensitive DRG cells to
menthol, CA, capsaicin, and various combinations
n ⫽ 239
%
IBA
Menthol
CA
Capsaicin
Total Number
%
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
239
100
⫹
⫹
⫹
⫹
71
29.7
⫹
⫹
⫹
⫹
69
28.9
⫹
⫹
⫹
⫹
158
66
38
13
25
8
2
31
5
117
239
15.9
5.4
10.5
3.3
0.8
13
2.1
49
100
created to calculate percentages of cells that responded to various
combinations of the applied chemicals. Data from the second experiment were excluded from this calculation, because the initial application of capsaicin may have cross-desensitized subsequent responses
to TRP agonists. Data from the third experiment with RR were also
excluded from the pivot table.
For studies of self-desensitization, peak responses (baseline-corrected) to the first and second application of IBA were compared by
paired t-test. For studies of cross-desensitization, IBA-sensitive DRG
cells were divided according to whether they responded or not to
capsaicin. Baseline-corrected peak responses to the first and second
application of IBA were compared by paired t-test. To evaluate
capsaicin cross-desensitization, unpaired t-tests were used to compare
responses to the initial application of capsaicin with responses to
capsaicin post-IBA.
For studies with RR, the incidence of responses of DRG cells to
application of IBA, menthol, CA, or capsaicin in the presence of RR
was compared with the incidence of responses of naïve DRG cells to
each of these agents using Fisher’s exact test with P ⬍ 0.05 considered to be statistically significant. Statistical analyses were done using
SPSS 9.0 software (SPSS, Chicago, IL).
Behavioral Studies
Adult male Sprague-Dawley rats (weight range: 297– 434 g) were
singly housed and given rodent chow and water ad libitum. Behavioral
studies were conducted at approximately the same time each day to
reduce circadian effects. This aspect of the study was approved by the
University of California Davis Institutional Animal Care and Use
Committee.
Evoked behaviors. Rats were first habituated in three daily sessions
to the recording arena that consisted of a glass plate through which the
animals could be videotaped from below. For testing, each rat received a 10-␮l intraplantar injection of either 2% 5-HT (94 mM;
Sigma); 0.1% capsaicin (3.3 mM; Sigma); and 0.1, 0.5, 1, 5, or 10%
(4.2– 424 mM) IBA or vehicle (propylene glycol; MWI Veterinary
Supply, Meridian, ID) using a 30-gauge hypodermic needle, and then
the rats were immediately placed onto the recording arena and
videotaped for 60 min. The rationale for these chemicals is that we
wished to compare behavioral responses associated primarily with
burning irritation (capsaicin) or itch (5-HT) with those associated with
tingle from IBA. Each videotape was analyzed offline by two independent observers who were blinded as to treatment. The following
behavioral responses were separately scored in 2-min intervals over
the 60-min period: time spent licking the injected paw, absolute
number of paw-shakes or flinches, time spent guarding the paw
(defined as holding the injected hindpaw against the body such that it
did not contact the floor), time spent biting the injected hindpaw, time
J Neurophysiol • VOL
spent grooming the face, time spent grooming the body, and absolute
number of wet-dog shakes. The scores provided by the two observers
were in good concordance and were averaged. For statistical analysis
of dose-related behavioral scores, the behavioral counts (time or
absolute numbers) were totaled over the 60-min recording period and
subjected to repeated-measures ANOVA with a P ⬍ 0.05 considered
to be significant.
Heat and mechanical paw withdrawals. Tests of hindpaw heat
withdrawal latency (Hargreaves et al. 1988) and mechanical paw
withdrawal threshold were conducted as described previously (Tsergareli et al. 2009, Klein et al. 2010). Heat or mechanical withdrawals
were tested before and 3, 15, 30, 45, 60, and 120 min following
unilateral intraplantar hindpaw microinjection of 10 ␮l of 0.05, 0.1,
0.25% IBA or vehicle (propylene glycol). IBA at 0.5% elicited
prolonged nocifensive responses that interfered with the thermal and
mechanical paw withdrawal tests. Heat withdrawal latencies and
mechanical withdrawal thresholds at each IBA concentration were
normalized to baseline, and subjected to one-way repeated-measures
ANOVA using SPSS 9.0 software (SPSS). Multiple comparisons
were done using the least significant difference post hoc test. A 95%
confidence interval was used for all statistical comparisons, and error
reported is SE.
RESULTS
Calcium Imaging
Incidence of response to IBA and TRP agonists. In the first
set of experiments, 701 DRG cells were tested with IBA.
Figure 1B shows examples of 3 IBA-responsive DRG cells.
Cell 1 (green cell in Fig. 1B; green trace in Fig. 1C) responded
to the initial application of IBA but did not respond to subsequent TRP agonists, while cells 2 and 3 (black and red cells in
Fig. 1B; black and red traces in Fig. 1C) responded to repeated
IBA as well as TRP agonists menthol, CA, AITC, and
capsaicin.
The incidences of responses of IBA-sensitive and IBAinsensitive DRG cells to menthol, CA, capsaicin, and various
combinations are shown in Tables 1 and 2, respectively.
Thirty-four percent (239 of 701) of all DRG cells responded to
IBA, 47.1% to capsaicin, 14.4% to menthol, and 13.3% to CA,
with many cells responding to various combinations. Only
5.4% of all DRG cells (38/701) responded exclusively to IBA
but not to any TRP agonists. Of IBA-responsive cells, 66%
additionally responded to capsaicin, 29.7% to menthol, and
28.9% to CA, with approximately half of menthol- and CAresponsive cells also responding to capsaicin (Table 1). Of the
Table 2. Coincidence of responses of IBA-insensitive DRG cells
to menthol, CA, capsaicin, and various combinations
n ⫽ 462
%
IBA
Menthol
CA
Capsaicin
Total Number
%
0
0
⫹
⫹
⫹
⫹
30
6.5
⫹
⫹
⫹
⫹
24
5.2
⫹
⫹
⫹
⫹
172
37.2
11
5
11
3
3
5
153
271
462
2.4
1.1
2.4
0.6
0.6
1.1
33.1
58.7
100
The ⫹ indicates that cells responded; - indicates lack of response to given
chemical.
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IBA, isobutylalkenyl amide; DRG, dorsal root ganglion; CA, cinnamic
aldehyde. The ⫹ indicates that cells responded; - indicates lack of response to
given chemical.
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SANSHOOL EXCITES DRG CELLS AND EVOKES NOCIFENSIVE BEHAVIOR
35%) compared with the first response (Fig. 2C). Figure 2B
shows mean responses to successive applications of IBA for
DRG cells whose response to the first application did not
recover to within 10% of prestimulus baseline before the
second IBA was applied. The mean response to the second IBA
application was also significantly lower (by 70%) compared
with the first (Fig. 2C), consistent with self-desensitization.
Less than 30% of cells that did not exhibit IBA self-desensitization also responded to capsaicin.
Capsaicin cross-desensitization. In a separate population of
461 DRG cells, capsaicin was applied first followed by IBA.
Thirty-two percent (147/461) responded to the initial application of capsaicin and only two (0.43%) responded to subsequently applied IBA. Figure 2D shows the mean response of
the capsaicin-sensitive DRG cells. Figure 2E shows the responses of 18 DRG cells that did not respond to initial
application of capsaicin (⬍20% of preceding baseline) but
responded to subsequent application of IBA. The mean response to IBA postcapsaicin (Fig. 2F, open bar) was significantly smaller compared with the mean response to IBA when
tested first in separate DRG cells (Fig. 2C, filled bars). However, the mean response to IBA postcapsaicin was not significantly different (P ⬎ 0.05, unpaired t-test) compared with the
second response to IBA when it was not preceded by capsaicin
(Fig. 2C, open bars), indicating that capsaicin had no effect that
was greater than self-desensitization by IBA.
Capsaicin also appeared to cross-desensitize DRG cell responses to menthol and CA. This is supported by observations
Fig. 2. Self- and cross-desensitization of IBA-evoked responses of DRG cells. A: averaged calcium responses of 118 DRG cells to 2 repeated applications of
IBA at a 5-min interstimulus interval. These cells exhibited partial or complete recovery (within 10% of initial baseline). Error bars ⫽ SE. B: averaged calcium
responses for 95 other DRG cells to 2 repeated applications of IBA at a 5-min interstimulus interval. These cells did not exhibit a recovery to within 10% of
initial baseline following the initial application of IBA. C: mean responses of DRG cells in A and B to IBA (baseline subtracted). *Significantly different from
IBA#1 (P ⬍ 0.05, paired t-test). D: response to capsaicin tested first. E: response to IBA postcapsaicin in Cap⫺ cells. F: mean response to capsaicin tested first
and to IBA postcapsaicin (Cap⫺ cells). $Mean response significantly different (P ⬍ 0.05, unpaired t-test) compared with IBA#1. Cells still exhibited robust
responses to IBA indicating only partial cross-desensitization by capsaicin.
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IBA-insensitive cells, 37% responded to capsaicin with much
lower percentages responding to menthol and/or CA (Table 2).
The percentage of capsaicin-responsive cells was significantly
greater for IBA-sensitive compared with IBA-insensitive cells
(P ⬍ 0.001, Fisher’s exact test). Similarly, the proportions of
cells responsive to menthol and CA were significantly greater for
IBA-sensitive vs. IBA-insensitive cells (P ⬍ 0.001, Fisher’s exact
test).
Figure 1D shows the distributions of cell diameters of all
cells (black bars); IBA-responsive cells that did not respond to
capsaicin, menthol, or CA (blue bars); IBA- and capsaicinsensitive cells (red bars); and IBA- and menthol-sensitive cells
(green bars). DRG cells responsive only to IBA were larger
than other cell groups; the size distribution of IBA-only responsive cells was significantly different compared with IBAand capsaicin-sensitive cells (P ⬍ 0.01, unpaired t-test) and to
IBA- and menthol-sensitive cells (P ⬍ 0.001).
Self-desensitization (tachyphylaxis) to repeated IBA. Of the
IBA-responsive cells described in the preceding section, 213
were tested with two successive applications of IBA at a 5-min
interstimulus interval, followed by the TRP agonists menthol,
CA, and capsaicin. With repeated application of IBA, the
second response was significantly smaller, indicating selfdesensitization or tachyphylaxis. Figure 2A shows responses to
successive applications of IBA for DRG cells whose initial
response recovered to within 10% of the prestimulus baseline
before the second IBA application. For these cells, the response
to the second application of IBA was significantly lower (by
SANSHOOL EXCITES DRG CELLS AND EVOKES NOCIFENSIVE BEHAVIOR
Table 3. Percentages of DRG cells responsive to IBA and TRP
RR. In the presence of RR, the percentages of DRG cells
responsive to IBA, menthol, CA, and capsaicin were significantly reduced (Table 3). RR reduced the incidence of capsaicin-evoked responses to the greatest extent (by 82%), followed by menthol (67%) and CA (42%). RR only reduced the
incidence of IBA responsiveness by 29%.
agonists without and in the presence of ruthenium red
IBA
Menthol
CA
Capsaicin
Naive
⫹RR
34.1
14.4
13.3
47.1
24.1*
4.8*
7.7*
8.4*
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Behavioral Studies
TRP, transient receptor potential; ⫹RR, in the presence of ruthenium red;
naïve, without RR. Naïve: n ⫽ 701 cells. RR: n ⫽ 510, 249, 261, and 155 DRG
cells for IBA, menthol, CA, and capsaicin, respectively. *Significantly different from naïve (P ⬍ 0.05, Fisher’s exact test).
Fig. 3. Intraplantar injection of IBA elicits nocifensive behavioral responses in rats. Rats were injected with 10 ␮l of IBA at doses ranging from 0.1 to 10%;
vehicle (Veh) was propylene glycol. Cumulative duration of grooming, paw licking, biting, and guarding behaviors was recorded in seconds, and cumulative
numbers of paw shakes and wet dog shakes were counted during the 60-min recording period. Video recordings were viewed by 2 independent reviewers blind
to animal treatment. A: cumulative time spent licking injected hindpaw vs. IBA concentration. Error bars ⫽ SE (n ⫽ 15). There was a significant [*F(5,84) ⫽
12.6; P ⬍ 0.001] effect of dose with all but 0.1% different from vehicle. Doses of 1, 5, and 10% IBA were not significantly different from each other. Mean lick
time for 0.1% capsaicin (algogen) and 2.0% 5-HT (pruritogen) was significantly different from vehicle (#,$ P ⬍ 0.01 for both, respectively) and are shown for
comparison (n ⫽ 6/group). Error bars ⫽ SE. B: number of paw shakes-flinches of injected hindpaw vs. IBA concentration. There was a significant [*F(5,84) ⫽ 24.5;
P ⬍ 0.001] effect of dose with all but 0.1% different from vehicle (n ⫽ 15/group). Injection concentration of 10% was not different from 0.5%; concentrations of 5%
not different from 1%. Mean number of paw shakes-flinches for capsaicin and 5-HT were also significantly different from vehicle (#,$ P ⬍ 0.01 for both; n ⫽ 6/group).
C: mean cumulative time spent guarding injected hindpaw vs. IBA concentration. There was a significant [*F(5,84) ⫽ 5.8; P ⬍ 0.001] effect of dose and all
concentrations except 10% different from vehicle (n ⫽ 15/group). Injected concentration of 5% was different from vehicle but not other concentrations. Mean time
guarding for capsaicin and 5-HT were also significantly different from vehicle (#,$ P ⬍ 0.01 for both; n ⫽ 6/group). D: mean cumulative time spent biting injected
hindpaw vs. IBA concentration. There was no significant dose effect of mean biting time postintraplantar injection with IBA. Mean time biting for capsaicin and 5-HT
also shown for comparison (n ⫽ 6/group) and were not significantly different from vehicle. E: mean cumulative time spent grooming body () or face (Œ) vs. IBA
concentration. There was no significant dose effect of body or facial grooming postintraplantar IBA injection. Mean time body or facial grooming for capsaicin (□, )
and 5-HT (⽧, 〫) also shown for comparison (n ⫽ 6/group) and were not significantly different from vehicle. F: mean number of wet dog shakes postintraplantar injection
with IBA. Number of wet dog shakes postintraplantar injection did not vary with IBA concentration (P ⬎ 0.05). Mean time guarding for capsaicin and 5-HT also shown
for comparison (n ⫽ 6/group) and were not significantly different from vehicle.
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that menthol activated a significantly (P ⬍ 0.05, Fisher’s exact
test) lower percentage of DRG cells when tested after vs.
before capsaicin (7.2 vs. 14.4%, respectively). Similar results
were obtained with CA (0.8 vs. 13.3%; P ⬍ 0.05). For this
reason, this cell population was not included in the distributions shown in Tables 1 and 2.
Intraplantar injection of IBA elicited significant, dose-dependent increases in behavioral counts for paw licking, paw
shakes-flinches, and paw guarding (Fig. 3, A–C) that peaked at
1–5% IBA and declined with higher doses. For comparison,
capsaicin (3 mM) and 5-HT (94 mM) elicited behavioral
counts that corresponded to IBA at concentrations of 0.1– 0.5%
(Fig. 3, A–C). Biting of the injected paw, facial and body
grooming, and wet dog shakes did not depend on the dose of
injected IBA, and comparable behavioral counts were elicited
by capsaicin and 5-HT (Fig. 3, D–F).
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SANSHOOL EXCITES DRG CELLS AND EVOKES NOCIFENSIVE BEHAVIOR
The time courses for paw licks, shakes-flinches, and guarding following intraplantar IBA are shown in Fig. 4, A, D, and
G, respectively. There was a degree of similarity in that IBA
(0.5%) elicited a biphasic response pattern reminiscent of
formalin-evoked behaviors, with an immediate initial response
followed by a lull and then another increase in responses at
⬃10 min (Fig. 4, A, D, and G). Licking ceased at 30 min while
shaking-flinching and guarding ceased at 40 –50 min postinjection. Intraplantar vehicle injections elicited very little licking and almost no shaking-flinching (Fig. 4, A and D, open
circles). There was a small increase in paw-guarding 10 –20
min postvehicle injection that was considerably less compared
with IBA (Fig. 4G).
For comparison, the time courses of capsaicin- and 5-HTevoked behavioral responses are also shown in Fig. 4, aligned
vertically for licking (Fig. 4, A–C), paw shaking-flinching (Fig.
4, D–F), and paw-guarding (Fig. 4, G–I). Both capsaicin and
5-HT elicited paw licking that ceased within about 20 min,
while IBA-evoked licking lasted 10 min longer. Both capsaicin
and 5-HT elicited considerably less paw shaking-flinching
compared with IBA (Fig. 4, D–F). Capsaicin and 5-HT elicited
a marked rise in paw-guarding that continued throughout the
60-min observation period, while IBA-evoked paw-guarding
ceased at 40 min (Fig. 4, G–I).
IBA had no significant effect on heat withdrawal latency
either on the treated (ipsilateral) or untreated (contralateral)
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Fig. 4. Time course of nocifensive responses postintraplantar injection of IBA. A: mean time spent licking injected hindpaw vs. time following intraplantar
injection of 0.5% IBA () or vehicle (Œ). Error bars ⫽ SE. B and C: mean time spent licking injected hindpaw vs. time following intraplantar injection of 0.1%
(3.3 mM) capsaicin (B) and 2% (94 mM) 5-HT (C). D, E, and F: mean number of paw shakes-flinches vs. time following intraplantar injection of 0.5% IBA
or vehicle (D), 0.1% capsaicin (E), and 2.0% 5-HT (F). G, H, and I: mean time spent guarding hindpaw after intraplantar injection of 0.5% IBA or vehicle (G),
0.1% capsaicin (H), and 2.0% 5-HT (I).
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SANSHOOL EXCITES DRG CELLS AND EVOKES NOCIFENSIVE BEHAVIOR
paw (Fig. 5, A and B), although there was a trend toward lower
latencies initially with a later increase. The highest concentration of IBA tested resulted in a significant short-lasting decrease in mechanical withdrawal threshold for the treated
(ipsilateral) paw (Fig. 5C) with a significant increase contralaterally (Fig. 5D) possibly reflecting greater weight-bearing on
the contralateral paw due to guarding of the injected paw.
DISCUSSION
Comparison with Human Tingle Sensation
IBA delivered to the tongue of human subjects elicits a
sensation initially dominated by tingle and accompanied by
cooling, pungent (burning), and numbing qualities with a
switch to predominantly cooling sensation after 10 –20 min
(Albin and Simons 2010). The present data are consistent with
this, since a substantial proportion of IBA-responsive DRG cells
additionally responded to capsaicin and/or menthol and hence
presumably coexpressed TRPV1 and/or TRPM8. TRPV1-expressing DRG cells are presumably nociceptors that convey pungency, while TRPM8 is expressed in cold receptors that likely
mediate the cooling quality elicited by IBA. It is currently not
clear if these same cells also signal tingle or if a select
subpopulation of IBA-responsive DRG cells (e.g., those unresponsive to TRP agonists) is critical for tingle sensation. That
multiple types of DRG cells signal tingle is supported by an
earlier study in which identified mechanoreceptor, cold receptor, and nociceptor afferents recorded in the rat lingual nerve
all responded to lingual application of hydroxy-␣-sanshool
(Bryant and Mezine 1999).
Our results showing significant self-desensitization of IBAevoked responses of DRG cells are consistent with our recent
report (Sawyer et al. 2009) of self-desensitization of spinal
wide dynamic range (WDR)-type spinal neurons to intraplantar
IBA. They are also consistent with psychophysical data,
whereby reapplication of IBA after a 30-min rest period elicited significantly weaker tingle sensation (Albin and Simons
2010). The latter study also reported that capsaicin failed to
cross-desensitize IBA-evoked tingle sensation. Our data with
capsaicin-insensitive DRG cells are largely consistent with
this, in that IBA still elicited robust responses following
application of capsaicin in capsaicin-insensitive DRG cells
(Fig. 2E), although the mean response was significantly less
than when IBA was tested first in a different cell population. If
IBA-sensitive capsaicin-insensitive DRG cells signal tingle
sensation, then this result implies that capsaicin should crossdesensitize tingle sensation to a limited extent that might not
have been detected in the human psychophysical paradigm.
Capsaicin did appear to cross-desensitize DRG cell responses
to CA, consistent with capsaicin cross-desensitization of irritant sensation elicited by a variety of chemicals (Green 1991).
Comparison with Previous studies of DRG Cells
In the present study, 47.1% of all DRG cells responded to
capsaicin, 14.4% to menthol, and 13.3% to CA, with smaller
Fig. 5. Enhanced mechanical sensitivity in
the absence of altered thermal sensitivity
ipsilateral to intraplantar injection of IBA.
A: graph plots mean normalized heat withdrawal latencies for treated (ipsilateral)
hindpaw before [baseline (BL)] and at various times following intraplantar microinjection of IBA at the indicated concentrations
or vehicle [propylene glycol (PG)]. There
was no significant effect (P ⬎ 0.05, repeated-measures ANOVA). Error bars ⫽ SE;
n ⫽ 8/group for all graphs. B: as in A for
contralateral (uninjected) hindpaw. There
were no significant treatment or time effects.
C: graph as in A plotting mean electronic
von Frey paw withdrawal threshold for the
ipsilateral (injected) hindpaw. The 0.25%
IBA group was significantly different from
all other groups (*P ⬍ 0.05, repeated-measures ANOVA). D: as in C for contralateral
hindpaw. The 0.25% IBA group was significantly different from all other groups (*P ⬍
0.05, repeated-measures ANOVA).
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The present study shows that about one-third of rat DRG
cells respond to the tingly alkylamide IBA and that two-thirds
of these cells are also activated by capsaicin and to a lesser
extent by menthol and/or CA. Intraplantar injection of IBA
elicited dose-related nocifensive behaviors consistent with the
presence of an aversive sensation. These results are discussed
in relation to human tingle sensation and to previous studies of
the peripheral receptors that transduce and transmit neural
signals related to tingle.
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SANSHOOL EXCITES DRG CELLS AND EVOKES NOCIFENSIVE BEHAVIOR
J Neurophysiol • VOL
2008) reported that sanshool excites mouse TG and DRG cells
via inhibition of two-pore potassium channels KCNK3, 9, and
18 that are widely expressed in central and peripheral neurons,
without directly activating TRPV1 or TRPA1. Moreover, TRP
channel antagonists did not reduce sanshool activation of
mechanoreceptor afferents (Lennertz et al. 2010). Our present
data are consistent with previous studies (Koo et al. 2007;
Bautista et al. 2008; Bhattacharya et al. 2008; Riera et al. 2009;
Lennertz et al. 2010) in that IBA activated both capsaicinsensitive and capsaicin-insensitive cells. We presently observed that the incidences of responses of DRG cells to
capsaicin, menthol, and CA were significantly reduced (by
42– 82%) in the presence of the general TRP channel blocker
RR. RR also significantly reduced the incidence of IBAresponsive DRG cells but to a much lesser extent (by 29%).
We assume that the main effect of RR was to block TRP
channels at the cell surface, although we cannot exclude the
possibility that RR also interfered with a previously unidentified action of IBA on intracellular calcium-signaling pathways
such as ryanodine receptor-mediated calcium release or mitochondrial calcium uptake. We interpret this to indicate that
IBA has a minor excitatory effect on DRG cells via action at
TRPV1 and TRPA1 consistent with Koo et al. (2007) but that
the main mechanism of action of IBA is independent of TRP
channels consistent with the findings of Bautista et al. (2008)
and Lennertz et al. (2010).
Neural Mechanisms of Tingle
As noted above, alkylamide activation of capsaicin-sensitive
neurons may contribute to pungency, and IBA activation of
menthol-sensitive cells observed presently may contribute to its
cooling sensation. However, it is not presently clear which cells
mediate tingle sensation. One possibility is that tingle results from
simultaneous input from many fiber types including mechanoreceptors, thermoreceptors, and nociceptors shown to be activated
by hydroxy-␣-sanshool (Bryant and Mezine 1999; Lennertz et al.
2010). The present data are wholly consistent with this possibility
since IBA-activated cells were also sensitive to capsaicin, menthol, and/or CA.
Alternatively, it may be argued that the tingle or buzzing
sensation elicited by alkylamides is largely a mechanical sensation, consistent with sanshool (Bautista et al. 2008) and IBA
(present data) activating larger-diameter capsaicin-insensitive
cells that may be mechanoreceptors. Buzzing or tingling paraesthetic sensations commonly occur during peripheral nerve
decompression and correlate with bursting activity in singlefiber recordings presumably from low-threshold mechanoreceptors (Ochoa and Tjorebork 1980). Hydroxy-␣-sanshool activates large-diameter sensory neurons that are candidate
mechanoreceptors since they respond to radial stretch and other
mechanical deformations of the cell membrane (Bhattacharya
et al. 2008). A very recent study (Lennertz et al. 2010) reported
that hydroxy-␣-sanshool elicited high-frequency firing in afferent fibers of rapidly adapting D-hair and A␤ guard-hair
mechanoreceptors and lower frequency activity in slowly
adapting type I (Merkel) afferents. Although there are no hair
follicle receptors in the tongue and oral mucosa, candidate
mechanoreceptors that may mediate oral tingle sensation include superficial low-threshold rapidly adapting (Type I;
Meissner) as well as deeper slowly adapting (Type I, II;
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subsets responding to various combinations of these agonists.
This is similar to our previous study (Klein et al., 2011) of rat
TG cells in which 46% responded to capsaicin, 17% to menthol, 18% to CA, and various combinations of these. In both
studies, about half of menthol-sensitive DRG and TG cells
responded to CA and vice versa. All of our studies used cells
from ⬃3-wk-old rats. In contrast, no mouse DRG cells were
observed to respond to both CA and menthol at any developmental stage up to adulthood, although otherwise comparable percentages of cells responsive to capsaicin, capsaicin ⫹ menthol, and
capsaicin ⫹ CA were observed (Hjerling-Leffler et al. 2007).
Thus our present observation that many rat DRG cells responded to both TRPM8 and TRPA1 agonists may be more
likely attributed to a species difference.
We presently found that 34% of rat DRG cells responded to
IBA and two-thirds of these also responded to capsaicin. Our
results are in reasonable agreement with a recent study (Bautista et al. 2008) reporting that 52% of mouse TG and DRG
cells responded to hydroxy-␣-sanshool, with 48% of them also
responsive to capsaicin. This indicates that while alkylamides
may activate many TRPV1-expressing nociceptors, they also
activate a substantial proportion of non-nociceptive (capsaicininsensitive) neurons. A novel finding is that a significant
proportion (29%) of IBA-sensitive DRG cells also responded
to menthol and presumably express TRPM8. This result differs
from previous studies reporting that very few (Bautista et al.
2008) or no (Riera et al. 2009) sanshool-sensitive cells respond
to menthol and that hydroxy-␣-sanshool did not activate HEK
cells transfected with TRPM8 (Koo et al. 2007). A possible
explanation for this discrepancy is that differences in chemical
structures of IBA vs. hydroxy-␣-sanshool may have targeted
different sets or size distributions of sensory neurons or that
species (rat vs. mouse) or methodological differences resulted
in differences in the cell samples. In any event, our data are
consistent with the psychophysical observation of a strong
cooling effect elicited by oral IBA that may be mediated via its
activation of TRPM8-expressing sensory neurons that signal
cold. Besides TRPM8, potassium leak channels (KCNK2)
participate in the detection of cold and a Kv1 channel helps to
establish the threshold of cold-sensitive sensory neurons (Viana et al. 2002; Belmonte et al. 2009; Madrid et al. 2009).
Sanshool was reported to block KCNK3, 9, and 18 but not
KCNK2 channels (Bautista et al. 2008). Nevertheless, we
cannot rule out the possibility that alkylamides might block
cold-sensitive potassium channels as a possible mechanism
underlying their perceptual cooling effects.
Presently, 29% of IBA-sensitive DRG cells also responded
to CA and presumably express TRPA1 (Table 1). This is again
higher compared with the low (1%) percentage of hydroxy-␣sanshool-sensitive mouse TG and DRG cells that were also
activated by AITC (Bautista et al. 2008). However, our data are
consistent with reports that hydroxy-␣-sanshool excites CAsensitive DRG cells (Riera et al. 2009). Since TRPA1 is
usually coexpresssed with TRPV1 (Tominaga et al. 1998;
Caterina et al. 2000; Story et al. 2003; Nagata et al. 2005),
activation of TRPA1-expressing sensory neurons may contribute to the pungency elicited by IBA.
Sanshool has been reported to depolarize DRG neurons via
a direct action at TRPV1 and TRPA1 in a manner that is
reduced by selective and nonselective TRP channel antagonists
(Koo et al. 2007). However, a subsequent study (Bautista et al.
SANSHOOL EXCITES DRG CELLS AND EVOKES NOCIFENSIVE BEHAVIOR
Nocifensive Behavior
Intraplantar injection of IBA elicited dose-dependent increases in paw licking, shaking-flinching, and guarding in a
biphasic temporal pattern reminiscent of formalin-evoked nocifensive behavior (e.g., Carlton and Zhou 1998). Capsaicin
and 5-HT also elicited these behaviors, with the time courses of
licking and shaking-flinching being shorter while paw-guarding was more prolonged compared with IBA-evoked behaviors. Our data are consistent with a previous study (Koo et al.
2007) reporting substantial licking behavior in mice over a
20-min period following intradermal hindpaw injection of
hydroxy-␣-sanshool (4 mg/20 ␮l); licking was markedly attenuated by the TRPV1 antagonist capsazepine or in TRPV1 null
knockout mice. These data are consistent the possibility that
sanshool and IBA elicit an aversive sensation of irritation or
pain via activation of nociceptors expressing TRPV1. Alternatively, these chemicals may elicit an unfamiliar, aversive but
nonpainful tingle sensation that is expressed behaviorally by
the limited nocifensive response repertoire available to the
animal. Mice avoid hydroxy-␣-sanshool in a one-bottle drinking paradigm (Lennertz et al. 2010), supporting the contention
that tingle is an aversive sensation in rodents.
Because hydroxy-␣-sanshool was reported to activate
TRPA1 and TRPV1 (Koo et al. 2007; Reira et al. 2009), we
investigated if intraplantar injection of IBA enhanced behavioral responses to mechanical and well as noxious heat stimuli,
as has been reported for capsaicin (Gilchrist et al. 1996) and
CA (Tsagareli et al. 2010). While IBA did not significantly
affect heat-evoked withdrawal latencies, it significantly lowered mechanical paw withdrawal threshold on the injected side,
implying enhanced mechanosensitivity. If IBA acts at least
partly via inhibition of KCNK channels similar to sanshool
(Bautista et al. 2008), then blockade of such channels, if they
were expressed in mechanoreceptors, could have a depolarizing action rendering them more sensitive to mechanical stimuli. There was also a reduction in mechanosensitivity on the
opposite side, possibly reflecting increased weight bearing by
the uninjected paw during guarding of the injected paw. It is
interesting that IBA had the opposite effect, reducing mechanical sensitivity on the human tongue (Albin and Simons 2010).
This effect occurred shortly after IBA application and is poorly
explained by the weaker numbing effect of IBA that occurs
later. It was suggested that IBA activates mechanoreceptors to
J Neurophysiol • VOL
increase “noise,” thereby reducing the ability to discriminate
weak mechanical stimuli via a Weber’s Law effect (Albin and
Simons 2010).
GRANTS
This work was supported by National Institutes of Health Grants DE013685 and AR-057194.
DISCLOSURES
C. T. Simons, S. Furrer, and J. P. Slack are employees of Givaudan Food
Flavors, who provided the sanshool derivative IBA used in the present study.
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