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JPET #196717 1
Central Mechanisms of Menthol-Induced Analgesia
Rong Pan, Yuzhen Tian, Ruby Gao, Haitao Li, Xianguo Zhao, James E. Barrett,
and Huijuan Hu
Department of Pharmacology and Physiology, Drexel University College of
Medicine, Philadelphia, USA (R. P., Y. T., R. G., H. T., J. B., H. H.); Center for
Teaching & Experiment, Nanjing University of Traditional Chinese Medicine,
Nanjing, China (H. L.); PharmaOn, LLC, Monmouth Junction, USA (X. Z)
JPET Fast Forward. Published on September 5, 2012 as DOI:10.1124/jpet.112.196717
Copyright 2012 by the American Society for Pharmacology and Experimental Therapeutics.
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Running Title: Mechanisms of Menthol’s Action
Corresponding author:
Huijuan Hu, Ph.D.
Department of Pharmacology and Physiology
Drexel University College of Medicine
245 N. 15th Street, M.S. #488
Philadelphia, PA 19102
Tel: 215-762-3566
Fax: 215-762-2299
Number of text pages: 34
Number of tables: 1
Number of figures: 9
Number of references: 40
Abstract: 247 words
Introduction: 493 words
Discussion: 1481 words
ABBREVIATIONS: Nav, voltage-gated sodium channel; Cav, voltage-gated calcium
channel; INa, sodium current; ICa, calcium current; I-V, current-voltage; TTX,
tetrodotoxin; EPSC, excitatory postsynaptic current; TRP, transient receptor
potential; CFA, complete Freund’s adjuvant; Veh, vehicle. IT, intrathecal injection;
IP, intraperitoneal injection; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione.
LC/MS/MS, liquid chromatography tandem mass spectrometry.
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ABSTRACT
Menthol is one of the most commonly used chemicals in our daily life, not only
because of its fresh flavor and cooling feeling but also because of its medical
benefit. Previous studies have suggested that menthol produces analgesic action
in acute and neuropathic pain through peripheral mechanisms. However, the
central actions and mechanisms of menthol remain unclear. Here, we report that
menthol has direct effects on the spinal cord. Menthol decreased both ipsilateral
and contralateral pain hypersensitivity induced by complete Freund’s adjuvant in
a dose dependent manner. Menthol also reduced both first and second phases of
formalin-induced spontaneous nocifensive behavior. We then identified the
potential central mechanisms underlying the analgesic effect of menthol. In
cultured dorsal horn neurons, menthol induced inward and outward currents in a
dose-dependent manner. The menthol-activated current was mediated by Cl- and
blocked by bicuculline, suggesting that menthol activates A-type gamma-
aminobutyric acid receptors. In addition, menthol blocked voltage-gated sodium
channels and voltage-gated calcium channels in a voltage-, state-, and use-
dependent manner. Furthermore, menthol reduced repetitive firing and action
potential amplitude, decreased neuronal excitability, and blocked spontaneous
synaptic transmission of cultured superficial dorsal horn neurons. LC/MS/MS
analysis of brain menthol levels indicated that menthol is rapidly concentrated in
the brain when administered systemically. Our results indicate that menthol
produces its central analgesic action on inflammatory pain likely via blockage of
voltage-gated Na+ and Ca2+ channels. These data provide molecular and cellular
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mechanisms by which menthol decreases neuronal excitability therefore
contributing to menthol-induced central analgesia.
Introduction
Menthol is an organic compound naturally occurring in peppermint oil from
mint plants. It is well known for its cooling sensation and is widely used in a
number of products including toothpaste, cough drops, mouthwash, chewing
gum, and topical analgesics. Menthol has complex peripheral effects. Topical
application of low-to-moderate concentrations of menthol reduces the irritancy of
capsaicin, heat hypersensitivity, sprains, and headaches (Green and McAuliffe,
2000; Borhani Haghighi et al., 2010; Higashi et al., 2010; Klein et al., 2010), but
intraplantar injection or topical application of a high concentration of menthol
induces cold allodynia and hyperalgesia (Hatem et al., 2006; Wasner et al., 2008;
Gentry et al., 2010; Cordova et al., 2011). Interestingly, menthol induces cold
allodynia in healthy volunteers while producing analgesic effects in patients with
neuropathic pain (Wasner et al., 2008). TRPM8 and TRPA1 have been identified
as the cold and menthol-sensitive channels (McKemy et al., 2002; Peier et al.,
2002; Karashima et al., 2007; Bianchi et al., 2012). The peripheral mechanism of
menthol-induced cold hyperalgesia is thought to be induced by activating TRPM8
and TRPA1 channels (Gentry et al., 2010; Knowlton et al., 2010). Although the
peripheral mechanism of the antinociceptive effect of menthol is not certain, it
has been suggested that menthol blocks voltage-gated calcium currents in
cultured sensory neurons (Swandulla et al., 1987). A previous study
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demonstrated that menthol inhibits Nav1.2 sodium currents heterologously
expressed in HEK293 cells (Haeseler et al., 2002). A recent report also showed
that menthol blocks voltage-gated sodium channels in dorsal root ganglion
neurons (Gaudioso et al., 2012).
Menthol-induced cold sensitivity have been studied intensively. In contrast,
only a few reports have directly assessed the analgesic properties of menthol.
Systemic menthol reduces acute pain induced by heat and acetic acid (Galeotti
et al., 2002). Intrathecal application of menthol decreases mechanical allodynia
and thermal hyperalgesia in animals with chronic constriction injury (Proudfoot et
al., 2006; Su et al., 2011). The mechanism underlying menthol-induced analgesia
is still controversial. It has been suggested that the analgesic effect of menthol is
mediated by activation of TRPM8 in the central terminals of sensory neurons
(Proudfoot et al., 2006). However, the role of TRPM8 in mechanical and thermal
hypersensitivity has not been established (Colburn et al., 2007; Su et al., 2011).
Whether the analgesic effect of menthol is restricted to these particular pain
conditions or whether menthol is a nonselective analgesic for pain
hypersensitivity is an open question.
Here, we examined the effect of menthol on pain hypersensitivity induced by
inflammation. We demonstrated that menthol attenuates complete Freund’s
adjuvant (CFA)-induced primary and secondary pain hypersensitivity, and
decreases formalin-induced first and second phases of spontaneous nociceptive
behavior. We further identified the central mechanism(s) of menthol-induced
analgesia. Our data indicate that menthol directly activates A-type gamma-
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aminobutyric acid (GABAA) receptors and blocks voltage-gated sodium and
calcium channels in dorsal horn neurons. The latter may account for the central
spinal mechanisms of menthol-induced analgesic action.
Materials and Methods
All behavioral tests were performed using 8- to 10-week-old CD-1 male mice
purchased from Charles River (Wilmington, MA). Experiments were done in
accordance with the guidelines of the National Institutes of Health and were
approved by the Animal Care and Use Committee of Drexel University College of
Medicine. Mice were housed in 12 h/12 h light/dark cycles. All experiments were
performed with the experimenter blind to the drug treatment.
CFA-Induced Mechanical and Thermal Hypersensitivity. CFA-induced
inflammatory pain was measured as previously described (Gao et al., 2010).
Twenty microliters of 50% CFA was injected subcutaneously into the plantar
surface of the right hind paw. Mechanical sensitivity was measured using a
series of von Frey filaments (North Coast Medical, Inc., San Jose, CA) as
previously described (Hu et al., 2006). The smallest monofilament that evoked
paw withdrawal responses on three out of five trials was taken as the mechanical
threshold. Thermal sensitivity was measured using the Hargreaves’ method as
previously described (Hu et al., 2006). The baseline latencies were set to around
10 s with a maximum of 20 s as the cutoff to prevent potential injury. The
latencies were averaged over three trials, separated by 15-min intervals.
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Formalin-Induced Spontaneous Nociceptive Behavior. The formalin test
was performed as previously described (Hu et al., 2006). Mice were habituated
for 1 h in a transparent Plexiglas test box (5 × 5 × 10 inches) before any
injections. Mice received i.p. injections of 100 mg/kg menthol 15 min prior to
injection with formalin. Ten microliters of 2% formalin solution was injected
subcutaneously into the plantar surface of the right hind paw, and the mouse was
returned to the test box immediately. The total time spent in spontaneous
nociceptive behavior (licking and lifting of the injected paw) was recorded in 5-
min intervals for 1 h.
Cell Culture. Primary cultures of spinal cord superficial dorsal horn neurons
were prepared from 2- to 4-day-old CD-1 mouse pups as described previously
(Hu et al., 2002). Cells were cultured for 1 to 2 days for voltage-gated sodium
and calcium current recordings, for 3 to 4 days for menthol-induced current
recordings, and for 5 to 7 days for action potential recordings.
Electrophysiological Recording. Standard whole-cell recordings were made
at room temperature using an EPC 10 amplifier and PatchMaster software
(HEKA Elektronik, Lambrecht, Germany) as previously described (Hu et al.,
2007). Electrode resistances were 3 to 5 MΩ with series resistances of 6 to 10
MΩ, which was compensated by > 60%. Membrane potentials were corrected for
the liquid junction potential. All neurons included in this study had resting
membrane potentials ≤ -45 mV, had stable input resistance, and had leak
currents < -80 pA (at -80 mV), which were not subtracted on line.
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Recordings of menthol currents, Na+ and Ca2+ currents were performed under
voltage-clamp configurations. For menthol-activated current recordings, the
membrane voltage was held at -65 mV; the bath solution was Tyrode’s solution
containing (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 10 Hepes, and 10
glucose. The electrode solution contained (in mM) 140 CsCl (or CsMeSO4), 2
MgCl2, 1 EGTA, 10 HEPES, 3 Na2ATP, and 0.3 Na2GTP, pH 7.4. For Na+ current
recordings, the membrane voltage was held at -80 mV, and the extracellular
solution contained (in mM) 105 NaCl, 5 KCl, 10 CsCl, 20 TEA-Cl, 1 MgCl2, 200
µM CdCl2, 1 CaCl2, 5 HEPES, and 10 glucose. The electrode solution contained
(in mM) 140 CsMeSO4, 2 MgCl2, 1 EGTA, 10 HEPES, 3 Na2ATP, and 0.3
Na2GTP, pH 7.4. For Ca2+ current recordings, the membrane voltage was held at
-70 mV, the same intracellular and extracellular solutions were used except that
CdCl2 was omitted and 500 nM tetrodotoxin (TTX) and 6 mM BaCl2 were added
in the extracellular solution.
To determine the current-voltage (I-V) relationship, steady-state activation and
inactivation, and use-dependent blockage of sodium and calcium channels, the
membrane potential was held at -80 mV (INa) or -70 mV (ICa). For calcium current
activation, voltage steps of 250 ms were applied at 10-s intervals in +10 mV
increments from -30 mV to +30 mV. For sodium current activation, a series of
voltage steps of 50 ms was applied at 5-s intervals in +10 mV increments from -
50 mV to +60 mV. To determine the voltage dependence of inactivation of Na+ or
Ca2+ channels, conditioning pre-pulses ranging from -100 to 10 mV (INa) or to 30
mV (ICa) were applied in 10-mV increments for 100 ms (INa) or 1 s (ICa), followed
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by a step to -20 mV for 25 ms (INa) or to 10 mV for 200 ms (ICa). To determine
use-dependent blockage of sodium and calcium channels, a series of 25-ms
pulses to -20 mV (INa) or 50-ms pulses to 10 mV (ICa) were applied at 20 Hz (INa)
or 10 Hz (ICa). For current-clamp recordings and excitatory postsynaptic current
(EPSC) recordings, the intracellular solution contained (in mM) 135 KMeSO4, 5
KCl, 2 MgCl2, 1 EGTA, 10 HEPES, 3 Na2ATP, and 0.3 Na2GTP, pH 7.4. The
bath solution was Tyrode’s solution.
Determination of the Concentration of Menthol in the Brain.
Sample and Calibrator Preparation. Three groups of mice were
administered 100 mg/kg, i.p. of menthol. Whole brain tissues were collected at 5,
30, and 60 min following menthol administration. Each brain sample was
weighed and homogenized with two volumes of Milli-Q water using a dounce
homogenizer, and sonicated using a Sonifier 250 (Branson Ultrasonics Co.,
Danbury, CT). Menthol stock solution was prepared at 25 mg/ml in DMSO. The
standard calibrators were prepared in control brain homogenates from this stock
containing 0, 0.006, 0.024, 0.10, 0.39, 1.56, 6.25, and 25 µg/ml menthol.
Extraction of Menthol and Preparation of Dimethylglycinyl Ester of
Menthol. The calibrators and brain homogenate samples from mice treated with
menthol were mixed with three volumes of hexane containing 100 ng/ml of
cyclohexanol (internal standard) using a vortex. The extracts were then
centrifuged at 10000x g for 10 min. Aliquots (300 µl) of the resulting supernatants
were mixed with 200 mg sodium sulfate (anhydrous) for 2 hr. The dehydrated
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hexane extracts (100 µl) were transferred into individual glass vial. Derivatization
of menthol with dimethylglycine was made prior to LC/MS/MS analysis using
modified method (Jiang et al., 2007). Briefly, thirty microliters of dimethylglycine
hydrochloride (0.5M) and N,N-dimethylaminopyridine (2M) in CHCl3 and 30 µl of
N-(3-dimethylaminopropyl)N’-ethylcarbodiimide hydrochloride (1M) were added
into the individual sample. The reaction was carried out at 37 ºC overnight. The
resulting solution was evaporated to dryness under nitrogen stream. The residue
was reconstituted with 400 µl of 0.1% acetic acid in water: methanol: acetonitrile
(100:225:75, v/v/v) and subjected to LC/MS/MS analysis.
Quantification of Menthol in Brain Tissue Using HPLC Mass
Spectrometry. LC/MS/MS analysis was performed on an API 3000 triple-
quadrupole mass spectrometer (Applied Biosystems/MDS Sciex, Foster City,
CA), equipped with a TurboIonSpray source. Chromatographic separation was
performed on Shimadzu HPLC system (Shimadzu, Piscataway, NJ) with a
Symmetry C18 analytical (3.5 µm, 4.6 x 75 mm) column (Waters, Milford, MA).
Mobile phase A was 0.1% acetic acid in water and mobile phase B was
methanol: acetonitrile (75:25, v/v). Mobile phase was delivered at an initial
condition of 100% A and a flow rate of 0.5 ml/min with a linear gradient to 20% B
in 2 min, then increased to 90% B in 5 min. The column was re-equilibrated with
100% A after washing. Under these conditions, the retention time was 6.3 min for
menthol ester and 3.9 min for cyclohexanol ester. The injection volume was 5 µl.
The transition is m/z 242→104 for menthol ester and m/z 242→104 for
cyclohexanol ester. The instrument was operated in positive-ion mode, with an
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ion spray voltage of 4500 V. The curtain gas, ion source temperature and
collision energy were set at 12 psi, 250 ºC, and 20, respectively.
Chemicals and Reagents. DL-Menthol and 6-cyano-7-nitroquinoxaline-2,3-
dione (CNQX) were purchased from Sigma-Aldrich (St. Louis, MO) and dissolved
in dimethyl sulfoxide as stock solutions. Bicuculline, TTX, and
tetraethylammonium chloride were purchased from Sigma-Aldrich and dissolved
in water as stock solutions. All of these solutions were further diluted to final
concentrations in bath solution (for in vitro, 0.5% DMSO) and in saline solution
(for intrathical injection, 2.5% DMSO; for intraperitoneal injection, 2%
DMSO/cremophor). Cyclohexanol was purchased from Sigma-Aldrich and used
as the LC/MS/MS internal standard. Dimethylglycine hydrochloride, N,N-
dimethylaminopyridine and N-(3-Dimethylaminopropyl)N’-ethylcarbodiimide
hydrochloride were purchased from Sigma-Aldrich and used for derivatization of
menthol. All other reagents were HPLC grade and purchased from Sigma-
Aldrich.
Coverslips were placed in a small laminar flow perfusion chamber. Chemical
agents were applied by local gravity-driven perfusion. Cells were continuously
perfused at approximately 5 to 6 ml/min.
Data Analysis. Off-line evaluation was done using PatchMaster (HEKA
Elektronik) and Origin 8.1 (OriginLab Corporation, Northampton, MA). Data were
expressed as original traces or as means ± SEM. For the I-V curves, peak
currents at different potentials were normalized to the maximal current (I/Imax).
For the activation curves, the peak current was converted to conductance (G) by
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the formula G = I/(Vm – Vrev), where Vm is the membrane voltage of depolarization
pulses and Vrev is the reversal potential of the current derived from I-V curves.
The voltage dependency of activation and inactivation was fitted with the
Boltzmann function: G/Gmax = 1/(1 – exp[(V1/2 – V)/k]) was used, where G/Gmax is
the normalized conductance, V1/2 is the potential of half-maximum channel
activation, and k is the slope factor. For inactivation, I/Imax = 1/(1 – exp[(V1/2 –
V)/k]) was used, where I/Imax is the normalized current. For the use-dependent
block, inhibition of current amplitudes at each voltage step was calculated as %
inhibition = 100 – 100 × (I/Imax), where I = the value of the current in the presence
of menthol; Imax = the value of current in the absence of menthol. Time constants
for Ca2+ current decay were obtained using a single exponential function: y = A(1
– exp(-t/ּז)), where A is the current amplitude, t is time (milliseconds), and 1ז is the
time constant. For the concentration-dependent curve, the value of the current in
the presence of menthol normalized to the current in the absence of menthol was
used to calculate the potency of the menthol for blocking the channels. The half
maximal inhibitory concentration (IC50) in the various experimental conditions
was determined by fitting the concentration–response curves with the Hill
equation: I/I0 = 1/(1 + (C/IC50)n), where I0 is the maximum peak current amplitude,
C is the menthol concentration, and n is the Hill coefficient. Treatment effects
were statistically analyzed with a one-way or two-way analysis of variance
(ANOVA). Data are presented as mean ± SEM. When ANOVA showed a
significant difference, pair wise comparisons between means were tested by the
post hoc Bonferroni method. Paired or two-sample t tests were used when
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comparisons were restricted to two means. Error probabilities of P < 0.05 were
considered statistically significant. The statistical software Origin 8.1 (OriginLab
Corporation, Northampton, MA) was used to perform fitting of the concentration–
response curves and all statistical analyses.
Results
Menthol Reduces CFA-Induced Thermal and Mechanical Hypersensitivity
To examine the effect of menthol on inflammatory pain, we first tested the
effect of menthol in a CFA-induced pain model in mice. Injection of 20 μl of 50%
CFA into the hind paw of a mouse produced thermal hyperalgesia in the
ipsilateral paw, which recovered completely after 3 weeks (Figure 1A). However,
CFA did not induce thermal hypersensitivity in the contralateral paw.
Administration (i.p.) of menthol 50, 100 mg/kg significantly decreased thermal
hyperalgesia in a time- and dose- dependent manner with the maximal effect
occurring after 30 min (Figure 1B).
CFA-treated mice fully developed robust mechanical allodynia in the ipsilateral
paw 3 h after CFA injection and in the contralateral paw 3 day post CFA injection.
CFA-induced mechanical allodynia lasted for 4 weeks (data not shown). Menthol
100 mg/kg i.p. reduced mechanical allodynia in both ipsilateral and contralateral
paws when measured at 96 hr following CFA injection (Figure 1C). To examine
its direct effect at the spinal level, menthol was administered intrathecally in 5 μl
at a concentration of 50 mM (250 nmol) and produced a marked analgesic effect
in both ipsilateral and contralateral paws (Figure 1D). These results suggest that
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menthol relieves CFA-induced inflammatory pain and has a direct action in the
spinal cord.
Menthol Reduces Formalin-Induced Spontaneous Nociception
The formalin test is a useful and reliable model of nociception. It allows for an
assessment of analgesic action and provides the ability to distinguish the site of
action of analgesics (Hunskaar and Hole, 1987; Shibata et al., 1989). Formalin
induces spontaneous biphasic nociceptive behavior. The first phase is due to
acute stimulation of nociceptors. The second phase of nociception is thought to
be involved in central sensitization of dorsal horn neurons (Coderre and Melzack,
1992). Injection of 2% formalin subcutaneously in the hind paw of a mouse
resulted in intense spontaneous licking or lifting of the injected paw with a classic
biphasic response. A single i.p. injection of 100 mg/kg menthol significantly
decreased both first and second phases of formalin-induced nociception (Figure
2A, B). This feature of blocking both phases is similar to that observed with
centrally acting drugs such as narcotics, further suggesting that menthol may
have a direct central action.
Concentration of Free Menthol in the Brain after a Single i.p. Administration
To further confirm that menthol has a direct central action, free menthol
concentrations in brain tissues were analyzed by LC/MS/MS. Mice received a
single i.p. administration of menthol at 100 mg/kg, the same dose used for the
assessment of behavioral tests. Whole brain samples were obtained at 5, 30 and
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60 min after menthol injection (i.p.). Menthol was rapidly absorbed and a high
concentration of menthol (54.6 µg/g) was detected in brain tissue at 5 min after
100 mg/kg i.p. injection. Its concentration was largely decreased to 12.1µg/g at
30 min, but was remained detectable after 60 min (Fig. 3). This result indicates
that menthol has great access to CNS.
Menthol Activates GABAA Receptors in Spinal Dorsal Horn Neurons
As demonstrated above, menthol has a direct central action and can cross the
blood brain barrier when administered systemically. We were interested in
exploring the central mechanisms of menthol-induced analgesia. It has been
shown that menthol activates GABAA receptors in hippocampal neurons (Zhang
et al., 2008). We hypothesized that the central mechanism of menthol-induced
analgesic effect may be mediated by the activation of GABAA receptors in dorsal
horn neurons. To test this hypothesis, we performed patch-clamp recordings in
cultured dorsal horn neurons. Neurons were held at -65 mV (the average resting
membrane potential is -56.2 ± 1.0 mV) with a continuous recording protocol. Bath
application of 2 mM menthol induced both inward and outward currents with
varying amplitudes in most neurons tested (45/48 neurons). The effects of
menthol were readily reversible upon drug washout (data not shown). We then
examined the concentration-response relationships for menthol. Menthol evoked
inward currents in a concentration-dependent manner and an EC50 value of 1.08
± 0.04 mM (n = 4; Figure 4A).
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To determine whether menthol-induced current is mediated by activation of
GABAA receptors, we pretreated neurons with bicuculline, a GABAA antagonist.
Menthol-induced currents were dramatically attenuated in a concentration-
dependent manner (Figure 4B). To confirm that menthol-induced current was
mediated by Cl- channels, we performed voltage-clamp recordings in cultured
dorsal horn neurons. Replacement of CsCl in the internal solution with CsMeSO4
resulted in a significant shift of the reversal potential (Figure 4C, D). These
results indicated that the menthol-activated currents are mediated by GABAA
channels.
Menthol Blocks Voltage-Gated Sodium Channels in Dorsal Horn Neurons
As we demonstrated above, activation of GABAA receptors by menthol
required a relatively high concentration. An effective dose (100 mg/kg) of menthol
may not be sufficient to reach the concentration required for GABAA activation in
the spinal cord. Therefore, other mechanisms may underlie the central analgesic
action of menthol. Previous studies indicated that the peripheral mechanism of
menthol-induced analgesia is mediated by blocking voltage-gated sodium
channels in dorsal root ganglion neurons (Gaudioso et al., 2012). We
hypothesized that menthol may block sodium channels in dorsal horn neurons.
We performed Na+ current recordings with K+ and Ca2+ currents eliminated
pharmacologically (see Materials and Methods). When neurons were held at -80
mV, ten steps of depolarization from -60 to +30 mV evoked fast activating, fast
inactivating voltage-dependent Na+ currents, which were completely blocked by
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500 nM TTX (data not shown). Sodium currents were activated at approximately
-50 mV and reached their peak around -20 mV (Figure 5A, B). Bath application of
menthol shifted the I-V curve upward and blocked the peak of Na+ currents with
IC50 297.1 ± 64.0 µM and a Hill coefficient of 1.6 ± 0.1 (Figure 5B-D). These
results show that menthol blocks Na+ channels in dorsal horn neurons in a
concentration-dependent manner.
Mechanism of Blocking Na+ Channels by Menthol
To explore the mechanism of menthol’s effect on Na+ channels, we first
examined a state-dependent block of sodium channels. Neurons were held at
-100 mV or -55 mV and stimulated by a brief test pulse of -20 mV once every 10
s. Bath application of 300 µM menthol blocked about 17% of peak Na+ current
with IC50 1936 ± 269 μM when neurons were held at -100 mV (resting state).
However, menthol blocked about 73% of peak Na+ current with IC50 89 ± 17 µM
when neurons were held at -55 mV (inactivated state) (Figure 6A, B). We then
studied the possible use-dependent block of sodium channels by menthol.
Neurons were depolarized to -20 mV from -80 mV (holding potential). Under this
condition, there was a 20% to 30% reduction in sodium currents at the end of
stimulation with 20 Hz. However, in the presence of 300 μM menthol, there was
substantial use-dependent block during 20-Hz stimulation (Figure 6C, D).
Menthol reduced the peak currents by 44 ± 3% at the first pulse and 64 ± 4% at
the 10th pulse. To determine the voltage-dependent block of sodium channels,
we tested the effect of menthol on steady-state activation and inactivation.
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Neurons were stimulated by activation or inactivation protocols as described in
Materials and Methods. Bath application of menthol did not alter the activation
curve. However, menthol negatively shifted the inactivation curve by 10 mV
without changing the slope factor (Figure 6E and Table 1). Together, these
results suggest that menthol blocks sodium channels in a state-, voltage-, and
use-dependent manner.
Menthol Blocks Voltage-Gated Calcium Channels in Dorsal Horn Neurons
Menthol has been shown to block calcium channels in dorsal root ganglion
neurons (Swandulla et al., 1987). To test whether menthol also blocked voltage-
gated Ca2+ currents in dorsal horn neurons, we performed Ca2+ current
recordings. To increase calcium currents, Ba2+ was used as the major charge
carrier. Neurons were depolarized to a series of test pulses: -30 mV to 60 mV
from -70 mV (holding potential). Ca2+/Ba2+ currents were evoked around -10 mV
and reached their maximal amplitude around 10 mV (Figure 7A, B), which were
resistant to 50 µM NiCl2 (a known T type calcium channel blocker) and
completely blocked by 200 µM CdCl2 (data not shown). Bath application of 300
µM menthol shifted the I-V curve upward and blocked the peak of Ca2+/Ba2+
currents with IC50 124.8 ± 16.0 µM (Figure 7 B-D). The decay rate of the maximal
Ca2+/Ba2+ current was slow and could be fitted with a single exponential in most
neurons (at 10 mV). In the presence of menthol, the time constant was
significantly decreased from 220 ± 19 to 86 ± 9 ms (n = 6, P < 0.05). These
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results suggest that menthol blocks Ca2+ channels and facilitates calcium
channel inactivation.
Mechanism of Blocking Ca2+ Channels by Menthol
To determine the mechanism of the effect of menthol on Ca2+ channels, we
also examined the state-dependent block of calcium channels. Neurons were
held at -100 mV or -70 mV and stimulated by a test pulse at 10 mV once every
15 s. Bath application of 300 µM menthol blocked the peak Ca+ current by 61%
at -100 mV or 81% at -70 mV (Figure 8A, B), suggesting that menthol may bind
to both resting and inactivated Ca2+ channels with preferential binding to
inactivated channels. To test the use-dependent block of calcium channels by
menthol, neurons were depolarized to 10 mV from -70 mV. Under this condition,
a 10-Hz train of 50 ms depolarization pulse produced about 50% reduction of the
calcium current at the end of the pulse train. Bath application of 150 μM menthol
produced a use-dependent block and reduced peak currents by 47% at the first
pulse and by 78% at the 10th pulse (Figure 8C, D). Increased concentrations of
menthol did not enhance the use-dependent block (data not shown). To study
activation parameters, Ca2+ currents were elicited by applying 250-ms voltage
steps ranging from -30 to 30 mV in 10-mV increments from a holding potential of
-70 mV. In the presence of 300 µM menthol, a small depolarizing shift in half-
activation voltage (V1/2) by 5.0 mV was observed (Figure 8E). To determine
inactivation parameters, we generated steady-state inactivation curves using the
protocols described in Materials and Methods. Menthol induced a hyperpolarizing
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shift of the inactivation curve by -8.3 mV (Figure 8E). Taken together, these
results suggest that the menthol blocks calcium channels in a voltage-, state-,
and use-dependent manner.
Menthol Reduces Neuronal Excitability and Blocks Synaptic Transmission
in Spinal Dorsal Horn Neurons
To examine the consequence of the block of voltage-gated channels by
menthol, we performed current-clamp recordings in cultured dorsal horn neurons.
Action potentials were evoked by depolarizing current injections from a holding
potential of -65 mV (the Cl- reversal potential in our recording conditions). Bath
application of 300 or 600 μM menthol dramatically decreased the number of
action potentials and slightly reduced the amplitude of action potentials (Figure
9A-B), but had no effect on the threshold and first latency (data not shown).
Menthol (600 μM) also increased the rheobase (the minimum current required to
elicit an action potential) (Figure 9B). These results indicate that menthol
decreases excitability in spinal cord dorsal horn neurons. To test the overall
effect of menthol on spontaneous activity in dorsal horn neurons, we recorded
spontaneous excitatory postsynaptic currents (EPSC). Neurons were held at -65
mV, and spontaneous EPSCs were recorded with a frequency of 20-30 Hz,
which were completely blocked by 10 µM CNQX, a competitive AMPA/kainate
receptor antagonist (data not shown). Interestingly, menthol completely shut
down the TTX-sensitive spontaneous EPSC at the concentrations of 100 µM, 300
μM (Figure 9C). In contrast, menthol had no effect on miniature EPSC (in the
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presence of TTX). This result indicates that menthol blocks spontaneous activity-
induced synaptic transmission in dorsal horn neurons.
Discussion
Results from the present study reveal that menthol produces an analgesic
effect on inflammatory pain. Menthol attenuated CFA-induced thermal and
mechanical hypersensitivity, indicating that the analgesic action of menthol is not
associated with a specific stimulus. Previous studies and our results suggest that
menthol reduces acute pain, neuropathic pain, and inflammatory pain (Swandulla
et al., 1987; Gaudioso et al., 2012), indicating that menthol is a nonselective
analgesic. Systemic administration of menthol reduced both ipsilateral and
contralateral mechanical allodynia. It is generally accepted that contralateral pain
(secondary hyperalgesia) is associated with plastic changes in the central
nervous system (Koltzenburg et al., 1999; Filipovic et al., 2012). The attenuation
of pain by menthol on the contralateral paw may suggest that it acts as a central
modulator. The results from the formalin tests demonstrated that menthol
decreased both the first and second phases of formalin-induced spontaneous
nociceptive responses. It has been suggested that centrally acting drugs such as
narcotics inhibit both phases; peripherally acting drugs such as indomethacin and
dexamethasone only inhibit the second phase (Hunskaar and Hole, 1987;
Shibata et al., 1989). The reduction of both phases may suggest that menthol
has a central action. Intrathecal application of menthol had a similar effect on
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mechanical allodynia, rendering further support for an action of menthol at the
spinal level.
The peripheral analgesic action of menthol has been recognized for decades.
The peripheral mechanism of menthol is thought to be mediated by blocking
voltage-gated calcium and sodium channels (Swandulla et al., 1987; Gaudioso et
al., 2012). Recent studies have suggested that menthol induces analgesic effect
at the spinal level (Proudfoot et al., 2006; Su et al., 2011). Our results provide
further evidence that menthol produces a central analgesic action. To explore the
central spinal mechanisms underlying this action, we performed a series of
electrophysiological experiments in cultured dorsal horn neurons. We found that
menthol induced Cl--mediated currents, which was blocked by bicuculline, a
GABAA receptor antagonist, indicating that menthol directly activates GABAA
receptors. This finding is consistent with results from previous studies implicating
menthol as a GABAA receptor modulator (Watt et al., 2008; Zhang et al., 2008).
We also demonstrated that menthol blocked TTX-sensitive Na+ channels in
spinal cord dorsal horn neurons. A higher concentration of Menthol (300 μM)
slightly blocked Na+ channels with IC50 about 1936 μM at a holding potential of -
100 mV, at which all channels are expected to be in the resting state. This small
effect of menthol could be defined as a weak resting channel block. Menthol
dramatically decreased Na+ currents with IC50 89 μM at -55 mV. The 20-fold
increase in blocking potency at a more depolarized holding potential, at which a
fraction of the channels are inactivated, suggests that menthol preferentially
binds to Na+ channels when the channels are inactivated. This finding is further
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supported by the evidence that menthol induced a hyperpolarizing shift in the
steady-state inactivation curve. Our data also showed that menthol-induced
inhibition of Na+ currents increases with repetitive depolarization, indicating use-
dependent block of Na+ channels. The use-dependent phenotype develops when
the interval between pulses is too short to completely recover from inactivation,
which represents an accumulation of inactivated channels. The menthol-induced
use-dependent block of Na+ channels is most likely due to the preferential
binding of menthol to Na+ channels in the inactivated state. Our findings are
consistent with results from a previous study in DRG neurons and DRG neuron-
derived F11 cells (Gaudioso et al., 2012). However, menthol blocks Na+ channels
in dorsal horn neurons with 2-fold more potency than in DRG neurons (at the
same holding potential). This difference could be due to differences in subunit
composition of Na+ channels between dorsal horn and DRG neurons.
Menthol blocks Ca2+ channels with a greater potency than Na+ channels. The
mechanisms involved in the blocking Ca2+ channels are similar to those of
blocking the Na+ channels. However, menthol accelerated the rate of Ca2+
current decay during longer test pulses. It produced a more pronounced block of
plateau Ca2+ currents and a weak block of the peak Ca2+ currents. Menthol-
induced acceleration of current decay may result from open channel blocking,
drug-induced inactivation or both (Lee and Tsien, 1983; Berjukow and Hering,
2001). Our results also showed that menthol blocks resting Ca2+ channels and
shifted the Ca2+ channel inactivation curves in the hyperpolarization direction
without changes in the slope factor, suggesting that menthol may enhance the
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transition of Ca2+ channels from the resting to the inactivated state. Furthermore,
menthol shifted steady state activation of Ca2+ currents, indicating that menthol
may act as a gating inhibitor. The detail mechanisms of menthol-induced calcium
channel block remain to be determined. In contrast to a previous report that
menthol reduces the amplitude of low voltage-gated but not high voltage-gated
calcium currents (Swandulla et al., 1987), we found that menthol reduces the
amplitude of high voltage-gated calcium currents. Calcium currents recorded in
the present study were activated at higher voltages from the holding potential of -
70 mV, at which most T-type Calcium channels are inactivated (Huang, 1989). In
addition, these calcium currents were resistant to 50 µM NiCl2, a known T-type
calcium channel blocker, further supporting that these currents are mediated by
high voltage-gated calcium channels. The discrepancy can be explained by the
expression of different subtypes of high voltage-gated calcium channels between
dorsal horn and DRG neurons.
As a consequence of the finding that menthol blocked the Na+ channels, we
observed that menthol decreased neuronal excitability. Menthol-induced
inhibition of repetitive firing is consistent with use-dependent block of Na+
channels. Inhibition of presynaptic Na+ and Ca2+ channels would be expected to
reduce neurotransmitter release at the synapse. Indeed, menthol completely
blocked the TTX-sensitive spontaneous EPSC but had no effect on the miniature
EPSC (in the presence of TTX), which agrees with results from a previous study
in monocultured dorsal horn neurons (Tsuzuki et al., 2004). Menthol blocked
spontaneous activity-induced synaptic transmission at 100 µM, at which menthol
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only inhibits 25% Na+ currents and 40% Ca2+ currents (at -80 mV). The increase
in potency could be due to the fact that a fraction of Na+ and Ca2+ channels are in
the inactivated state at -65 mV. The inhibitory effect of menthol increases at more
depolarized potentials. In addition, both Na+ and Ca2+ channels are essential for
activity-induced synaptic transmission, and menthol hit both Na+ and Ca2+
channels in the same neuron. This double block may account for increasing in
potency. It is generally accepted that excessive depolarization and abnormal
excitability occur in the chronic pain state (Latremoliere and Woolf, 2009).
Menthol blocks Na+ and Ca2+ channels with enhanced state-dependent and use-
dependent block by preferentially binding to inactivated channels. These features
are critical for the suppression of abnormal excitability.
Menthol has been proposed as an activator of TRPM8 (McKemy et al., 2002;
Peier et al., 2002), and a modulator of TRPA1 (Karashima et al., 2007; Xiao et
al., 2008). To date, there is no report suggesting functional expression of TRPM8
and TRPA1 in dorsal horn neurons. Therefore, the effects of menthol on GABAA
receptors and Na+ and Ca2+ channels are caused by direct interaction with these
channels and are not likely mediated by the activation of TRPM8 or TRPA1.
GABAA receptors and voltage-gated sodium and calcium channels are the major
players in pain signal transmission. However, activation of GABAA receptors by
menthol requires a much higher concentration than that needed for blocking
calcium and sodium channels. We observed a GABAA agonist-like sedative effect
when menthol was administered at a higher dose (200 mg/kg, data not shown).
Therefore, the central mechanisms of the analgesic action of menthol are unlikely
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mediated by blocking GABAA receptors at the doses used for this study. Our
LC/MS/MS result shows that menthol has great brain penetration. Menthol was
detected with a relative high concentration (12.1µg/g) at 30 min, but its
concentration was largely decreased to 3.1µg/g at 60 min. This result is
consistent with our in vivo data that the efficacy of high-dose menthol (100
mg/kg) is better at 30 min than that at 60 min. In addition, low-dose menthol (50
mg/kg) significantly reduces CFA-induced thermal hypersensitivity at 30 min, but
has no effect at 60 min. The relationship between the brain concentration and
behavioral efficacy of menthol further supports that menthol has a central
analgesic action. The brain menthol level may also correlate with the
concentration required for blocking voltage-gated Na+ and Ca2+ channels.
In summary, our results indicate that menthol produces a central analgesic
action by blocking Na+ and Ca2+ channels in dorsal horn neurons, an effect that
has also been demonstrated in peripheral neurons (Swandulla et al., 1987;
Gaudioso et al., 2012). It appears that menthol acts as a nonselective analgesic
agent through multiple peripheral and central pain targets, which is probably
therapeutically beneficial in treating chronic pain. It may be used as an effective
analgesic in chronic pain as well as a topical analgesic.
Acknowledgments
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JPET #196717 27
We would like to thank Dr. Seena Ajit, Dr. Alessandro Graziano, and Ms Pamela
W. Fried for critically reading and providing valuable comments on the
manuscript.
Authors’ Contributions
Participated in research design: Huijuan Hu, Rong Pan and Xianguo Zhao.
Conducted experiments: Rong Pan,Yuzhen Tian, Ruby Gao, Haitao Li, Xianguo
Zhao and Huijuan Hu.
Performed data analysis: Huijuan Hu, Rong Pan and Xianguo Zhao.
Wrote or contributed to the writing of the manuscript: Huijuan Hu and James E.
Barrett
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Figure Legends
Fig. 1. Menthol reverses CFA-induced mechanical allodynia and thermal
hyperalgesia. A, time course of CFA-induced thermal hyperalgesia (n = 9, *P <
0.05 compared to baseline). B, time-dependent effect of menthol on CFA-
induced thermal hyperalgesia measured 24 h after CFA injection (n = 6-7, *P <
0.05 compared to vehicle control). C, effects of menthol on CFA-induced
mechanical allodynia measured 96 h after CFA injection, by intraperitoneal
injection (IP, n = 6-7) or by intrathecal injection (IT, n=6-8). *P < 0.05 compared
to pre-menthol. Data are presented as mean ± S.E.M.
Fig. 2. Menthol reduces formalin-induced nociceptive behavior. A, the time
course of nociceptive behavior following subcutaneous formalin (2%, 20 μl)
injection into the hind paw following pretreatment with vehicle or 100 mg/kg
menthol (n = 8-9). B, total time spent in nociceptive behavior during the first (0-10
min) and second (10-50 min) phases of the formalin response with vehicle or
menthol. *P < 0.05 compared to vehicle control.
Fig. 3. Menthol concentrations in the brain. Mice were injected with 100 mg/kg
i.p. menthol and then sacrificed at 0, 5, 30, or 60 min. Brain menthol
concentrations are presented as mean ± S.E.M. n = 3 mice/group.
Fig. 4. Menthol activates GABAA receptors in spinal dorsal horn neurons. A,
concentration response curve of menthol-induced currents (n=5). Inset,
representative recordings of menthol-induced inward currents with four different
concentrations at a holding potential of -65 mV. B, representative menthol-
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induced inward and outward currents recorded in control condition, in the
presence of bicuculline. C, representative recordings of menthol-induced currents
with 140 mM CsCl or 120 mM CsMeSO4 (20 mM CsCl) based internal solutions.
D, current-voltage (I-V) relationships of menthol-induced currents recorded with
CsCl or CsMeSO4 based internal solutions.
Fig. 5. Menthol blocks voltage-gated sodium channels in cultured spinal dorsal
horn neurons. A, representative Na+ currents evoked by test pulses ranging from
−50 to +60 mV for 50 ms from the holding potential of −80 mV before and after
application of 300 µM menthol. B, current-voltage (I-V) relationships of the Na+
currents in A (n=6). The currents measured at the peak were plotted vs. test
potentials. C, representative sodium currents activated by a voltage step to -20
mV from the holding potential of -80 mV before and after application of 30, 100,
300, and 1000 µM menthol and 5 min after washout of the drug. D, concentration
response curve of menthol-induced inhibition of sodium currents at a holding
potentials of -80 mV (n=5-6).
Fig. 6. Menthol blocks voltage-gated sodium channels with use-, state- and
voltage- dependent manners. A, representative sodium currents evoked by a
voltage step to -20 mV from the holding potential of -100 mV or -70 mV before
and after application of 300 µM menthol. B, summary of inhibition of Na+ currents
by menthol at the holding potential of -100 mV and -70 mV (n = 5, *P < 0.05
compared to the holding potential of -100 mV). C, representative sodium currents
activated by a 20-Hz train of 50 ms depolarization pulses to -20 mV from the
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JPET #196717 33
holding potential of -80 mV before and after application of 300 µM menthol. D,
summary of inhibition of Na+ currents by menthol at the first pulse and at the 10th
pulse (n = 5, *P < 0.05 compared to P1). E, steady-state activation and
inactivation curves before and after application of 300 µM menthol.
Fig. 7. Menthol blocks voltage-gated calcium channels in cultured spinal dorsal
horn neurons. A, representative Ca2+ currents evoked by test pulses ranging
from −30 to +60 mV for 200 ms from the holding potential of −70 mV before and
after application of 300 µM menthol. B, current-voltage (I-V) relationships of the
Ca2+ currents in A. The currents measured at the peak were plotted vs. test
potentials. C, representative calcium currents activated by a voltage step to 10
mV from the holding potential of -70 mV before and after application of 30, 100,
300, and 1000 µM menthol, and 5 min after washout of the drug. D,
concentration response curve of menthol-induced inhibition of calcium currents
(n=6).
Fig. 8. Menthol blocks voltage-gated calcium channels with use-, state- and
voltage- dependent manners. A, representative sodium currents activated by a
voltage step to 10 mV from the holding potential of -100 mV or -70 mV before
and after application of 300 µM menthol. B, summary of inhibition of Na+ currents
by menthol at the holding potential of -100 mV and -70 mV (n = 5, *P < 0.05
compared to the holding potential of -100 mV). C, representative calcium
currents activated by a 10-Hz train of 50 ms depolarization pulses to 10 mV from
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JPET #196717 34
the holding potential of -70 mV before and after application of 300 µM menthol.
D, summary of inhibition of calcium currents by menthol at the first pulse and at
the 10th pulse (n = 5, *P < 0.05 compared to P1). E, steady-state activation and
inactivation curves before and after application of 300 µM menthol (n=5-6).
Fig. 9. Menthol reduces neuronal excitability and blocks spontaneous synaptic
transmission in cultured dorsal horn neurons. A, representative action potentials
recorded from dorsal horn neurons before and after application of 300 and 600
µM menthol and 5 min after washout of the drug. B, summary of changes in
action potential amplitude, spike frequency, threshold, and rheobase induced by
menthol in dorsal horn neurons (n=6, *P < 0.05 compared to pre-menthol). C,
representative spontaneous EPSC recorded before and during application of 100
µM menthol and after washout in the absence or in the presence of 500 nm TTX.
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JPET #196717 35
TABLE 1. Effects of menthol on the voltage dependency of INa and Ica activation and inactivation
Activation Inactivation Sample V1/2 k n V1/2 k n
INa Pre-menthol -36.4 ± 3.0 2.3 ± 0.4 6 -42.2 ± 2.8 4.4 ± 0.2 6 Menthol -33.8 ± 2.9 3.2 ± 0.4 6 -52.8 ± 2.5* 4.6 ± 0.6 6
ICa Pre-menthol -3.9 ± 2.2 4.4 ± 0.1 7 -18.7 ± 5.1 16.8 ± 1.3 6
Menthol 1.1 ± 2.1* 5.1 ± 1.2 7 -27.0 ± 6.0* 18.5 ± 1.7 6
Values are means ± SE; n = number of neurons. V1/2, voltage of half-maximal activation or inactivation; k, slope factor. *p<0.05 compared to pre-menthol.
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Fig. 1
Paw
with
draw
al th
resh
old
(g)
Paw
with
draw
al th
resh
old
(g)
Menthol100 mg/kg
VehicleMenthol 50 mg/kg
Paw
with
draw
al th
resh
old
(g)
Paw
with
draw
al la
tenc
y (s
)
Ipsilateral
Contralateral
Baseline 0 5 10 15 20
Time after CFA injection (days)
*** *0
5
10
15
Paw
with
draw
al la
tenc
y (s
)
Baseline Vehicle Menthol0.0
0.2
0.4
0.6
0.8
1.0 IpsilateralCFA
Baseline
IP injection
*
0.0
0.2
0.4
0.6
0.8
1.0 Contralateral*
*
Baseline 0 10 20 30 40 50 60
*
Time after menthol injection (min)
*
0
5
10
15
A B
C
0.0
0.2
0.4
0.6
0.8
1.0
IT injection
Ipsilateral
*
Vehicle or
Menthol
CFA
CFA
Pre-
CFA
Pre-
CFA
CFA
Baseline Vehicle MentholBaseline Pre-Pre-
CFA
CFA
Paw
with
draw
al th
resh
old
(g)
0.0
0.2
0.4
0.6
0.8
1.0
Contralateral
*
CFA
CFA
Baseline Vehicle MentholBaseline Pre-Pre-Baseline Vehicle MentholBaseline Pre-Pre-
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1
Fig. 2
0 10 20 30 40 50 60
0
50
100
150
200
250
* *Tim
e S
pent
Lic
king
/Lift
ing
(sec
)
Time after formalin injection (min)
Control, Menthol
**
*
A
Tot
al T
ime
Spe
nt L
icki
ng/L
iftin
g (s
ec)
Control Menthol Control Menthol0
100
200
300
400
500First phase Second phase
**
B
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0 min 5 min 30 min 60 min
0
10
20
30
40
50
60
70M
enth
ol c
once
ntra
tion
(µg/
g)
Fig. 3
Time post menthol injection
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Fig. 4
20 40 600-20 -40-60 -80
20 mM [Cl-]
140 mM [Cl-]
30 s
30 s
300
pA40
0 pA
mV
-100 -80 -60 -40-20 0 20 40 60 80
-400
-200
0
200
400
600
Voltage (mV)
Cur
rent
(pA
)2 mM Menthol
100
pA
20 s
50 µM Bicu
10 µM Bicu2 mM Menthol Menthol
10 µM Bicu
Menthol
0.3 mM 3 mM1 mM 9 mM
Inw
ard
Cur
rent
s (p
A)
Menthol (mM)0.1 1 10
0
200
400
600
A B
C D
MentholMenthol
Menthol
-65 mV
-65 mV
+65 mV
20 mM [Cl-]140 mM [Cl-]
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Fig. 5
0.1 1 10 100 1000 10000
0
50
100
Nor
mal
ized
I Na
Menthol (µM)
25
75
500
pA
5 ms
1 mM
300 µM
Control
30 µM
100 µM
Washout
-80 mV
-20 mV
C
D
-60-50-40-30-20-10 010 20 30 40 50 60 70
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
I/I m
ax
Voltage (mV)
WashoutPre-menthol Menthol
500
pA
5 ms
-80 mV
A
B
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Pre-menthol
Menthol
Fig. 6
-100 -80 -60 -40 -20 0
0.0
0.2
0.4
0.6
0.8
1.0
AVoltage (mV)
I/Im
ax
-80 mV
1 nA
100 ms
Pre-menthol Menthol
-20 mV
P1 P100
20
40
60
*
% In
hibi
tion
of I N
a
C D
-100 mV-20 mV
Control
Menthol
-55 mV-20 mV
500
pA
5 ms
Control
Menthol
A B
E Inactivation
-60 -50 -40 -30 -20 -10 0 10 20
0.0
0.2
0.4
0.6
0.8
1.0
G/G
max
Voltage (mV)
Pre-menthol
Menthol
Activation
-100 mV0
20
40
60
*
-55 mV
% In
hibi
tion
of I N
a
80
5 ms
1 nA
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Fig.7
0.2
-30 -20 -10 0 10 20 30 40 50 60 70
-1.0
-0.8
-0.6
-0.4
-0.2
0.0Voltage (mV)
Pre-Menthol
300 μM Menthol
Washout
I/ I M
ax
250
pA
200 ms
Control Menthol
-70 mV
250
pA
100 ms
1 mM
300 µM
Control30 µM
100 µM
Washout
10 mV-70 mV
Nor
mal
ized
I Ca/
Ba
Menthol (µM)0.1 1 10 100 1000 10000
0.0
0.2
0.4
0.6
0.8
1.0
A
B
C
D
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Fig. 8
G/G
max
Voltage (mV)Voltage (mV)
I/Im
ax
-100 -80 -60 -40 -20 0 20 40
0.0
0.2
0.4
0.6
0.8
1.0
Pre-menthol
Menthol
-50 -40 -30 -20 -10 0 10 20 30 40 50
0.0
0.2
0.4
0.6
0.8
1.0
Pre-menthol
Menthol
Pre-menthol
200 ms
100
pA
Menthol
-70 mV
10 mV
P1 P10
P1 P100
20
40
60
80
100
% In
hibi
tion
of I C
a *
C D-100 mV -70 mV
0
20
40
60
80
100*
% In
hibi
tion
of I C
a
-70 mV
Control
Menthol
-100 mV10 mV 10 mV
Control
Menthol
100 ms10
0 pA
A B
E Inactivation Activation
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Fig. 9
Pre-menthol 300 μM 0
20
40
60
Ampl
itude
(mV)
0 2 4 6 8
10 12 14
Freq
uenc
y (H
z)
* *
*
*
600 μM Pre-menthol 300 μM 600 μM
0
5
10
15
20
25
Rhe
obas
e (p
A)
-30
-20
-10
0
Thre
shol
d (m
V)
Pre-menthol 300 μM 600 μM Washout
0.1 mM menthol
-65 mV
200
pA
40s
-65 mV 25 pA
0.1 mM menthol + 500 nM TTX
A
B
C
Pre-menthol 300 μM 600 μM
Pre-menthol 300 μM 600 μM
0.3 mM menthol
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