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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.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on September 5, 2012 as DOI: 10.1124/jpet.112.196717

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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

[email protected]

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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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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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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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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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

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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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