Carvacrol Decreases Neuronal Excitability by ... - ACS Publications

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Carvacrol Decreases Neuronal Excitability by Inhibition of VoltageGated Sodium Channels Humberto Cavalcante Joca,† Yuri Cruz-Mendes,† Klausen Oliveira-Abreu,† Rebeca Peres Moreno Maia-Joca,† Roseli Barbosa,‡ Telma Leda Lemos,§ Paulo Sergio Lacerda Beiraõ ,⊥ and José Henrique Leal-Cardoso*,† †

Laboratório de Eletrofisiologia, Instituto Superior de Ciências Biomédicas, Campus do Itaperi, Universidade Estadual do Ceará, Fortaleza, CE, Brazil ‡ Mestrado em Bioprospecçaõ Molecular, Universidade Regional do Cariri, Crato, CE, Brazil § Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Fortaleza, CE, Brazil ⊥ Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil ABSTRACT: The monoterpenoid carvacrol (1) is present in many essential oils of plants and has attracted attention because of its beneficial biological activities, especially analgesic activity. However, the mechanism of action of 1 remains unknown. The present study aimed to explore the mechanisms whereby 1 produces its effects on the peripheral nervous system. Carvacrol reversibly blocked the excitability of the rat sciatic nerve in a concentration-dependent manner with an IC50 value of 0.50 ± 0.04 mM. At 0.6 mM, 1 increased the rheobase from 3.30 ± 0.06 V to 4.16 ± 0.14 V and the chronaxy from 59.6 ± 1.22 μs to 75.0 ± 1.82 μs. Also, 1 blocked the generation of action potentials (IC50 0.36 ± 0.14 mM) of the intact dorsal root ganglion (DRG) neurons without altering the resting potential and input resistance. Carvacrol reduced the voltage-gated sodium current of dissociated DRG neurons (IC50 0.37 ± 0.05 mM). In this study it has been demonstrated that 1 blocks neuronal excitability by a direct inhibition of the voltagegated sodium current, which suggests that this compound acts as a local anesthetic. The present findings add valuable information to help understand the mechanisms implicated in the analgesic activity of carvacrol.

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Analgesic action in neurogenic pain of many terpenes and terpenoids is related to depression of excitability in the peripheral nervous system.18−22 Our group recently reported that two compunds, the monoterpenoid linalool and the phenylpropanoid eugenol, blocked excitability in the sciatic nerve and the generation of action potentials in peripheral neurons.19,22 Both substances inhibited voltage-gated sodium currents in isolated neurons of the dorsal root ganglia (DRG).19,23 Despite previous studies indicating that 1 blocks compound action potentials (CAP) in the sciatic nerve, to date there is no systematic study aimed at determining the mechanisms whereby 1 elicits its effect in the peripheral nervous system.24 Therefore, the present study was designed to test the hypothesis that carvacrol (1) impairs action potential generation by blocking voltage-gated sodium channels.

erpenes and terpenoids are the main components of the essential oils of aromatic plants and are considered important agents in the food industry and for medicinal use.1−3 The monoterpenoid carvacrol (1; 5-isopropyl-2-methylphenol) is present in the essential oils of many plants, especially in the genera Origanum and Thymus.2,4 Carvacrol is approved as a safe food additive in the United States and Europe and has been reported as safe for topical use.5,6 Carvacrol (1) has attracted attention because of its wide variety of beneficial biological activities such as antibacterial, antifungal, and antioxidant effects.4,7−10 This monoterpenoid also has potent antiinflammatory activity by nonselective inhibition on both cyclooxygenase (COX) isoforms11 and on the suppression of COX-2 expression.12 In tracheal and aortic smooth muscle preparations, 1 at low concentrations relaxes contractions induced by high-potassium extracellular concentrations or a receptor agonist, indicating that this substance has great potential as a therapeutic agent.13−15 Extracts of plants with carvacrol as a major constituent have shown analgesic action in several animal models.16,17 Carvacrol when pure has reduced acetic acid-induced abdominal writhing and inhibited both the early (neurogenic pain) and the late (inflammatory pain) phases of formalin-induced licking.7 © 2012 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Effects of Carvacrol (1) on Compound Action Potentials of the Isolated Rat Sciatic Nerve. First recorded Received: January 16, 2012 Published: September 11, 2012 1511

dx.doi.org/10.1021/np300050g | J. Nat. Prod. 2012, 75, 1511−1517

Journal of Natural Products

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The full blockade of CAP demonstrates that carvacrol exerts its inhibitory effect on all types of myelinated fibers that contribute to the first and second CAP components of the sciatic nerve. Under the recording conditions used, two major components could be distinguished according to their differences in conduction velocity. These differences are related to the two distinct groups of nerve fibers present in the sciatic nerve trunk. The first component has an average conduction velocity of 90.3 ± 3.3 m/s (n = 25), which is compatible with Aα fibers.25,26 The second component showed a mean conduction velocity of 29.6 ± 1.3 m/s, in agreement with values reported for the myelinated sensorial fibers Aβ and Aγ.25 When exposed to the highest concentration of 1 used in the present study (1 mM), the positive amplitude of both CAP components was fully inhibited, but the time to develop this effect was different. While the second component was completely inhibited within 60 min, the inhibition of the first component amplitude was remarkably slower, and only after 180 min was full blockade reached. Also, to determine if carvacrol (1) affects the conduction velocity of the first and second CAP components, the respective conduction velocities were measured using submaximal concentrations of 1. Carvacrol, in a concentration-dependent manner, significantly decreased conduction velocity for both components. The conduction velocity cannot be measured when using 1 mM carvacrol due to the complete blockade of the CAP waveform. To evaluate the effect of 1 on the excitability of sciatic nerve fibers, a series of strength-duration curves were performed to determine the rheobase and chronaxy (n = 5). The rheobase was measured as the minimal stimulus voltage that can elicit an active response with a long-duration pulse (1000 μs), and the chronaxy as the threshold pulse duration with a pulse for which the voltage is twice the rheobase. In this experimental

were the compound action potentials, which reflects the overall electrical activity resulting from the action potentials of the nerve axons. Under control conditions (in the absence of 1) the CAP showed two components (Figure 1A, black trace) with a mean peak-to-peak amplitude of 10.8 ± 0.8 mV (n = 25). When 1 (1 mM) was applied, the peak-to-peak amplitude of CAP was gradually and slowly reduced until total blockade (Figure 1A, light gray trace). After removal of 1 and following a 180 min washout period, the CAP waveform had a similar shape and amplitude when compared to the control (Figure 1A, dark gray trace). Figure 1 also depicts the time course (Figure 1B, n = 5 for each concentration) and concentration−response relationship (Figure 1C, n = 5) of carvacrol-induced reduction of CAP amplitude. At the lowest concentration used (0.03 mM), 1 had no significant effect on CAP peak-to-peak amplitude even after 180 min of exposure. As the concentration of 1 was increased, the inhibition was enhanced, allowing the calculation of the concentration at which a 50% block was observed. Figure 1C shows the concentration−response curve after 180 min of carvacrol application and data points fitted to logistic equation in the form: Y = Min + (Max − Min)/(1 + (X/IC50)slope). The calculated IC50 was 0.50 ± 0.04 [0.41−0.59] mM, with a fitted slope coefficient of −3.82.

Figure 1. Effects of carvacrol (1) on compound action potentials. Panel A shows representative CAP tracings for control (black trace), after 180 min of nerve exposure to 1 mM 1 (light gray trace), and after a 180 min washout period (dark gray trace). Note that there is a good recovery of amplitude and shape of CAP, but the waves of CAP last longer than in the control due to an incomplete recovery of conduction velocity. Panel B represents the time course of the effects of 1 on CAP peak-to-peak amplitude. The concentration−response relationship effects on CAP peak-to-peak amplitude and the IC50 determination are shown in panel C. Data are reported as means ± SEM (n = 5 for each concentration). 1512



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A previous study has reported that carvacrol (1) blocked excitability in the sciatic nerve at a 10 mM concentration.24 However, the present results demonstrate that 1-induced inhibition in peripheral nerve fibers can be achieved with a 10-fold lower concentration (1 mM), and compared with other essential oil components such as eugenol, estragole, and 1,8cineole, 1 has similar effects on the CAP amplitude, time course, and pharmacological potency, at a submillimolar range.18,20,22 In frog sciatic nerve preparations stimulated at a similar frequency to that used in the present study, lidocaine inhibited 46% of CAP amplitude at 0.5 mM, while the same concentration calculated for 1 induced 50% CAP blockade.27 Also, 1 is slightly more potent than lidocaine when comparing their IC50 values (0.50 and 0.74 mM, respectively).28 When the present results are taken together, one can conclude that carvacrol blocks nervous conduction, and this may explain its reported antinociceptive activity in models related to neurogenic pain.7 Carvacrol (1) showed an inhibitory effect on all myelinated fibers that contribute to the first and second CAP components of the sciatic nerve. However, when considering the conduction velocity of both CAP components, fibers responsible for the second component (slower conduction) were more sensitive to effects of 1. This difference is also described for the monoterpenoid linalool and other classical local anesthetics such as lidocaine and bupivacaine.19,29,30 The inhibitory action of linalool at a similar concentration (0.8 mM) is more evident on the second CAP component, but this difference is more subtle compared to that observed in experiments with 1.19 Classical local anesthetics, such as lidocaine and bupivacaine, have a minor inhibitory effectiveness for fast Aα fibers, while for sensorial slow Aδ and C fibers, their effect is greatly enhanced.29,30 In addition, changes in threshold values in the presence of carvacrol (1) indicate that this monoterpenoid effectively blocks excitability in the peripheral nervous system. Our group has reported previously that other compounds share most of this pharmacological profile.18−22,31 However, the underlying mechanisms seem not to be the same. Alterations in resting membrane potential and/or in input membrane resistance would be implicated in a reduction of neuronal excitability. These mechanisms have been described already to explain how 1,8-cineole inhibits action potentials in peripheral neurons.20,31 On the other hand, other compounds such as eugenol and linalool block neuronal action potential generation by acting

procedure carvacrol was used at 0.6 mM based on an estimate of the IC50 of the CAP peak-to-peak amplitude inhibition. Under control conditions, the rheobase and chronaxy were 3.30 ± 0.06 V and 59.6 ± 1.2 μs, respectively. In the presence of 1 there was a clear reduction in nerve excitability, as evidenced by a significant increase in both parameters to 4.16 ± 0.14 V for the rheobase and 75.0 ± 1.8 μs for the chronaxy (Figure 2B). After removal of 1, both the rheobase and chronaxy showed a partial recovery, but these were not statistically significant.

Figure 2. Effects of carvacrol (1) on conduction velocity and threshold. Panel A shows the reduction on conduction velocity of the CAP component at the end of 180 min of exposure to 1. The carvacrol-induced increase of rheobase and chronaxy values is shown in panel B. Data are expressed as means ± SEM (n = 5 for each concentration); *p < 0.05 compared to control and **p < 0.05 compared to the first component at the same concentration (ANOVA followed by Tukey's posthoc test).

Table 1. Effects of Carvacrol on Action Potential Parameters and Passive Membrane Properties of Dorsal Root Ganglia Neuronsa carvacrol (mM) control (n = 108) amplitude (mV) resting potential (mV) input resistance (MΩ) maximum ascendant inclination (V/s) maximum descendant inclination (V/s) duration (ms)

0.03 (n = 7)

0.1 (n = 10)

0.3 (n = 16)

1 (n = 19)

3 (n = 37)

79.0 ± 1.3 −60,0 ± 1.1

77.0 ± 5.4 −62.6 ± 3.7

53.9 ± 12.3b −60.2 ± 4.6

43.9 ± 10.2b −59.7 ± 1.8

32.1 ± 7.6b −60.2 ± 1.7

0.6 (n = 20)

4.2 ± 4.2b −60.2 ± 1.2

0.0 ± 0.0b −57.5 ± 1.7

25.5 ± 2.1

29.3 ± 5.1

26.7 ± 3.1

27.1 ± 4.3

23.3 ± 4.1

26.3 ± 5.1

20.8 ± 2.2

151.2 ± 6.2

128.6 ± 16.5

94.8 ± 25.4b

84.4 ± 21.6b

56.5 ± 15.0b

4.4 ± 4.4b

0.0 ± 0.0b

−104.7 ± 3.6

−102.2 ± 11.2

−78.9 ± 27.8b

−62.7 ± 15.8b

−48.0 ± 11.8b

−4.5 ± 4.4b

0.0 ± 0.0b

1.6 ± 0.1

1.6 ± 0.3

1.5 ± 0.5

0.9 ± 0.3b

0.6 ± 0.1b

0.2 ± 0.2b

0.0 ± 0.0b

Data are reported as mean ± SEM with (n) indicating the number of experiments. bp < 0.05 compared to control (ANOVA followed by Tukey's posthoc test). a

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Figure 3. Carvacrol(1)-induced action potential blockade on dorsal root ganglia neurons. Panel A shows intracellular recordings of action potentials of neurons for a control (black trace), after exposure to 3 mM 1 during 5 min or less (light gray trace), and after a washout period (dark gray trace). Dashed straight line shows zero voltage level. The concentration−response relationship and the IC50 curve for carvacrol-induced action potential blockade are shown in panel B. Panel C shows the percentage increase of action potential current threshold values in the presence of 1. Data are expressed as means ± SEM (n = 108 cells). *p < 0.05 compared to control (ANOVA followed by Tukey's posthoc test).

ship between concentration of 1 and action potential blockade had an IC50 value of 0.36 ± 0.14 [0.11−0.61] mM with a fitted slope coefficient of 1.23 (n = 108). Also, 1 induced a progressive elevation in current threshold level as a function of concentration, reaching more than 60% (62.4 ± 10.1%, n = 37) of control threshold value at the highest concentration tested (Figure 3C). However, despite the small depolarization in resting potential and the slight reduction in input resistance in some experiments, 1 did not induce statistically significant changes in these membrane properties at all concentrations used, whereas all other action potential parameters (amplitude, duration, maximum ascendant inclination, and maximum descendant inclination) decreased as the carvacrol concentration increased. Data for action potential parameters, the resting potential, and the input resistance in the presence of carvacrol are presented in Table 1. The results mentioned above are compatible with other studies using isolated chemical compounds from aromatic plants tested on DRG neurons.19,22,31 Eugenol, for instance, completely blocked DRG action potentials at 2 mM concentration, being in this respect slightly more potent than carvacrol.21 In contrast, linalool and 1,8-cineole demonstrated a full blockade of action potentials only when used at 6 mM, approximately 2-fold higher in concentration than carvacrol.19,31 Action potential blockade is also a fundamental step for local anesthetic activity, with lidocaine completely blocking single and/or bursts of action potentials in DRG neurons at a concentration of 1 mM.32,33 It is well known that voltage-gated sodium channels are responsible for the depolarization phase of action potentials.34 Lidocaine, which inhibits these channels, caused an increase in current threshold level to elicit an action potential, as did carvacrol.32

directly in voltage-gated sodium channels without any changes in resting potential or input resistance.19,22,23 Effects on Action Potentials, Passive Membrane Properties, and the Voltage-Dependent Sodium Current in Neurons Isolated from Dorsal Root Ganglia. To further investigate the decrease of neuronal excitability elicited by carvacrol (1) in peripheral neurons and to elucidate its mechanism of action, experiments were performed using conventional sharp microelectrodes in the current-clamp mode in intact DRGs to measure resting membrane potential, input resistance, and action potential parameters. Current pulses were used to depolarize the neuronal membrane and to generate action potential, and also to determine current threshold levels. To avoid small variations in experimental conditions that could affect analysis, positive current pulses 25% higher than threshold value were used. After impalement, DRG neurons had a mean resting potential of −60.0 ± 1.2 mV, an input resistance of 25.5 ± 2.1 MΩ, and a current threshold of 1.43 ± 0.1 nA (n = 108). All action potential parameters are summarized in Table 1. In the presence of high carvacrol concentrations (3 mM), all tested neurons showed an action potential blockade within 2 to 5 min of exposure, while the membrane passive response was maintained (Figure 3A, light gray trace). After washing carvacrol, the action potential slowly recovered its usual shape and all measured parameters reached similar values to the control (Figure 3A, dark gray trace). The percentage of neurons that showed a blockade in action potential generation was dependent on carvacrol concentration used in each experiment (Figure 3B). At the lowest concentration used (0.03 mM), there was no action potential blockade and no significant change in any of the measured parameters. When the carvacrol concentration was raised to 3 mM, DRG neurons failed to generate action potentials (Figure 3B). The relation1514



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attributable to the several layers that envelop the nerve (the peri-, meso-, and endoneurion), since in dissociated neurons without any enveloping layers the steady state is reached in a few minutes. Carvacrol (1) did not change the time-to-peak at any of the concentrations tested. Like the other preparations, carvacrol induced no changes at low concentrations (0.01 mM), showing a progressive increase of effect until full inhibition was attained at the highest concentrations tested (3 and 10 mM). The calculated IC50 value was 0.37 ± 0.05 [0.27−0.46] mM with a fitted slope coefficient of 0.97 (Figure 4B, n = 50). In a recent review, Araújo and colleagues emphasized that terpenoids derived from plants are very promising compounds that target voltage-gated ion channels.35 This also applies for carvacrol, for which several studies have provided evidence indicating that it modulates diverse types of ion channels.36−39 In TRP channels, 1 acts as an activator in TRPV3 and hTRPA1 and has an inhibitory effect on Drosophila TRPL and mammalian TRPM7 channels.36,38,39 In these cases, carvacrol modulation in TRP channels occurs at a similar concentration range to that found in the present study for inhibition of voltage-gated sodium current.36,38,39 Futhermore, 1 blocks voltage-gated calcium currents in canine and human ventricular cardiomyocytes, accelerates the inactivation time course, and causes leftward shift in the voltage dependence of steady-state inactivation without alteration in activation kinetics.37 Acting on the voltage-gated sodium current, blockade of excitability without changes in resting potential is a feature of local anesthetics, which directly affect voltage-gated sodium channels.34,40 Carvacrol (1) showed these characteristics and, when compared with lidocaine, is slightly less potent in inhibiting the sodium current under the same set of conditions. Lidocaine at 1 mM causes a complete inhibition of sodium current in DRG neurons, while the same concentration of carvacrol reduced approximately 70% of the total current.32 Compared with other constituents of aromatic plants that inhibit sodium current, 1 had a biological potency similar to eugenol but higher than linalool.19,23 Thymol, a plant-derived compound and structural analogue of 1, also has inhibitory effects on neuronal and skeletal muscle isoforms of voltagegated sodium channels expressed heterologously in HEK293 cells.41 For the neuronal voltage-gated sodium channel isoform (rat brain IIA), thymol showed an IC50 of 366 μM at −100 mV holding potential (as used in the present work).41 This value is virtually the same as was calculated for carvacrol inhibitory effect (approximately 370 μM). In conclusion, carvacrol (1) reversibly blocks excitability and nerve conduction in peripheral neurons in a concentrationdependent manner. Carvacrol-induced blockade of neuronal excitability occurs without altering the resting potential and/or the input resistance. Futhermore, this effect is the result of direct inhibition of the voltage-gated sodium current, which suggests that 1 acts as a local anesthetic. This work along with previous studies adds new and valuable information in understanding the underlying mechanisms implicated in the analgesic activity of carvacrol (1).

Contrary to information reported for 1,8-cineole, carvacrol did not alter resting potential nor input resistance in DRG neurons.31 Therefore, 1 showed a similar behavior to those previously observed for natural compounds and local anesthetics that do not change passive membrane properties at the concentration range clearly inhibiting action potentials and voltage-dependent sodium channels in DRG neurons.19,32,33 So far, all the collected evidence indicates a strong relationship between carvacrol/local anesthetics and plant compounds that block neuronal excitability by inhibition of voltage-dependent sodium channels. To test the hypothesis that 1 blocks excitability by inhibition of the voltage-gated sodium current, a series of voltage-clamp experiments were performed using the patch-clamp technique (whole-cell mode) with dissociated DRG neurons. To elicit a macroscopic sodium current, pulse steps were used to 0 mV of 100 ms duration from a holding potential of −100 mV with 5 s intervals. For these experiments, a total of 50 cells were used with capacitance ranging from 20 to 60 pF (estimated diameter of 25.23 to 43.70 μm) with an average of 39.1 ± 3.1 pF, and the stimulation protocol generated a rapid sodium current with a time-to-peak of 1.7 ± 0.5 ms and a current density of 56.6 ± 6.9 pA/pF (Figure 4A, n = 50 cells).

Figure 4. Carvacrol(1)-induced inhibition of the voltage-gated sodium current of dissociated dorsal root ganglia neurons. Panel A shows sodium current traces for a control (black trace), after 120 s of exposure to 1 (light gray trace), and after a washout period (dark gray trace). The concentration−response relationship and IC50 curve for carvacrol-induced inhibition of the sodium current is shown in panel B. Data are reported as means ± SEM (n = 50 cells).

Carvacrol (1) provoked a considerable inhibition of sodium current. At 3 mM, this monoterpenoid almost completely abolished the sodium current after 120 s of perfusion with 1 (Figure 4A, light gray trace). After returning to the perfusion solution without carvacrol, the amplitude of sodium current rapidly returned to a value near that of the control (Figure 4A, dark gray trace). The difference in time course of effect of 1 from the intact ganglia and dissociated neurons to the nerves is

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EXPERIMENTAL SECTION

General Experimental Procedures. The carvacrol (1) used was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and had a minimum purity of 98% as determined by HPLC. Locke’s solution was used for extracellular and intracellular recording, whose composition was as follows (in mM): NaCl 140, KCl 5.6, MgCl2 1.2, CaCl2 2.2, 1515



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tris(hydroxymethylaminomethane) 10, and glucose 10. In dissociation protocol, Hanks’ balanced salt solution was used with the following composition (in mM): NaCl 137.93, KCl 5.33, KH2PO4 0.44, NaHCO3 4.0, Na2HPO4 0.3, and glucose 5.6. For patch-clamp recordings, the composition of the bath solution was as follows (in mM): NaCl 140, KCl 5, CaCl2 1.8, MgCl2 0.5, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid) (HEPES) 5, and glucose 5. For the isolation of the voltage-dependent sodium current, the external test solution had the following composition (in mM): NaCl 20, choline-Cl 90, CsCl 10, CaCl2 2, MgCl2 1, tetraethylammonium-Cl 20, CdCl2 0.2, HEPES 10, and glucose 10. All solutions above had a pH adjusted to 7.4 with HCl. The pipet internal solution to measure sodium currents contained (in mM) NaCl 10, CsCl 100, HEPES 10, ethylene glycol tetraacetic acid 11, tetraethylammonium-Cl 10, and MgCl2 5, and pH adjusted to 7.2 with CsOH. To isolate voltage-gated sodium currents, tetraethylammonium and cesium (Cs+) was used to block voltagegated potassium channels and cadmium (Cd+) was used to block voltage-gated calcium channels. Due to the low solubility of carvacrol (1) in water, it was dissolved in DMSO with a maximal concentration of 0.2% v/v. At this concentration, this solvent did not alter electrophysiological parameters and was added in all solutions used.42 The stock solutions of 1 were prepared daily and added to chambers containing modified Locke’s solution for extracellular and intracellular recordings and external solution for patch-clamp recordings. The concentrations of 1 used in extracellular recordings were 0.03, 0.1, 0.3, 0.6, and 1 mM, for intracellular recordings, 0.03, 0.1, 0.3, 0.6, 1, and 3 mM, and for patchclamp recordings, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 mM. Experiments were carried out at room temperature (18 to 22 °C). Other salts and reagents were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Animals and Tissue Dissection. Wistar rats (200−300 g) of both sexes were used. They were kept under conditions of constant temperature (22 ± 2 °C) with a 12 h light/12 h dark cycle and free access to food and water. All animals were handled in compliance with the Guide for the Care and Use of Laboratory Animals, published by the United States National Institutes of Health (NIH Publication 2789, revised 1996; http://www.nap.edu), and all efforts were made to minimize animal suffering. All procedures described herein were first reviewed and approved by the local animal ethics committee (Committee on Ethics of Use of Animals for Research of the Universidade Estadual do Ceará). The sciatic nerve and DRG were dissected from rats sacrificed by carbon dioxide inhalation. The tissues were immediately placed in a container containing ice cold (4 °C) modified Locke’s solution for experimental recording in the same day for intracellular and extracellular recordings, and ice cold Ca2+-, Mg2+ -free Hanks’ balanced salt solution for patch-clamp recording. Dissociation Protocol. For patch-clamp recording the DRG were placed in dissociation solution, which consisted of Hanks’ balanced salt solutions supplemented with 1 mg/mL collagenase type I for 75 min and 2.5 mg/mL trypsin for 15 min, both at 37 °C. After exposure to the dissociation solutions, the DRG neurons were freed from connective tissue by gentle trituration in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 U/mL streptomycin, and 0.1 mg/mL penicillin. The cells were plated on coverslips coated with 0.01% poly D-lysine. The neurons were incubated in an air atmosphere containing 5% CO2 maintained at 37 °C and were used 12 to 48 h after the dissociation procedure. Extracellular Recordings. The sciatic nerve was mounted and the evoked compound action potentials were recorded as previously described.18,20,21 Briefly, the sciatic nerve was mounted in a moist chamber, and one of its ends was stimulated with a stimulus isolation unit connected to a stimulator (model S48, Astro-Med Industries, West Warwick, RI, USA). Evoked compound action potentials were recorded with platinum electrodes placed 40 to 50 mm from the stimulating electrodes (5 mm of distance between them) and continuously monitored using an oscilloscope (model 547, Tektronix, Inc., Portland, OR, USA) and recorded by computer acquisition hardware (sampled at 20 kHz) for further analysis. A segment of the sciatic nerve (15 to 20 mm) was suspended between the stimuli and

recording electrodes and immersed in Locke’s solution, which was used to maintain chamber humidity and to administer carvacrol (1). Exposure to 1 was performed only when stable peak-to-peak compound action potential amplitude was achieved for at least 30 min, and the exposure time was set to 180 min. This period was followed by a 180 min washout/recovery period. Electrophysiological parameters measured in extracellular recording were the rheobase, chronaxy, peak-to-peak amplitude, and conduction velocity of the CAP components. Intracellular Recording. The intact DRG was used and transmembrane responses were recorded as reported in previous articles from our laboratory.19,22,31 Briefly, the ganglia were fixed in an acrylic chamber designed to permit superfusion with Locke’s solution or carvacrol (1)-containing solution. The chamber was placed under a magnifying glass (2 to 40×), and the microelectrode movement and impalement were done with a hydraulic micromanipulator (MWO-3; Narishige International, Long Island, NY, USA). The microelectrode was filled with 3 M KCl solution and connected to an Axoclamp 2B amplifier (Molecular Devices, Sunnyvale, CA, USA) via a silver−silver chloride wire. The response signal was visualized continuously on an oscilloscope. The cells were acceptable for study when the neurons had a stable resting potential more negative than −48 mV and with input resistance greater than 10 MΩ for 3 to 5 min after impalement. The intact ganglia were perfused with 1 until an action potential blockade was established or the upper limit of 5 min was reached, followed by a washout/recovery period. The electrophysiological parameters measured in intracellular recording were resting potential, input resistance, action potential amplitude and duration (measured at half-maximum amplitude), maximum ascendant inclination, and maximum descendant inclination, that is, the minimum value of negative inclination. Patch-Clamp Recordings. Recording of total sodium current in dissociated DRG neurons was performed as described in a previous study.19 The neurons were placed in a chamber on an inverted phasecontrast microscope and maintained in bath solution. The cell under examination was continuously perfused via a perfusion pipet positioned in its vicinity. External test solutions without (control) or with carvacrol (1) were changed by an electric command to a solenoid valve (The Lee Co., Essex, CT, USA) that controlled the perfusion. To make the patch pipet, thick-walled flint glass tubing (outside diameter 1.5 mm, inside diameter 1.1 mm, Perfecta, SP, Brazil) was pulled with a Flaming/Brown type puller (P-97 micropipet puller, Sutter Instruments, Novato, CA, USA) and had resistance ranging from 1.5 to 3.0 MΩ when filled with internal solution. Patch-clamp recordings were performed under a voltage-clamp in the whole-cell configuration, using an Axopatch 200B amplifier driven by Clampex 10.2 software (Molecular Devices, Sunnyvale, CA, USA). To elicit the total sodium current, a 100 ms voltage step to 0 mV from a −100 mV holding potential was used with a 5 s interval between pulses. Before exposure to 1, the neurons were allowed to stabilize at least 5 min (control period). Afterward, the neurons were exposed to carvacrol for 90 to 180 s and then back to test solution perfusion (washout/recovery period). Leakage and capacitive currents subtraction were performed using a P/4 subtraction protocol. Series resistance compensation (70% to 90%) was routinely employed to reduce voltage error. The liquid junction potential was not corrected in this set of experiments. The sodium currents were sampled at 20 kHz and low-pass filtered at 3 kHz, and data acquisition and storage were performed using computer-based acquisition hardware (Digidata 1440A, Molecular Devices). Statistical Analysis. All the results are expressed as means ± SEM and in some cases (IC50 values) with a confidence interval (95%), where n indicates the number of experiments. The unpaired Student’s t test and one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test were used when appropriate; p < 0.05 indicates statistical difference. 1516



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AUTHOR INFORMATION

Corresponding Author

*Tel: +55 85 3101 9814. Fax: +55 85 3101 9810. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank R. Vieira-do-Nascimento, P. Vicente de Cassia Lima Pimenta, and P. Militão AlbuquerqueNeto for technical support and Dr. J. S. Cruz for reading the manuscript. This work was supported by Conselho Nacional de ́ Desenvolvimento Cientifico e Tecnológico (CNPq), Fundaçaõ Cearense de Apoio ao Desenvolvimento Cienti fí co e Tecnológico (FUNCAP), Coordenaçaõ de Aperfeiçoamento ́ de Pessoal de Nivel Superior (CAPES) and Universidade Estadual do Ceará (UECE).

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