Eucalyptol induces hyperexcitability and epileptiform activity in snail neurons by inhibiting potassium channels.

European Journal of Pharmacology 764 (2015) 70–78

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

Eucalyptol induces hyperexcitability and epileptiform activity in snail neurons by inhibiting potassium channels Zahra Zeraatpisheh 1, Jafar Vatanparast n Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran

art ic l e i nf o

a b s t r a c t

Article history: Received 10 March 2015 Received in revised form 31 May 2015 Accepted 25 June 2015 Available online 30 June 2015

The effects of eucalyptol (1,8-cineole) were studied on the activity of central neurons of land snail Caucasotachea atrolabiata. Eucalyptol (3 mM) depolarized the membrane potential and increased the frequency of spontaneous activity in a time dependent and reversible manner. These effects were associated with suppression of afterhyperpolarization and significant reduction of amplitude and slope of rising and falling phases of action potentials. While the eucalyptol-induced suppression of action potential amplitude and rising slope were essentially dependent on membrane depolarization, its actions on repolarization slope and afterhyperpolarization were not affected by resetting the membrane potential close to the control value. These findings suggest an inhibitory action on the potassium channels that underlie repolarization and afterhyperpolarization. Eucalyptol also increased the frequency of driven action potentials but suppressed the post stimulus inhibitory period, indicating an inhibitory action on calcium-activated potassium channels. A higher concentration of eucalyptol, 5 mM, reversibly changed the pattern of activity to burst firing associated with paroxysmal depolarization shift (PDS). Low doses of eucalyptol and potassium channel blockers, tetraethylammonium and 4-aminopyridine, synergistically acted to induce burst firing. At high concentration (30 mM), tetraethylammonium was able to induce burst firing and PDS. The sodium currents and ion channel phosphorylation by protein kinases A and C were not required for the eucalyptol-induced epileptiform activity, but calcium currents were essential for this action. Our findings show the excitatory and epileptogenic action of eucalyptol, which is most likely mediated through direct inhibitory action on potassium channels. & 2015 Elsevier B.V. All rights reserved.

Keywords: Eucalyptol Snail neuron Burst firing Excitability Potassium channel Calcium channel Chemical compounds studied in this article: Eucalyptol (PubChem CID: 2758) Tetraethylammonium (PubChem CID: 5413) 4-Aminopyridine (PubChem CID: 1727) Nifedipine (PubChem CID: 4485) Chelerythrine chloride (PubChem CID: 72311) H-89 (PubChem CID: 449241)

1. Introduction Despite of the common assumption that natural substances are safe, they can have adverse effects when used inappropriately. Convulsion is one of the most hazardous symptoms that have been associated with indiscriminate usage of some plant essential oils (Burkhard et al., 1999). Many reports have described the convulsive properties of some essential oils, but the knowledge about the effective components and their mechanism(s) of action are usually incomplete. Eucalyptol (C10H18O), known also as 1,8-cineole, is a monoterpenoid cyclic ether that constitutes the major component of Eucalyptus essential oil (Zhang et al., 2014). It is used as flavoring and fragrance in various products because of its fresh and pleasant n

Corresponding author. Fax: þ98 7132280916. E-mail address: [email protected] (J. Vatanparast). 1 Present address: Shiraz Neuroscience Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. http://dx.doi.org/10.1016/j.ejphar.2015.06.050 0014-2999/& 2015 Elsevier B.V. All rights reserved.

smell, or spicy taste and cooling effect. It is widely used in many brands of toothpaste, mouthwash, shampoo and, as a food grade flavor ingredient, it can be found in the list of additives to cigarettes and even some food products. The effects of eucalyptol on transient receptor potential (TRP) and cyclic nucleotide-gated channels of olfactory and gustatory receptors provide a basis for its special taste and odor (Chen et al., 2006; Leffingwell, 2009). It has also a broad range of applications in therapeutics and especially used in the folk medicine as a remedy for respiratory problems because of its antiseptic, decongestant and expectorant properties (Juergens et al., 2003). Eucalyptol reduces inflammation and pain when applied topically (Ximenes et al., 2013). The agoniztic action of this compound on TRPM8 channels underlies its cooling action. The analgesic and anti-inflammatory effect of eucalyptol is possibly due to its inhibition of TRPA1 and desensitization of TRPV3 channels (Kolassa, 2013). In addition to interaction with ion channels on the peripheral sensory cells, eucalyptol has been reported to show smooth muscle relaxant, hypotensive and bradycardic properties. It also

Z. Zeraatpisheh, J. Vatanparast / European Journal of Pharmacology 764 (2015) 70–78

reduces the excitability of rat sciatic nerve and superior cervical ganglion neurons (Lima-Accioly et al., 2006; Ferreira-da-Silva et al., 2009). On the other hand, it has been suggested that eucalyptol and some other oxygenated monoterpenoids, contributes to the convulsant action of several essential oils (Burkhard et al., 1999; Culić et al., 2009; Kolassa, 2013; Waldman 2011). The effect of eucalyptol on the central neurons that underlie its convulsive action may involve interaction with different ion channels, but these mechanisms have not been described yet. Snail neurons have the essential substrates to develop and sustain epileptiform activity that in many aspects resemble those that happening in vertebrate neurons. These neurons have often been used as model system both for studying the neurophysiological basis of epilepsy and also for primary screening of compounds with potential efficacy in inducing or preventing epileptiform activity (Altrup et al., 1992; Onozuka et al., 1991). In this work, intracellular recording techniques under current clamp conditions were carried out on snail neurons to determine the possible epileptogenic action of eucalyptol and the ionic mechanisms that may underlie this effect.

2. Material and method 2.1. Animals and preparations Adult specimens of land snail Caucasotachea atrolabiata were collected from gardens in Babol, on the Caspian Sea coast, and kept under laboratory conditions. Animals were treated in accordance with the EC guidelines and the experimental protocols were reviewed and approved by the animal care committee of the Shiraz University. Snails were activated by wetting once or twice per week and fed with lettuce and carrot. All experiments were performed on neurons in the subesophageal ganglia. Animals were activated in cold water and the ganglionic mass with its main peripheral nerves and aorta was rapidly dissected out. The subesophageal ganglia were pinned by the nerve and edges of the connective tissue into a small (1 ml) Sylgard-grounded recording chamber (Dow Corning Midland, MI, USA) containing normal snail Ringer. To expose neurons, the overlying connective tissue was gently torn out using fine forcipes without any proteolytic enzyme pretreatment. 2.2. Intracellular recording Recording electrodes were fabricated from thin-wall (outer diameter 1.5 mm, inner diameter 1.12 mm) borosilicate glass capillaries using a horizontal micropipette puller (P-97, Sutter Instrument Co., USA), filled with 3 M KCl and those with a resistance of 1–5 MΩ were used for recording. SEC-10LX amplifier (npi, Germany) was used to record the membrane potentials and to inject current under current clamp conditions. Data were digitized using a LIH 8 þ8 data acquisition interface (HEKA, Germany) and analyzed by Fitmaster software (HEKA, Germany). Neurons with stable resting membrane potentials (RMP) more negative than 38 mV were studied. The RMP was defined as the mean voltage during interspike intervals, excluding the decaying phase of afterhyperpolarization and the depolarizing ramp that precedes each action potential. Hyperpolarizing current steps (1–5 nA, 500 ms) were injected into the neurons and steady-state voltage changes from RMP were plotted against the injected currents. The Rin was calculated from a linear fit of the current–voltage plot. Spike amplitude was defined as the change in voltage from the RMP to the peak of spike. The amplitude of afterhyperpolarization was measured from the RMP to the peak negativity after a spike and the duration was measured as the time required declining to 80% of its peak value. Spike threshold was estimated by eye as the

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sharp inflection point at which the action potential began. The slope of rising phase was measured as the mean slope between the threshold potential and the peak of the action potential, and the slope of falling phase was measured as the slope between peak of action potential and peak of afterhyperpolarization. 2.3. Solutions and drugs The normal snail Ringer solution contained (in mM): NaCl 80, KCl 4, CaCl2 10, MgCl2 5, glucose 10, HEPES 5; pH adjusted to 7.4 with TRISMA-base. The Na þ -free Ringer was prepared by substituting NaCl with equimolar quantities of Tris–HCl. Eucalyptol, tetraethyl ammonium (TEA), 4-aminopyridine (4-AP), dimethyl sulfoxide, nifedipine, N-[2-(p-Bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and chelerythrine were purchased from Sigma (St. Louis, MO, USA). Other chemicals were obtained from Merck (Darmstadt, Germany). Eucalyptol was prepared as 0.5 M stock in 60% ethanol and diluted (3–5 mM) daily in snail Ringer. Stock solutions of H-89 and chelerythrine were made in dimethyl sulfoxide and stored at 20 °C in single-use aliquots for direct bath application. Nifedipine (a blocker of L-type Ca2 þ channel) was prepared in a stock solution of 10 mM in 95% ethanol and 1 M stock solutions of TEA and 4-AP were daily prepared in double distilled water. TEAcontaining Ringer was prepared by replacing NaCl with equimolar amounts of TEA. 2.4. Statistical analysis Data were presented as mean 7S.E.M. with n being the number of neurons on which the measurements were done. The statistical differences were analyzed with either paired t-tests, or repeated measures ANOVA followed by Bonferroni post-hoc tests. A value of P o0.05 was considered as statistically significant.

3. Results 3.1. The effects of eucalyptol on neuronal firing, action potential characteristics and pattern of activity All of the studied neurons showed rhythmic spontaneous firing in control condition. Extracellular application of eucalyptol (3 mM) increased the frequency of action potentials in a time dependent manner (Fig. 1A). This effect was along with membrane depolarization and modulatory actions on action potential characteristics, which has been summarized in Table 1. Especially, the excitatory effect of eucalyptol was associated with partial suppression of afterhyperpolarization and significant reduction of amplitude and the slope of rising and falling phases of action potentials (Fig. 1B). The effects of eucalyptol on firing frequency, RMP and action potential configurations showed recovery after 1–2 min washing with normal Ringer at a rate of approximately 3 ml/min (Table 1, Fig. 1B). Since the effects of eucalyptol on the frequency and the waveform of action potentials were associated with a significant membrane depolarization, Pearson's correlation test was used to determine if correlations existed between RMP and Rin, action potential parameters and excitability. The RMP was not correlated with Rin but significant positive correlations between RMP (mean voltage of interspike interval) and firing frequency, action potential threshold and integral was detected. The action potential amplitude, rising and falling slope and the afterhyperpolarization duration and amplitude showed significant negative correlations with RMP (Table 2). The correlation found between RMP and some variables does not necessarily imply that RMP changes underlie


10 mV

72

10 ms

EUC (10 min)

20 mV

Washout Control 1s Fig. 1. Eucalyptol enhanced the frequency of action potentials, reduced the slope of rising and falling phase and suppressed amplitude of action potential and afterhyperpolarization in a time dependent and reversible manner. Spontaneous activity recorded from a representative neuron in control (A), 5 min (B), and 10 min after application of 3 mM eucalyptol (C) and 2 min after washout (D). The small horizontal bars right to each graph show 0 mV. E: Superimposed action potentials from a neuron in control condition, 10 min after application of eucalyptol (3 mM) and 2 min after washout with normal snail Ringer. Action potential traces from eucalyptol treated condition and after washout were vertically adjusted in respect to the control trace.

the modulatory action of eucalyptol on these variables. To determine whether there is a true causal relationship or not, the membrane potentials of some neurons were monitored during experiment and manually maintained close to the control level by injecting negative direct current (DC) in the presence of eucalyptol. The accuracy of membrane potential resetting was also verified offline during analysis by Fitmaster software. Interestingly, the effect of eucalyptol on the frequency, threshold, amplitude and upward slope of action potentials was recovered to a great extent when RMP was restored to control level by DC injection, although the action potential frequency and amplitude was still significantly different from control condition (Fig. 2). These findings suggest that the increased action potential threshold and suppressed rising slope are most likely consequent to eucalyptol-induced membrane depolarization. It seems that membrane depolarization partially contributes to the effects of eucalyptol on the action potential frequency and amplitude. On the other hand, repolarization slope, afterhyperpolarization and the area under action potential curve were not significantly affected by DC injection in the presence of eucalyptol (Fig. 2), and seem to be directly affected by eucalyptol through mechanisms that are not necessarily dependent on membrane potential. The firing frequency was also negatively

correlated with afterhyperpolarization duration (r ¼ 0.842, Po 0.001, n ¼8) and this negative correlation was still detected after DC injection to reset the RMP close to the control value (r ¼ 0.931, Po 0.001, n¼ 7). The action potential integral was negatively correlated with absolute magnitude of falling slope (r ¼ 0.775, P o001, n ¼8) and even an almost perfect reverse correlation was observed after DC injection (r ¼ 0.998, P o001, n¼ 7). In control condition, injection of depolarizing current pulses (2 nA, 500 ms) evoked high frequency action potentials that were followed by prominent post stimulus inhibitory periods. Within 5 min of exposure to 3 mM eucalyptol, the frequency of evoked action potentials significantly increased from a control value of 9.6 70.59 Hz to 11.3370.67 Hz (P o0.01). Eucalyptol also decreased the post stimulus inhibitory period from 1.57 0.12 s to 0.7 70.14 s (P o0.05) (Fig. 3). Exposure to 5 mM eucalyptol induced a more dramatic change in the firing pattern: in 10 out of 12 studied neurons, within 2– 5 min of exposure to 5 mM eucalyptol, the pattern of activity changed from regular spiking to burst firing. In the pre-convulsive phase, eucalyptol enhanced the frequency of spontaneous activity that was associated with membrane depolarization and modulatory actions on the action potential characteristics that, as

Table 1 The effects of eucalyptol on resting membrane potential, input resistance and action potential frequency and waveform properties. Parameter RMP (mV) Rin (MΩ) Action potential Frequency (Hz) Threshold (mV) Amplitude (mV) Rising slope (V/s) Falling slope (V/s) AHP duration (ms) AHP amplitude (mV) Peak AHP (mV) Integral (mVs)

Control (8)

EUC 5 min (8)

ECU 10 min (8) a

Washout (5)

a,b

42.647 2.83a,c 10.22 7 1.61 2.117 0.38c 31.067 1.97c 74.34 7 5.36a,c 12.38 7 3.17a,c 6.98 7 1.66b,c 65.89 7 22.49c 4.92 7 0.96c 48.127 3.1a,c 248.6 7 62.8c

46.647 1.93 10.08 7 1.22

41.75 7 2.63 11.45 7 2.04

38.96 73.26 11.06 72.15

1.98 7 0.21 32.52 7 1.83 77.55 7 2.95 17.067 2.02 8.487 1.15 82.377 19.27 6.28 7 0.39 53.08 7 2.14 256.9 7 23.5

2.79 7 0.22a 30.377 2.01a 69.58 7 4.02a 10.667 2.09a 4.167 0.9a 46.9 7 13.31a 2.75 7 0.78a 45.23 7 2.91a 366.17 44.3a

2.82 70.21a 26.98 72.08a 60.28 76.12a,b 6.56 71.87a,b 2.11 70.44a,b 28.48 711.48a 1.25 70.48a 41.4 73.19a,b 415.1 745.7a

Data present mean7 standard error of the mean. The numbers in parentheses indicate the number of neurons assayed. a b c

Significantly different from control; Significantly different from EUC 5 min; Significantly different from EUC 10 min. EUC, eucalyptol (3 mM); RMP, resting membrane potential; Rin, input resistance; AHP, afterhyperpolarization.


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Table 2 Pearson correlation coefficients (r) and associated p-Values between resting membrane potential and input resistance, frequency and action potential characteristics. Measure

Rin

Frequency

Threshold

Amplitude

Rising slope

Falling slope

AHP duration

AHP amplitude

Integral

r-Value p-Value

0.06 0.863

0.684 0.00087

0.885 0.00001

0.921 0.00001

0.835 0.00001

0.49 0.028

0.718 0.00015

0.61 0.0021

0.678 0.001

Absolute magnitude of falling slope and AHP amplitude values were used to determine the correlation between these variables and resting membrane potential. Measurements made in 7–8 neurons in different experimental conditions. Rin, input resistance; AHP, afterhyperpolarization.

**

20 mV

*

200 ms

* **

*† *

-60

**

Rdep

Thr

Int

Frq

Amp

-80 -100

EUC

†

-40

*

* *

*

*

Fig. 2. The effects on action potential frequency and characteristics within 5 min exposure to 3 mM eucalyptol (EUC) and after direct current (DC) injection in the presence of eucalyptol to restore the membrane potential to control level (EUC– DC). Values are shown as percent change from control but statistical comparisons were made on the original data. *Po 0.05 and **P o 0.01 compared to control; †Po 0.05 compared to eucalyptol. Frq, frequency; Int, integral; Thr, threshold; Amp, amplitude; Rdep, depolarization slope; Rrep, repolarization slope; AHPdur, afterhyperpolarization duration; AHPamp, afterhyperpolarization amplitude.

expected, were similar but more pronounced than those observed in the presence of 3 mM eucalyptol (data not shown). The burst firing induced by 5 mM eucalyptol was associated with paroxysmal depolarization shift (PDS). The eucalyptol-induced epileptiform activity showed gradually evolving changes (Fig. 4). The burst events usually emerged as two-spike burst doublets (Fig. 4B), but the duration of burst activity and the number of spikes in the burst gradually increased. The PDSs were superposed by action potentials and showed considerable variation in duration. The first action potential in each burst usually had the largest amplitude that showed a progressive decrease in time (Fig. 4C and D). At the end of some PDSs, the superposed action potentials were suppressed and a period of sustained depolarization was observed (Fig. 4D). During longer periods of exposure (more than 15 min), a phase of diminution of bursting was observed (Fig. 4E). The recent effect seemed to be secondary to the progressive membrane depolarization as it could be reversed by negative DC injection that restored the interburst potential close to control RMP level (not shown). The effects of eucalyptol (5 mM) on the pattern of activity of snail neurons were reversible. In 6 out of 10 bursting neurons, within 2–4 min of washout with normal snail Ringer, the epileptiform activity changed to regular firing and the RMP returned close to control level (Fig. 4F). In 2 out of 10 neurons the recording electrode came out of the neurons during washout before a sufficient solution change could be accomplished. Two neurons, which were so depolarized as to be completely silenced after a period of bursting in the presence of 5 mM eucalyptol, did not show recovery with washout (not shown). 3.2. Eucalyptol synergistically acts with potassium channel blockers

500 ms

2

15

** 10

1.5

PSIP (s)

†

2 nA

0 -20

Frequency (Hz)

20

** †

EUC EUC-DC

AHPamp

40

Control

**

AHPdur

60

Rrep

Change from control (%)

80

5

1 0.5

0

0 Control

EUC

Control

EUC

Fig. 3. Eucalyptol (3 mM) increased the frequency of driven activity and reduced post stimulus inhibitory period (PSIP). The pattern of spontaneous firing and delay in resumption of spontaneous firing after a high frequency evoked activity by a constant depolarizing current step (2 nA, 500 ms) in control condition and 5 min after exposure to 3 mM eucalyptol (EUC) (A). Pooled data of 6 neurons shows that eucalyptol increases the frequency of driven activity (B). Scatter plots which show the normalized post stimulus inhibitory period duration in the control condition (left) and in the presence of eucalyptol (right) for each cell that were linked by connecting lines (C). **P o0.01 compared to control.

to induce burst firing The finding that hyperexcitability and burst-inducing effect of eucalyptol was associated with decreased afterhyperpolarization and repolarization rate led us to investigate the possible involvement of K þ channel blockade to this action. We found that exposure to a bathing Ringer that contains 30 mM TEA, a blocker of the big-conductance (BK) Ca2 þ -activated K þ channels and delayed rectifier K þ channels, can elicit burst firing in snail neurons. Of 14 neurons that were exposed to 30 mM TEA, 9/14 neurons exhibited burst firing (Fig. 5A). In a subpopulation of these bursting neurons (4/9), the action potentials showed dramatically increased durations and an augmented plateau phase (Fig. 5E). In 2 out of 14 neurons that were treated with 30 mM TEA, the frequency of action potentials was considerably increased but the pattern of activity was not affected (not shown). The remaining neurons (3/14) were strongly hyperpolarized and displayed slow up and down fluctuations that rarely led to action potentials in the up state (not shown). Lower dose of TEA (5 mM) did not induce burst firing in any of the studied neurons (Fig. 5C, n¼ 7). Bath application of 4-AP,


20 mV

74

2s Fig. 4. Eucalyptol (5 mM) induced reversible epileptiform activity in snail neurons. Regular activity of a representative neuron in control condition (A). Two-spike bursts appeared within 2 min of eucalyptol application (B). The number of action potentials per burst was increased within 5 min (C) and sustained depolarization plateaus occurred after 7 min of exposure to 5 mM eucalyptol (D). Within 18 min of exposure and following progressive membrane depolarization, the number of overshooting action potentials reduced (E). The regular firing was resumed after 3 min washout with normal snail Ringer (F). The small horizontal bars right to each graph show 0 mV.

Control

Control

TEA (30 mM)

4AP (1mM) TEA (5mM) Washout

2s

20 mV

20 mV

Washout

+

3s

TEA (5 mM)

4AP (1 mM)

+ EUC (3 mM)

+ EUC (3 mM)

2s

20 mV

Control

20 mV

Control

3s

Control

TEA (30 mM, 2min)

20 mV

Washout

2s

20 mV

TEA (5min)

200 ms

Fig. 5. Potassium channel blockers reversibly induce burst firing and show synergic action with eucalyptol to induce burst firing. Spontaneous activity of neurons in control condition and in the presence of 30 mM TEA (A), co-application of 1 mM 4-AP plus 5 mM TEA (B), co-application of 5 mM TEA plus 3 mM eucalyptol (C) and co-application of 1 mM 4-AP plus 3 mM eucalyptol (D). Extracellular application of TEA reversibly widened action potentials and induced burst activity and sustained plateau depolarizations (E). The area within dashed rectangles has been shown at higher time resolution in the right panel.


75

Control

Na+ free Ringer

20 mV

EUC (5 mM)

2s

Control

EUC (5 mM)

EUC (5 mM)

+ NIF (60 µM, 3 min)

+ NiCl (4 mM, 2 min)

NIF (7 min)

NiCl (5 min)

20 mV

20 mV

Control

2s

2s

Fig. 6. Calcium inward currents are required for eucalyptol-induced epileptiform activity but sodium current is not. A: Spontaneous regular activity was maintained, although at a lower frequency, in the Na þ -free Ringer, and burst activity was elicited by 5 mM eucalyptol in this medium. B: Nifedipine (60 mM) suppressed paroxysmal depolarization shift and burst activity induced by 5 mM eucalyptol. C: Application of NiCl (4 mM) abolished the epileptiform activity induced by eucalyptol and changed the pattern of activity to regular single spike firing.

even at concentrations up to 5 mM, also did not induce burst firing in any of the 6 neurons tested (not shown). In the presence of both TEA (5 mM) and 4-AP (1 mM) action potential bursts were developed (Fig. 5B, n ¼7/8). This pattern of activity was also observed when eucalyptol (3 mM) was co-administered with either 5 mM TEA (Fig. 5C, n ¼6/8) or 1 mM 4-AP (Fig. 5D, n ¼7/7). 3.3. The calcium inward current is required for eucalyptol-induced burst firing, but the sodium current is not The contribution of major inward currents, Na þ and Ca2 þ currents, to the induction of burst firing were tested. To examine

the role of Na þ currents in the generation of burst firing, the extracellular Na þ was replaced by Tris–HCl. In 6 out of 8 neurons examined, action potential bursts were elicited after application of 5 mM eucalyptol in the Na þ -free snail Ringer (Fig. 6A). On the other hand, bath application of nifedipine (60 mM), or a non-selective T-type Ca2 þ channel blocker, NiCl (4 mM), consistently abolished the burst firing and PDS observed in the presence of 5 mM eucalyptol (Fig. 6B and C; n ¼6 for each blocker). 3.4. The eucalyptol-induced epileptiform activity is not dependent on phosphorylation through protein kinase A (PKA) and protein kinase

Control

EUC (5 mM)

EUC (5 mM)

H-89 (60 µM)

CHT (15 µM) 20 mV

20 mV

Control

2s

2s

Fig. 7. The effects of PKA and PKC inhibitors on eucalyptol-induced epileptiform activity. Application of H-89 (60 mM) did not induce a detectable effect (A.) Chelerythrine (15 mM) slightly decreased but did not block the eucalyptol induced epileptiform activity (B).

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(PKC) A set of experiments were conducted to examine the possible contribution of PKA and PKC mediated phosphorylation to the burst induction by eucalyptol. H-89 (60 mM), a selective blocker of PKA, did not induce a considerable effect on the eucalyptol-induced epileptiform activity (Fig. 7A, n ¼6). Chelerythrine chloride (15 mM), a potent inhibitor of PKC, depolarized the membrane potential and slightly suppressed but did not abolish the epileptiform activity induced by eucalyptol in snail neurons (Fig. 7B, n ¼6).

4. Discussion In the present study, the effects of eucalyptol on the spontaneous and driven activity of snail neurons were studied. We found that eucalyptol at 3 mM reversibly increases the neuronal excitability and at a higher concentration, 5 mM, induces epileptiform activity. There are a few reports that describe the effects of eucalyptol on neuronal excitability. It has been shown that eucalyptol reduces the excitability of rat sciatic nerve (Lima-Accioly et al., 2006) and superior cervical ganglion neurons (Ferreira-da-Silva et al., 2009) in a dose dependent and reversible manner. All parameters that were measured by Lima-accioly and colleagues as indices of neuronal excitability were highly dependent on proper activation of Na þ channels. Ferreira-da-Silva and colleagues also concluded that Na þ channel inactivation, which was consequent to membrane depolarization, essentially contributes to the suppressive action of eucalyptol on superior cervical ganglion neurons. Our finding similarly shows that eucalyptol-induced membrane depolarization contributes to the reduced Na þ channels availability, which is evidenced by the elevated action potential threshold and reduced amplitude and depolarization slope of action potentials. Despite of this, the final effect of eucalyptol on snail neurons was always excitatory. The minor contribution of Na þ current to the generation of action potential in snail neurons may explain this difference. In sciatic nerve, the inward current is exclusively carried by voltage dependent Na þ channels (Matzner and Devor, 1994). Both Na þ current and high voltage activated (but not low voltage activated) Ca2 þ current have been detected in superior cervical ganglion neurons, but still Na þ current is essentially required for the initiation of action potential (Schofield and Ikeda 1988). The Ca2 þ currents, which are carried by both low voltage and high voltage activated Ca2 þ channels, participate more intensively in the generation of action potentials in snail neurons (Senatore and Spafford, 2010; Standen, 1974). As we have shown in this study and earlier works (Vatanparast et al., 2006a, 2006b), neurons of C. atrolabiata are able to generate action potentials in Na þ -free bathing solution. The availability of Ca2 þ channels decreases as a function of membrane potential, in the same way as M

for the Na þ← channels, but slower and at more depolarized potential ranges (Sperelakis, 2012). Expectedly, the availability of essential mediator of inward current in snail neurons is less affected by eucalyptol-induced membrane depolarization than in sciatic nerve and superior cervical ganglion neurons. Furthermore, the moderate depolarization induced by eucalyptol also reduces the amount of depolarization required to reach action potential threshold. Several criteria support the hypothesis that K þ channels blockade contributes to the eucalyptol-induced hyperexcitability and epileptiform activity. The blockers of K þ channels were able to mimic the excitatory and epileptogenic effects of eucalyptol to a great extent. Potassium channels mediate the outward K þ

currents that underlie the action potential falling phase and afterhyperpolarization (Cloues and Sather, 2003; Thompson, 1977), which both were significantly reduced by eucalyptol. We found that the duration of afterhyperpolarization was reversely correlated with the neuronal excitability induced by eucalyptol. A similar reverse correlation has been shown for different neurons, including snail neurons (Cloues and Sather 2003; Janahmadi et al., 2008; Vatanparast et al., 2006a). So, the increased firing frequency in the presence of 3 mM eucalyptol and in pre-convulsive period after exposure to 5 mM eucalyptol could be related to the inhibition of K þ channels that underlie afterhyperpolarization. Furthermore, as the area under action potential curve was negatively correlated with the absolute magnitude of action potential falling slope, the K þ channel blockade may also underlie the increased action potential integral in the presence of eucalyptol. In snail neurons, the Ca2 þ current is activated mainly during the repolarizing phase of the action potential (Barra 1996; van Soest and Kits, 1998). The reduction of repolarization slope can indirectly augment the Ca2 þ inward current by prolonging the duration of repolarization phase (Holden et al., 1982). We found that the action potentials were widened in the presence of TEA and this effect was so dramatic in some neurons that a plateau phase was added to the action potential waveform. TEA can effectively block both delayed rectifier and BK potassium channels from the extracellular side. The K þ outward current through both of these channels contribute to the action potential repolarization (Gola et al., 1986). Upregulation of Ca2 þ currents may drive neurons to generate epileptiform activity (Cain and Snutch, 2012). Our results suggest that eucalyptol-induced blockade of K þ channels and consequent upregulation of Ca2 þ current may contribute to the action potential broadening and finally drive neurons into burst firing. Meanwhile, eucalyptol may also augment Ca2 þ currents directly. With the significant membrane depolarization in the presence of eucalyptol, which is most likely mediated by the blockade of K þ channels, a significant increase in Rin would be expected, but did not occur. This is possible when eucalyptol also increases the conductance of other ion(s) with positive equilibrium potentials, like Ca2 þ . This, of course, requires further studies. In contrast to the Na þ channels, both L-type and T-type Ca2 þ channels were essentially involved in eucalyptol-induced epileptiform activity. Comparatively, T-type channels require smaller depolarization to open and thus critically regulate excitability by influencing when cells reach action potential threshold (Senatore and Spafford, 2010). These channels are also essential for the formation of lowthreshold Ca2 þ potentials that trigger rhythmic burst firing, a hallmark of epileptiform activity. On the other hand, while T-type channels conduct more rapid and shorter Ca2 þ influxes, the L-type channels inactivate much more slowly than T-type channels and generate longer lasting Ca2 þ influxes (Cain and Snutch, 2012). Considering our result and the general properties of T-type and Ltype Ca2 þ channels, it may be reasonable to assume that in the presence of 5 mM eucalyptol, the T-type channels primarily induce the slow wave membrane depolarization and the following activation of L-type channels contributes to the consequent sustained depolarization. The Ca2 þ ions modulate the function of different ion channels, both directly and indirectly through interaction with cytoplasmic enzymes including kinases, and thereby modify the neuronal excitability and pattern of activity (Bond et al., 2005; Feng and Jaeger, 2008; Onozuka et al., 1991; Rouchet et al., 2008; Vatanparast et al., 2006a, 2006b). It was previously shown that the post stimulus inhibitory period is almost linearly correlated with the mean frequency of preceding train of evoked action potentials in snail neurons. Furthermore the post stimulus inhibitory period could be suppressed by Ca2 þ channel blockade or iontophoretic injection of a Ca2 þ chelator, showing that the post stimulus


inhibitory period is underlined by Ca2 þ -activated K þ currents (Vatanparast and Janahmadi, 2009). In the current study, exposure to eucalyptol (3 mM) increased the frequency of driven action potentials, but the consequent post stimulus inhibitory period was significantly reduced. This finding suggests that eucalyptol inhibits Ca2 þ -activated K þ currents. These currents play important roles in the regulation of neuronal excitability (Bond et al., 2005; Brodie et al., 2007). In some neurons the inhibition of Ca2 þ -activated K þ currents is involved in the transition to burst firing (Feng and Jaeger, 2008; Rouchet et al., 2008). Furthermore, these currents may be active at the RMP and can contribute to setting the level of membrane potential (Zhong et al., 2013). Accordingly, the inhibition of Ca2 þ -activated K þ currents may be involved in the eucalyptol-induced membrane depolarization and to the burst induction. Several studies have implicated that protein phosphorylation may contribute to the induction of epileptiform activity (Funase et al., 1993; Onozuka et al., 1991). Both PKA and PKC can affect the excitability and pattern of activity of snail neurons through modulatory actions on ion channels (Golowasch et al., 1995; Vatanparast et al., 2007a, 2007b). These kinases have been found critically involved in the induction of burst firing by some compounds in snail neurons (Chen and Tsai, 2000; Onozuka et al., 1991). Bath application of specific blockers of PKA or PKC was not able to block the eucalyptol-induced epileptiform activity. This finding suggests that channel phosphorylation, at least through these kinases, is not necessary for the eucalyptol-induced epileptiform activity. The time course of eucalyptol actions on neuronal excitability and pattern of activity was fast and comparable to those of ion channel blockers that were bath-applied. Furthermore these effects showed rapid recovery upon washout. These facts also support the idea that eucalyptol induces its excitatory and epileptogenic activities through direct interactions with ion channels, rather than indirect modulatory actions involving intracellular cascades. In conclusion, this study shows that eucalyptol has stimulatory and epileptogenic potential that seems to be mediated through inhibitory action on K þ channels. The Ca2 þ currents are essentially required for these actions of eucalyptol while the Na þ currents are not. The effects of eucalyptol on the membrane properties and excitability seem to be mediated through direct modulatory actions on ion channels and the phosphorylation of ion channels by PKA and PKC is not involved. While the excitatory action of eucalyptol at low doses may give it potential beneficial effects as a natural stimulant, it may induce epileptiform activity at high doses and should be avoided by patients with epilepsy. The recent action of eucalyptol may underlie the epileptogenic action reported after overdose/poisoning with products containing this compound.

Acknowledgment This work was supported by a grant (No. 87041302) from Iran National Science Foundation (INSF).

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