Burst firing of action potentials in central snail neurons elicited by d-amphetamine: effect of anticonvulsants

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Burst firing of action potentials in central snail neurons

elicited by d-amphetamine: effect of anticonvulsants

Yi-Hung Chen

a,b

_{, Cheng-Hsuan Chang}

b

_{, Gow-Jaw Liang}

b

_,

Shiang-Suo Huang

a

_{, Hung-Ming Hsieh}

a

_{, Chen-Ming Teng}

a

_,

Ming-Cheng Tsai

a,

_*

a_{Department of Pharmacology, College of Medicine, National Taiwan Uni}_{6ersity, No.}₁_{., Sec.}₁_{., Jen-Ai Road, Taipei, Taiwan, ROC} b_{Department of Pharmacy, Kaohsiung Military General Hospital, Kaohsiung, Taiwan, ROC}

Received 2 May 2000; received in revised form 20 June 2000; accepted 26 June 2000

Abstract

The effect of anticonvulsants on the burst firing of action potentials in snail central neuron elicited by d-amphetamine was studied in the identified RP4 neuron of the African snail, Achatina fulica Ferussac. Oscillation of membrane potential and burst firing of action potentials were elicited by d-amphetamine in a concentration-dependent manner. Voltage clamped studies revealed that d-amphetamine elicited a negative slope resistance (NSR) in steady-state I – V curve between − 40 and − 10 mV. The burst firing of action potentials was alleviated following extracellular application of phenytoin, but was not affected after ethosuximide, carbamazepine, and valproic acid. The NSR elicited by

d-amphetamine was blocked by phenytoin. However, the NSR was not altered if carbamazepine was added. These

results suggest that of the four anticonvulsants tested, only phenytoin could alleviate the burst firing of action potentials elicited by d-amphetamine in snail neuron. © 2000 Elsevier Science Inc. All rights reserved.

Keywords:Amphetamine; Anticonvulsant; Drug abuse; Neuron; Bursting; Potential; Invertebrate; Phenytoin; Epilepsy; Comparative pharmacology

1. Introduction

Convulsant such as pentylenetetrazole induced bursting activity of action potentials in the central neurons of snails. Factors altering the effects were well studied (Sugaya et al., 1985a; Onozuka et al., 1986; Tsai and Chen, 1989; Wiemann et al., 1996). Both d- and l-amphetamines also elicited burst firing of action potentials in a central neuron located on the right parietal ganglion (RP4 neu-ron) of the African snail, Achatina fulica Ferussac

(Tsai and Chen, 1995; Huang et al., 1999) and in the central thalamic neurons of new born rats (Tsai et al., 2000). The molecular mechanism un-derlying pentylenetetrazole-induced bursting ac-tivity is well studied (Onozuka et al., 1983, 1986, 1991a; Sugaya et al., 1985a,b). Factors altering the burst firing of action potentials elicited by d-amphetamine were tested. The effect was not blocked by high magnesium media, a number of drugs including propranolol, prazosin, haloperi-dol, phenobarbital, hexamethonium,

d-tubocurarine, atropine, calcium-free solutions, or verapamil (Tsai and Chen, 1995). The bursting activity elicited by d-amphetamine is not due to:

* Corresponding author. Tel.: + 886-2-23966786; fax: + 886-2-23915297.

E-mail address:mctsai@ccms.ntu.edu.tw (M.-C. Tsai).

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(a) the synaptic effects of neurotransmitters; or (b) the activity of cholinergic or adrenergic receptors of the excitable membrane (3). However, it has been associated with intracellular calcium ions (Chen and Tsai, 1996; Chen et al., 1998), second messengers (Chen and Tsai, 1997), and ionic cur-rents (Chen and Tsai, 2000) of the neuron. d-Am-phetamine elicits a negative slope resistance of the steady state I – V curve (NSR) in the neuron (Chen and Tsai, 2000). The NSR is closely associ-ated with the burst firing of action potentials in the neurons (Gillette, 1983; Funase, 1990; Onozuka et al., 1991b). Anticonvulsant drug af-fected on the spontaneous thalamocortical rhythm and it represented a potentially valuable in vitro model of generalized seizure discharges, with marked pharmacological and physiological simi-larities to various forms of clinical epileptic seizure activity (Zhang et al., 1996a,b,c). How-ever, which anticonvulsants can alleviate the burst firing of action potentials elicited by d-am-phetamine remained unknown. The aims of the present study were to assess the effects of anticon-vulsants, i.e. phenytoin, ethosuximide, carba-mazepine and valproic acid, on the burst firing of action potentials elicited by d-amphetamine. The results revealed that only phenytoin alleviated the burst firing of action potentials elicited by d-amphetamine.

2. Methods

2.1. Electrophysiological recordings

Experiments were performed on the identified RP4 neuron from the subesophageal ganglia of the African snail Achatina fulica Ferussac (Tsai and Chen, 1995). The ganglia were pinned to the bottom of 0.7 ml sylgar-coated perfusion chamber and the connective tissue sheath was removed to allow easy identification and penetration by croelectrodes. For voltage-clamp study, two mi-croelectrodes were penetrated into the neuron. The recording electrode (5 – 6 MV) and the cur-rent electrode (1 – 5 MV) were filled with 3 M KCl. The experimental chamber was perfused with a control saline. Solution composed of (mM): NaCl 85, KCl 4.0, CaCl2 8, MgCl2 7,

Tris – HCl 10 (pH 7.5) at room temperature of 23 – 24°C. Neurons were studied only if they ex-hibited resting membrane potentials more

nega-tive than − 50 mV (4). The ionic currents of the RP4 neurons were recorded using two-electrode voltage-clamp method. For voltage clamping, the neurons were clamped by means of Gene Clamp 500 amplifier (Axon Instrument). All potentials and currents were recorded on magnetic tape via a digitizing unit (Digidata 1200) and analyzed using the pCLAMP system. The steady state currents of the neurons were measured at the end of 5 s pulses (Chen and Tsai, 2000).

Data obtained after various treatments were compared with the pre-drug control by means of Student’s two-tailed t-test and paired t-test. Stu-dent’s t-test was used when the samples in the control and experimental conditions were from different groups of preparations. Student’s paired

t-test was used when the samples in the control

and experimental condition were from the same groups of preparations. Differences were consid-ered significant at PB0.05. Phenytoin (5,5-diphenylhydantoin sodium salt), carbamazepine, valproic acid, ethosuximide and d-amphetamine were purchased from Sigma (St Louis, MO).

3. Results

3.1. Electrophysiological properties of RP4

neurons

The resting membrane potential (RMP) of the identified RP4 neuron was − 60.190.6 mV (n= 15, mean9S.E.M.) and it showed a spontaneous firing of action potential at a frequency of 37.49 1.5 pulses/min (n = 15). The action potential showed a regularly spaced single spike. No burst firing of action potential was observed in control RP4 neuron. The mean amplitude of the sponta-neously generated action potentials was 83.290.8 mV (n = 15). The electrical characteristics of the RP4 neuron were quite similar to those reported previously (Tsai and Chen, 1995).

3.2. Effects of d-amphetamine on spontaneous

action potentials of RP4 neurons

The identified RP4 neuron showed a prominent response to d-amphetamine. Twenty minutes after extracellular perfusion of d-amphetamine (27 mM), the frequency of spontaneously firing action potentials was reduced. Higher concentration of

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d-amphetamine, e.g. 80mM, farther decreased the frequency of the action potentials. No burst firing of action potential was observed even after 4 h of application. However, 20 min after application of 270 mM, 94 neurons (out of 120 tested) showed the firing pattern changed from regularly spaced single spikes to one with bursts of 2 – 40 action potentials which were separated by a period of hyperpolarization. Oscillation of membrane po-tentials with a phasic depolarization followed by a sustained depolarization with burst of action po-tentials was found in d-amphetamine (270 mM) treated preparations. A sudden depolarization with a train of spikes on its rising phase was observed. Furthermore, 40 min after application of d-amphetamine (270mM), the bursting activity was found in 117 out of 120 neurons tested. Forty minutes after application of d-amphetamine (270 mM), each burst contained 10–40 action poten-tials and was terminated by hyperpolarization of the membrane. There were 1 – 5 bursts of action potentials in 1 min (3.290.7 bursts/min, n=5). The number of action potentials in each burst were in the range of 2 – 40 depending on individ-ual neuron tested. However, in the same neuron, the number of action potentials in each burst were reach steady after 40 min of d-amphetamine ad-ministration. In five neurons calculated, the burst

were 18.890.6 pulses/burst (n=5). The sub-tained depolarization depolarized reached a level of − 39.892.1 mV (n=5). This was followed by a long fall to a hyperpolarized level (Fig. 1C). The resting membrane potentials of the neurons were changed from − 60.190.6 mV (n=15) to − 67.490.9 mV (n=5) during the hyperpolariza-tion. There was no single-spike action potential 40 min after d-amphetamine (270 mM) treatment. The effect of d-amphetamine on the neuronal activities was reversible. After 120 min of continu-ous washing, the spontanecontinu-ously generated spikes of the central neuron returned to control level albeit with a lower frequency of spontaneous firing. An example of the effects of d-am-phetamine on the action potentials of the snail neuron is shown in Fig. 1.

3.3. Effects of phenytoin on the d-amphetamine

elicited bursting acti6ity of RP4 neuron

The effects of phenytoin on the

d-am-phetamine-elicited bursting activity of RP4 neu-ron are shown in Figs. 2A and 3A. At 10 mM, phenytoin significantly decreased the number of action potentials during the burst elicited by d-amphetamine. The number of action potentials in the presence of d-amphetamine (270 mM) and 30 min after perfusion with d-amphetamine contain-ing phenytoin (10mM) were 18.890.6 pulse/burst and 2.690.4 pulse/burst (n=5, PB0.05), respec-tively. Spaced single spikes were at the rate of 4.490.5 pulses/min were observed when d-am-phetamine (270mM) and phenytoin (10 mM) were co-administered. At higher concentration, pheny-toin (50 mM) significantly decreased (a) the fre-quency of burst firing of action potentials and (b) the number of action potentials during the burst elicited by d-amphetamine. The frequency of bursting activity in the presence of

d-am-phetamine (270 mM) and 30 min after perfusion with d-amphetamine containing phenytoin (50 mM) was 3.290.7 and 1.290.2 burst/min (n=5,

PB0.05), respectively, and the number of action

potentials in each burst was 18.890.6 and 2.29 0.2 pulse/burst (n = 5, PB0.05), respectively. The number of spaced single-spike potentials increased to 11.491.1 pulse/min. However, bursting activ-ity still appeared in phenytoin treated preparation although most of the action potentials changed its pattern to the spaced single spike (Figs. 2A and 3A). The effect of phenytoin on the burst firing of

Fig. 1. Effects of d-amphetamine on a central RP4 neuron of snail. A, B, C and D were from the same neuron. (A) Control, the neuron showed spontaneous firing of action potentials. (B) Potentials of RP4 neuron after 40 min of d-amphetamine (80 mM) administration. (C) Bursting firing of action potentials of the neuron 40 min after d-amphetamine (270_mM) administra-tion. (D) The potentials after 75 min washing off d-am-phetamine (270mM). The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that d-am-phetamine at 80mM did not, while d-amphetamine at 270 mM did, elicit burst firing of action potentials of RP4 neuron.

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Fig. 2. Effects of phenytoin, carbamazepine, ethosuximide and valporic acid on the d-amphetamine elicited burst firing of action potentials in RP4 neuron. A, B, C and D were from different neurons. A1, B1, C1 and D1: Controls, the spontaneous action potentials from four RP4 neurons, respectively. A2, B2, C2 and D2: The burst firing of action potentials 40 min after perfusion with d-amphetamine (270 mM) from A1, B1, C1 and D1, respectively. A3, B3, C3 and D3: Thirty minutes after further perfusion with d-amphetamine (270mM) containing phenytoin (50 mM, A3), carbamazepine (100 mM, B3), ethosuximide (100 mM, C3) and valproic acid (100_{mM, D3) from A2, B2, C2 and D2, respectively. The horizontal bar on the top left side indicated the membrane potential at} 0 mV. Note that the pattern of burst firing of action potentials was alleviated following extracellular application of phenytoin and the burst firing of potentials was changed into regularly spaced single spikes. The pattern of burst firing of action potentials was found in d-amphetamine with ethosuximide, carbamazepine and valproic acid treated preparations.

action potentials elicited by d-amphetamine was reversible. Thirty minutes after washing off phenytoin with d-amphetamine, the bursting ac-tivity resumed. Thus, phenytoin (50 mM) re-versibly alleviated the bursting firing of action potentials elicited by amphetamine in a concentra-tion dependent manner.

3.4. Effects of phenytoin on the spontaneously

firing action potentials of RP4 neuron

The RMP, the frequency and amplitude of the action potentials in control RP4 neuron were − 60.190.6 mV (n=15), 37.491.5 pulses/min (n = 15) and 83.290.8 mV (n=15), respectively. Thirty minutes after phenytoin (10mM) perfusion, the RMP and the frequency and amplitude of the action potentials were − 60.390.9 mV (n=3,

P\0.05) 39.690.4 pulses/min (n=3, P\0.05)

and 84.390.3 mV (n=3, P\0.05), respectively. Thirty minutes after phenytoin (50mM) perfusion, the RMP and the frequency and amplitude of the

action potentials were − 59.391.5 mV (n= 3, P\0.05) 39.391.6 pulses/min (n=3, P\ 0.05) and 83.690.9 mV (n=3, P\0.05), respectively. It appeared that phenytoin (10 – 50 mM) did not alter the RMP, the frequency and amplitude of the action potentials of the RP4 neuron.

3.5. Effects of carbamazepine on the

d-amphetamine elicited bursting acti6ity of RP4

neuron

Forty minutes after perfusion with d-am-phetamine (270 mM), the bursting firing of action potentials was elicited in RP4 neuron. The pattern of bursting activity did not change and there was no single spike action potentials 30 min after perfusion with carbamazepine (100 mM) contain-ing d-amphetamine (270 mm). However, the fre-quency of bursting in the presence of

d-amphetamine and 30 min after perfusion with

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d-am-phetamine (270mM) were 2.690.3 burst/min and 0.690.3 burst/min (n=3, PB0.05), respectively. Similar results were found in three other prepara-tions. Examples of the effects of carbamazepine on d-amphetamine elicited bursting firing of ac-tion potentials were shown in Figs. 2B and 3B. It appears that carbamazepine (100 mM) did not alter the pattern of bursting firing of action poten-tials elicited by d-amphetamine.

3.6. Effects of ethosuximide on the

neuron

Forty minutes after perfusion with d-am-phetamine (270 mM), the bursting firing of action potentials was elicited in the RP4 neuron (Figs. 2C and 3C). The pattern of bursting activity did not change and there was no single spike action potentials 30 min after perfusion with ethosux-imide (100 mM) containing d-amphetamine (270 mM). Similar results were found in another three neurons. It appears that ethosuximide (100 mM) did not alter the pattern of bursting firing of action potentials elicited by d-amphetamine.

3.7. Effects of 6alproic acid on the

neuron

Forty minutes after perfusion with d-am-phetamine (270 mM), the bursting firing of action potentials was elicited in the RP4 neuron. The pattern of bursting activity did not change and there was no single spike action potentials 30 min after perfusion with valproic acid (100 mM) con-taining d-amphetamine (270 mM). Similar results were found in five other preparations. Examples of the effects valproic acid on the d-amphetamine elicited bursting firing of action potentials were shown in Figs. 2D and 3D. It appears that val-proic acid (100 mM) did not affect the pattern of bursting firing of action potentials elicited by

d-amphetamine.

3.8. The steady-state currents of RP4 neuron

Currents in response to test potentials of − 50, − 30, − 10 and 10 mV are shown in Figs. 4.1 and 5.1. The currents were elicited by 5 s command pulses from a holding potential of − 60 mV to potentials ranging from − 100 to 10 mV. The

Fig. 3. The expanded pictures showing individual action potentials related to the effects of phenytoin, carbamazepine, ethosuximide and valporic acid on the d-amphetamine elicited burst firing of action potentials in RP4 neuron. A, B, C and D were from different neurons. A1, B1, C1 and D1: Controls, the spontaneous action potentials from four RP4 neurons, respectively. A2, B2, C2 and D2: The burst firing of action potentials 40 min after perfusion with d-amphetamine (270_{mM) from A1, B1, C1 and D1, respectively. A3, B3, C3 and} D3: Thirty minutes after further perfusion with d-amphetamine (270_{mM) containing phenytoin (50 mM, A3), carbamazepine (100 mM,} B3), ethosuximide (100mM, C3) and valproic acid (100 mM, D3) from A2, B2, C2 and D2, respectively. The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that the pattern of burst firing of action potentials was alleviated following extracellular application of phenytoin and the burst firing of potentials was changed into regularly spaced single spikes. The pattern of burst firing of action potentials was found in d-amphetamine with ethosuximide, carbamazepine and valproic acid treated preparations.

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Fig. 4. Effects of phenytoin on the d-amphetamine-elicited current changes in RP4 neuron. Steady-state currents were elicited by 5 s command steps from holding potentials of − 60 mV to stepping potentials of − 50, − 30, − 10 and 10 mV. (1) Control. The neuron was perfused with normal physiological saline. (2) Forty minutes after subsequent d-amphetamine (270 mM) administration. (3) Thirty minutes after phenytoin (10 mM) and d-amphetamine (270 mM) was subsequently perfused. (4) Phenytoin (50 _{mM) and d-amphetamine (270 mM) was} subsequently perfused. (5) Thirty minutes after washing off with d-amphetamine (270_mM).

− 50 mV. The steady-state outward currents re-versed its polarity into inward currents and the steady-state I – V curve revealed an N-shaped ap-pearance in the range from − 40 to − 10 mV after d-amphetamine (270 mM) treatment (6) (Figs. 4 – 6). The phenomenon revealed as the NSR of the steady state I – V curve. Both the burst firing of action potentials and NSR elicited by d-amphetamine were found 40 min after drug administration. The effects of d-amphetamine on the NSR response were reversible. After washing off d-amphetamine (270 mM) for 120 min, the outward steady-state membrane currents were re-turned with no NSR in the steady state I – V curve.

3.10. Effects of phenytoin on the

d-amphetamine-elicited steady-state currents

The effects of phenytoin on the

d-am-phetamine-elicited steady-state currents changes and the I – V relationships are shown in Figs. 4 and 6A and summarized in Table 1. Forty min-utes after d-amphetamine (270 mM) treatment, NSR of the steady-state I – V curve was found in potentials of − 40 to − 10 mV. Thirty minutes after further perfusion with phenytoin (10 mM) containing d-amphetamine (270mM), the outward currents during steps to − 30 and − 10 mV were

Fig. 5. Effects of carbamazepine on the d-amphetamine-elic-ited current changes in RP4 neuron. Steady-state currents were elicited by 5 s command steps from holding potentials of − 60 mV to stepping potentials of − 50, − 30, − 10 and 10 mV. (1) Control. The neuron was perfused with normal physiological saline. (2) Forty minutes after subsequent d-amphetamine (270 mM) administration. (3) Thirty minutes after carbamazepine (100 mM) and d-amphetamine (270 mM) was subsequently perfused.

steady-state current – voltage (I – V) relationships are shown in Fig. 6. The steady-state outward currents were observed with pulse potentials more positive than − 50 mV. The total outward current increased with more positive potential (Chen and Tsai, 2000).

3.9. Effect of d-amphetamine on the steady-state

currents of RP4 neuron

The effects of d-amphetamine on the steady-state currents of RP4 neuron are shown in Figs. 4 and 5. Compared with pre-drug controls, the steady-state currents of the neurons were signifi-cantly decreased 40 min after d-amphetamine (270 mM) treatment at potentials more positive than

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Fig. 6. (A) Effects of phenytoin on the d-amphetamine-elicited negative slope resistance (NSR) of the steady-state I – V curve. The currents were elicited by 5 s command steps from holding a potential of − 60 mV to stepping potentials ranging from − 100 to 10 mV at intervals of 10 mV. The steady-state currents were measured 5 s after initiation of stepping poten-tials. The closed circle ( ), closed diamond ("), open triangle () and open square ( ) points represented the steady-state I – V relationship before ( ), after d-amphetamine (270 mM) application ("), after 30 min further perfusion with phenytoin (10mM) and d-amphetamine (270 mM) () and 30 min after further perfusion with phenytoin (50mM) and d-amphetamine (270mM) ( ). Note that d-amphetamine (270 mM) elicited a NSR in the steady-state current – voltage relationships, which was reduced by phenytoin (10 and 50 mM). (B) Effects of carbamazepine on the d-amphetamine-elicited NSR of the steady-state I – V curve. The closed circle ( ), closed diamond (_{") and open triangle () points represented the steady-state} I – V relationship before (_{), after d-amphetamine (270 mM)} application (_{"), after 30 further perfusion with carbamazepine} (100 mM) and d-amphetamine (270 mM). Note that d-am-phetamine (270mM) elicited a NSR in the steady-state cur-rent – voltage relationships, and it was not affected by carbamazepine (100mM). Note that after d-amphetamine ad-ministration, the steady state currents elicited by depolarizing command steps in the range of − 50 – 10 mV became more inward than those in the control. The effects of d-am-phetamine were partially reversed if phenytoin (10mM) was perfused and were further reversed if phenytoin (50mM) was perfused. However, the NSR was not altered in carbamazepine (100mM) perfused preparations.

increased (Fig. 4 and Table 1). The NSR of the steady-state I – V curve was decreased (Fig. 6A). Thirty minutes after further perfusion with a higher concentration of phenytoin (50 mM) con-taining d-amphetamine (270 mM), the outward currents during steps to − 30 and − 10 mV were further increased (Fig. 4 and Table 1). The NSR of the steady-state I – V curve was further de-creased (Fig. 6A, Table 1). The NSR of the steady-state I – V curve returned after phenytoin was washed off for 30 min.

It appears that extracellular perfusion of pheny-toin decreased the NSR of the steady state I – V curve elicited by d-amphetamine in a concentra-tion dependent manner.

3.11. Effects of carbamazepine on the

d-amphetamine-elicited steady-state currents

The effects of carbamazepine on the d-am-phetamine-elicited steady-state currents changes and the I – V relationships are shown in Figs. 5 and 6B. Forty minutes after damphetamine (270 mM) treatment, NSR of the steady-state I–V curve appeared in potentials of − 40 to − 10 mV. Thirty minutes after further perfusion with carba-mazepine (100 mM) containing d-amphetamine (270 mM), the outward currents during steps to − 30 and − 10 mV did not significantly change compared to the currents in d-amphetamine (270 mM) alone (Figs. 5 and 6B). It appears that extracellular perfusion of carbamazepine did not affect the NSR of the steady-state I – V curve elicited by d-amphetamine.

4. Discussion

Convulsant such as pentylenetetrazole induced bursting activity of action potentials in the central neurons of snails (Sugaya et al., 1985b; Tsai and Chen, 1989; Wiemann et al., 1996). Both d- and

l-amphetamines also elicited burst firing of action

potentials in the central RP4 neuron of the African snail, A. fulica Ferussac (Tsai and Chen, 1995; Huang et al., 1999). However, pentylenete-trazole elicited bursting firing of potentials in most of the central neurons in the giant African snail (Tsai and Chen, 1989) while d-amphetamine elicited only to some specific neurons, such as RP4 neuron of the African snail (Tsai and Chen, 1995; Huang et al., 1999). In the present study, it was found that the burst firing of action

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poten-tials was alleviated following extracellular applica-tion of phenytoin, but was not affected after ethosuximide, carbamazepine, and valproic acid. A previous study also revealed that phenobarbital sodium (0.43 mM) did not alleviate the frequency and firing pattern of the central neuron elicited by

d-amphetamine (Tsai and Chen, 1995).

Antiepileptic drugs decreased membrane ex-citability by interacting with neurotransmitter re-ceptors or ion channels. Barbiturates enhanced GABAa receptor-mediated inhibition. Phenytoin and carbamazepine and possibly valproate de-creased high frequency repetitive firing of action potentials by enhancing sodium channel inactiva-tion. Ethosuximide and valproate acid reduced a low threshold (T type) calcium channel current (Macdonald and Kelly, 1995).

Phenytoin suppressed the sodium current of squid giant axon when applied internally or exter-nally (Morello et al., 1984). It appears that pheny-toin and carbamazepine possess membrane stabilization effect on squid giant axons. They blocked the sodium currents of the excitable membrane. However, in the present study, pheny-toin (10 – 50 mM) did not alter the resting mem-brane potentials, the frequency and amplitude of the action potentials of the RP4 neuron. The pattern of the spontaneously generated action po-tential of the RP4 neuron was not altered even after 2 h of high concentration of phenytoin ap-plication. These results suggest that phenytoin did not inhibit the generation and propagation of action potentials on the excitable membrane of the RP4 neuron. The RP4 neurons possess tetrodotoxin insensitive sodium current and

cal-cium current, and tetrodotoxin can not block the burst firing of action potentials elicited by d-am-phetamine (Chen and Tsai, 2000). Therefore, the alleviation effect of phenytoin on the burst firing elicited by d-amphetamine may not mainly due to its effect on the sodium currents of the membrane. Phenytoin and carbamazepine also potentiated g-aminobutyric acid (GABA) induced chloride currents in human embryonic kidney cells tran-siently expressing the a1 b2 g2 subtype of the GABAAreceptor and in cultured rat cortical

neu-rons (Granger et al., 1995). However, the burst firing of action potentials elicited by d-am-phetamine was not due mainly to the activation of these receptors (Tsai and Chen, 1995). Besides, phenobarbital and carbamazepine which potenti-ated GABA induced chloride currents, did not alleviate the d-amphetamine elicited bursting firing of action potentials in RP4 neuron. Thus, the alleviation effect of phenytoin on the burst firing of action potentials elicited by d-am-phetamine may not mainly due to the potentiation of the GABA induced chloride current.

The bursting activity elicited by d-amphetamine is not due to (a) the synaptic effects of neuro-transmitters or (b) the activity of cholinergic or adrenergic receptors of the excitable membrane because the burst firing of action potentials elic-ited by d-amphetamine is not blocked by high magnesium media, a number of drugs including propranolol, prazosin, haloperidol, phenobarbital, hexamethonium, d-tubocurarine and atropine, calcium-free solutions, or verapamil (Tsai and Chen, 1995). The bursting firing of potential changes elicited by d-amphetamine has been

asso-Table 1

Effects of d-amphetamine and phenytoin on the steady-state currents of RP4 neuronsa

c

a b d

Stepping potentials Control (nA) Amphetamine Phenytoin (10mM)+amphetamine Phenytoin (50 (mM)+amphetamine

(mV) (nA) (nA) (nA)

n = 3 n = 3 n = 3 n = 4 5.191.0 −30 −6.491.8* −1.190.4** 0.190.2*** 0.690.4*** −3.190.8** −9.592.2* 22.491.5 −10

a_{The steady-state currents were elicited by a 5-s pulse from a holding potential of −60 mV to potentials (−30 and −10 mV). The}

amplitudes of steady-state currents were measured at 5 s in (a) normal saline, (b) d-amphetamine (270_{mM) treatment for 40 min, (c)} further perfusion with phenytoin (10_{mM) containing d-amphetamine (270 mM) from (b), (d) further perfusion with phenytoin (50 mM)} containing d-amphetamine (270mM) from (b). Values were expressed as the mean9S.E.M. (n, the number of neurons tested).

* PB0.05, statistically significant by use of the t-test compared with a and b. ** PB0.05, statistically significant by use of the t-test compared with b and c. *** PB0.05, statistically significant by use of the t-test compared with c and d.

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ciated with (a) calcium ions (Chen and Tsai, 1996; Chen et al., 1998), (b) intracellular second messen-gers (Chen and Tsai, 1997), and (c) ionic currents (Chen and Tsai, 2000) of the neuron.

The intracellular calcium ion was associated with the action potential bursts elicited by d-am-phetamine. The occurrences of the action poten-tial bursts elicited by d-amphetamine was decreased following intracellular injection with ei-ther EGTA or magnesium ions or extracellular application of lanthanum (Chen and Tsai, 1996). The bursting firing of action potentials was also decreased following extracellular application of (1) H8 (N-(2-methyl-amino) ethyl-3-isoquinoline sulphonamide dihydrochloride), a specific protein kinase A inhibitor and (2) anisomycin, a protein synthesis inhibitor. However, the bursting firing of action potentials were not affected after (1) extracellular application of H7 (1,(5-isoquinoline-sulphonyl)-2 methylpiperasine dihydrochloride), a specific protein kinase C (PKC) inhibitor, or (2) intracellular application of GDPbS, a G protein inhibitor. The oscillation of membrane potential of the bursting activity was blocked after intracel-lular injection of 3%-deoxyadenosine, an adenylyl-cyclase inhibitor. These results suggested that the bursting firing of action potentials elicited by

d-amphetamine in snail neuron may be associated

with the cyclic AMP second messenger system; on the other hand it may not be associated with the G protein and PKC activity (Chen and Tsai, 1997). The intracellular cyclic nucleotide and cal-cium ion also play an important role on the pentylenetetrazole-induced bursting activity in

Euhadra neurons (Onozuka et al., 1983; Sugaya et

al., 1985a). Voltage clamp studies on the RP4 neuron revealed that d-amphetamine decreased the steady-state K+_{current and elicited a NSR in}

the steady-state I – V curve between − 50 and − 10 mV. The amplitude of NSR was decreased if either Na+_{-free saline or Co}2 +_{-substituted Ca}2 +

free saline were perfused (Chen and Tsai, 2000). In the present study, it was found that phenytoin did, while carbamazepine did not, block the NSR elicited by d-amphetamine. Its interesting to note that there are two serotonin-sensitive potassium channels in the identified heart excitatory neuron (PON) of the same snail. The activities of both channels could be recorded in the steady state and those activities disappeared on application of serotonin (Furukawa and Kobayashi, 1988). It is likely that the inhibitory effect of phenytoin on

the burst firing of action potentials elicited by

d-amphetamine may be due to its effects on

inhi-bition of (a) intracellular calcium concentration, (b) cAMMP activity and (c) NSR elicited by

d-amphetamine in the neuron. It is interesting to

note that pentylenetetrazole elicited bursting ac-tivity in the central neuron of the Japanese land sn4 Euhadra peliomphala, phenytoin inhibited the calcium release. Phenytoin also inhibited the pentylenetetrazole elicited changes in (1) the intra-cellular calcium binding state change near the cell membrane, (2) the intracellular protein changes induced by pentylenetetrazole and the intracellu-lar protein and (3) the increase in calcium depen-dent protein kinase activity during pentylenetetrazole elicited bursting activity (Sug-aya et al., 1985a,b). Phenytoin also inhibited the NSR in the bursting firing of potential changes elicited by injection of a specific 70k protein in the central neurons of snail (Onozuka et al., 1991a). Both pentylenetetrazole and amphetamine elicited bursting firing of potential changes. However, pentylenetetrazole elicited bursting firing of po-tentials in most of the central neurons (Tsai and Chen, 1989) while d-amphetamine elicited only to some specific neurons, such as RP4 neuron of the African snail, A. fulica Ferussac (Tsai and Chen, 1989; Huang et al., 1999). The reasons for the different effects of both compounds on the neuron remained unclear. However, the peptides in the neuron may play an important role on the func-tion. Anisomycin, the inhibitor of protein synthe-sis, did not alter the resting membrane potentials of the neuron. However, anisomycin significantly decreased the frequency of the spontaneously gen-erated action potentials of the neuron. An-isomycin pretreatment prevented the bursting firing of action potentials elicited by d-am-phetamine (Chen and Tsai, 1997). The new pep-tides and neuronal signaling in the neuron after different convulsants administration remained an interesting subject for further study.

In the present study, it was found that pheny-toin did (a) alleviate the burst firing of action potentials and (b) block the NSR elicited by

d-amphetamine in snail neuron while

carba-mazepine did not. It is concluded that phenytoin can alleviate the burst firing of action potentials elicited by d-amphetamine in snail neuron. The alleviation by phenytoin on the burst firing of action potentials may be due to its effects on (a) calcium ion, (b) cAMP activity, and (b) NSR

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elicited by d-amphetamine on the excitable membrane.

Acknowledgements

This work was supported by grants from Na-tional Science Council, NSC-88-2314-B-002-098, Taipei and Kaohsiung Military General Hospital, Kaohsiung, Taiwan.

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