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Bursting firing of action potentials in central snail neurons elicited by d-amphetamine: role of cytoplasmic second messengers
Neuroscience Research 27 (1997) 295 – 304
Bursting firing of action potentials in central snail neurons elicited by d-amphetamine: role of cytoplasmic second messengers Yi Hung Chen, Ming Cheng Tsai * Department of Pharmacology, College of Medicine, National Taiwan Uni6ersity, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan Received 15 October 1996; accepted 27 December 1996
Abstract The role of the intracellular second messengers on the bursting firing of action potentials in central snail neurons elicited by d-amphetamine was studied in the identified RP4 neuron of the African snail Achatina fulica Ferussac. Oscillation of membrane potential and bursting firing of action potentials were elicited by d-amphetamine in a concentration dependent manner. The bursting firing of action potentials was 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-isoquinolinesulphonyl)-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 intracellular injection of 3%-deoxyadenosine, an adenylyl-cyclase inhibitor. These results suggested that the bursting firing of action potentials elicited by d-amphetamine in snail neurons may be associated with the cyclic adenosine monophosphate (cAMP) second messenger system; on the other hand it may not be associated with the G protein and protein kinase C activity. © 1997 Elsevier Science Ireland Ltd. Keywords: Amphetamine; Second messenger; Drug abuse; Snail; Central neuron; Anisomycin; Action potential; cAMP
1. Introduction The central ganglia of the snail (Rees, 1950) contains many identifiable neurotransmitters and receptors (Kerkut et al., 1975; Takeuchi et al., 1996). Convulsants such as pentylenetetrazole-induced bursting activity of action potentials in the central neurons of the snail (Ferrendelli and Kinscherf, 1977; Sugaya and Onozuka, 1978a,b; Sugaya et al., 1987; Onozuka et al., 1983, 1986; 1991a; Tsai and Chen, 1989; Arvanov et al., 1994). The response strongly resembled the pentylenetrazole-induced seizure changes in cerebral cortical neurons of mammals (Sugaya et al., 1964). We have shown that * Corresponding author. Tel.: +886 2 3966786; fax: + 886 2 3915297; e-mail: [email protected]
amphetamine elicited bursting firing of action potentials in the central RP4 neuron of the African snail Achatina fulica Ferussac (Tsai and Chen, 1995). This bursting activity was not blocked in high magnesium medium or after continuous perfusion of propranolol, prazosin, haloperidol, phenobarbital, hexamethonium, d-tubocurarine, atropine or calcium free solution containing EDTA or verapamil. These results suggested that the bursting activity of potentials elicited by d-amphetamine was not due to (1) the synaptic effects of neurotransmitters or (2) the activity of cholinergic or adrenergic receptors of the excitable membrane (Tsai and Chen, 1995). However, it was associated with intracellular calcium ions because the bursting firing of action potentials elicited by amphetamine was reduced following intracellular injection with EGTA (Chen and Tsai, 1996).
0168-0102/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 8 - 0 1 0 2 ( 9 7 ) 0 1 1 5 9 - 0
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The cytoplasmic concentration of Ca2 + is regulated by regulation of several distinct Ca2 + -specific channels in the plasma membrane and its release from intracellular storage sites. Ca2 + channels may be opened by electrical depolarization, phosphorylation, cyclic adenosine monophosphate (cAMP)-dependent protein kinase, protein kinase C (PKC), G protein, etc. Since cytoplasmic second messengers play important roles in the functions of the neuron, the aim of the present study was to elucidate the interaction between the amphetamine-elicited bursting firing of action potentials and the second messengers in the snail central neuron. Evidence suggests that cAMP may alter membrane properties (Hausdorff et al., 1990). For example, cAMP elicited biphasic current in snail neurons whose activation was mediated through protein phosphorylation (Watanabe and Funase, 1991); cAMP elicited gating currents in toad olefactory cells (Kurahashi and Kaneko, 1993). In addition, cAMP-dependent protein kinase and PKC have been shown to modulate membrane Ca2 + and K + channels (Penner et al., 1988). Forskolin, a known activator of adenylate cyclase (Seamon and Daly, 1981; Levitan and Levitan, 1988), was found to activate the Ca2 + current of the motor nerve terminal (Tsai et al., 1992). To elucidate the role of adenylate cyclase and cAMP dependent protein kinase in the generation of the bursting activity induced by amphetamine, the effects of adenylyl cyclase activator, e.g. forskolin and inhibitor, e.g. 3%-deoxyadenosine on the bursting potentials were examined. The effects of a specific protein kinase A (PKA) inhibitor, N-(2-methylamino) ethyl-3-isoquinoline sulphonamide dihydrochloride (H8) (Levistre et al., 1995) and 8-bromo-cyclic AMP, a nonhydrolyzable analog of cAMP (Price and Goldberg, 1993) on the amphetamine-elicited bursting potentials were studied. Release of Ca2 + from intracellular stores may be modulated by inositol 1,4,5-triphosphate (IP3) which is produced from the membrane lipid, catalyzed by phospholipase C (Rhee and Choi, 1992). Both the PKC and IP3 pathways are branches of the PI pathway. To test whether PKC was involved in the generation of the bursting firing of action potentials mediated by amphetamine, the effects of a specific PKC inhibitor, 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride (H7) (Duraj et al., 1995), on the amphetamine elicited bursting activity of potentials were tested. G-proteins has been demonstrated to modulate the activity of adenylyl cyclase (Taussig and Gilman, 1995). Therefore, the roles of G protein and newly synthesized proteins on the bursting of action potentials induced by amphetamine were also examined with guanosine 5%-O2-thiodiphohsphate (GDPbS), an inhibitor of G protein (Chiba et al., 1992; Magoski et al., 1995) and
with anisomycin, an inhibitor of protein synthesis (Schacher et al., 1988). Our results indicated that the d-amphetamine elicited bursting of action potentials in the snail neuron may be associated with intracellular activity of cAMP and newly synthesized protein(s). In contrast, they may not be associated with G protein or PKC.
2. Methods
2.1. Electrophysiological recordings Experiments were performed on identified RP4 neuron from the subesophageal ganglia of the African snail A. fulica. The ganglia were pinned to the bottom of a 0.7 ml sylgard-coated perfusion chamber and carefully freed from the connective tissue sheath to allow easy identification and penetration by microelectrodes. For measuring the potentials, two microelectrodes penetrated into the neuron. The recording electrode (5 MV) was filled with 3 M potassium acetate (KAc) and the other electrode was filled with injection solutions for intracellular injection. The experimental chamber was perfused with control saline i.e. NaCl (mM) 85, KCl 4.0, CaCl2 8, MgCl2 7, Tris–HCl 10 (pH, 7.5) at room temperature of 23–24°C. The potentials were amplified using Axoclamp 2A amplifier and monitored on Tektronic oscilloscopes (Type 5441 and 5110). Neurons were studied only if they exhibited resting membrane potentials more negative than − 50 mV (Tsai and Chen, 1989; 1995), the time constant at around 5–8 ms and the rate of rise of the action potentials at around 5–8 DV/s. For the voltage clamp study, the neurons were clamped by means of an Axoclamp 2A amplifier. For the recording of membrane currents, the neurons were maintained at a standard potential of −75 mV. Current and voltage electrodes (5 MV) were filled with 3 M KAc. In order to eliminate the chloride effects from the electrodes during recording, acetate was used for the intracellular recording of the whole cell preparation (Tsai and Chen, 1995). Microperfusion was used for the rapid application of agonists. The perfusion tube, 0.9 mm inside diameter, was placed on the top of the neuron under microscopic control. This system allows rapid exchange of the bathing medium without apparent mechanical disturbance of the neuron under study (Slater et al., 1984). All potentials were recorded on tape via a digitalizing unit (Digidata 1200) and analyzed using a pCLAMP system. The mean amplitude of the potentials and the resting membrane potentials after various treatments were compared with the pre-drug control by means of Student’s two-tailed t-test. Differences were considered significant at PB 0.05.
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2.2. Intracellular injection For intracellular injections, 3%-deoxyladenosine or guanosine 5%-O-2-thiodiphohsphate (GDPbS) was dissolved in 100 mM KCl (Watanabe and Funase, 1991), and the intracellular injection solutions were filled into the micropipettes with a tip diameter of about 1 mm adjusted with a microforge (Narishige MF-83). Drugs were buffered to pH 7.5 with KOH or HCl. Injections were made with a pulse under manual control over the course of the experiment. The duration of each injection pulse is 2 s with a pressure of 200 mmHg. The diameter of the neuron was measured with a micrometer, assuming the shape of the neuron was spherical. The volumes of solutions injected into the neuron were measured by the application of similar pressure and duration to the pipettes containing solutions before impalement. The droplets on the edge of the micropipettes was measured with a micrometer, assuming the shapes of droplets were spherical. According to our previous work, the diameters of the RP4 neuron and the droplets were about 400 and 80 mm, respectively. The volume of the neuron and the volume of solution injected into the neuron were around 3.26 × 10 − 8 and 2.6 × 10 − 10 l, respectively (Tsai and Chen, 1989).
3. Results
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3.2. Effect of extracellular d-amphetamine on RP4 neuron The identified neuron in the right parietal ganglion, RP4 neuron had a prominent response to d-amphetamine. Twenty min after extracellular perfusion of d-amphetamine (0.027 mM), the frequency of spontaneously firing action potentials was reduced by 28% (from 39.591.8 pulses/min, n= 10) to 28.391.2 pulses/min, n=3, P= 0.003). Higher concentration of d-amphetamine, e.g. 0.08 mM, further decreased the frequency of the action potentials by 45% to 21.6 91.8 pulses/min, n =3, PB 0.001). An intermittent firing pattern was also observed. No bursting firing of action potentials was observed even after 4 h of incubation. However, 20 min after increasing the extracellular damphetamine concentration to 0.27 mM, the firing pattern changed from regularly spaced single spikes to one in which bursts of 2–20 action potentials were separated by large hyperpolarization of membrane potential (up to 8 mV) lasting 1–40 s (Fig. 1). Oscillation of membrane potentials with a phasic depolarization was followed by a sustained depolarization. The resting membrane potential was phasically depolarized from − 61.49 0.9 mV (n=11) to − 57.69 1.0 mV (n=11, PB 0.05). The effect of d-amphetamine continued throughout its application (up to 3 h). The membrane potential also showed a phasic depolarization followed by a sustained depolarization. The sustained depolar-
3.1. Some electrical characteristics of the identifiable RP4 neuron The resting membrane potential (RMP) of the identified RP4 neuron was −60.0 91.3 mV (n= 10, mean9 S.E.M.) and it showed a spontaneous firing frequency of about 39.591.8 pulses/min (n= 10) (Tsai and Chen, 1995). The mean amplitude of the spontaneously generated action potentials was 87.3 9 1.8 mV (n=10). The time constant of the action potential was 6.690.7 ms (n =5) and the rate of rise of the action potentials was 5.7 9 0.7 DV/s (n = 5). The neuron was sensitive to several neurotransmitters. Acetylcholine and g-aminobutyric acid (GABA) induced hyperpolarization of the membrane potential and decreased the frequencies of the spontaneous firing of action potentials (results not shown). GABA and ACh also elicited inward currents in the voltage clamped neuron. Glutamic acid and dopamine increased the frequency of the spontaneous action potentials. Serotonin induced biphasic responses, i.e. an initial hyperpolarization of membrane potential followed by an increase in the frequency of action potentials.
Fig. 1. Effects of d-amphetamine on a central RP 4 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 RP-4 neuron after 40 min of d-amphetamine (80 mM) administration. (C) bursting firing of action potentials of the neuron after 40 min of d-amphetamine (0.27 mM) administration. (D) the potentials after 75 min washing off d-amphetamine (0.27 mM). The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that d-amphetamine at 80 mM did not, while d-amphetamine at 0.27 mM did, elicit bursting firing of action potentials of RP4 neuron.
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ization reached −24.8 92.3 mV (n =9) 3 h after 0.27 mM d-amphetamine application. A sudden depolarization with a train of spike changes on its rising phase was observed. This was followed by a long fall to a hyperpolarization level ( − 69.4 9 1.4 mV, n = 11, PB 0.001). The train of spike changes on the rising phase was reduced after 2 or 3 h of d-amphetamine perfusion. The effect of d-amphetamine on the neuronal activities was reversible. After 120 min, of continuous washing, the spontaneously generated spikes of the central neuron almost returned to the control level albeit with a slower frequency of spontaneous firing. An example of the effects of d-amphetamine on the action potentials of the snail neuron are shown in Fig. 1.
3.3. Effects of H8 on d-amphetamine elicited bursting acti6ity of the RP4 neuron To examine the role of cAMP-dependent protein kinase in the generation of the bursting activity elicited by d-amphetamine, the effects of H8, an inhibitor of PKA, were studied. H8 did not alter the resting membrane potentials of the RP4 neuron. The resting membrane potentials of the RP4 neuron in saline was − 60.0 9 1.3 mV (n= 10, mean 9S.E.M.). Sixty min after 10 mM H8 treatment, the RMP of the neuron was −59.0 90.6 mV (n= 10, P= 0.5). However, H8 decreased the frequency of the spontaneously generated action potentials of the RP4 neuron from normal saline control of 39.59 1.8 (n= 10) to 8.79 0.7 pulses/min (n =10, P B0.001) 60 min after 10 mM H8 treatment. The frequency returned to the control level after 40 min of washing. It should be pointed out that H8 did not alter the amplitudes of the action potentials. An example of the effects of H8 on the spontaneously generated action potentials of the RP4 neuron are shown in Fig. 2. H8 blocked the bursting firing of action potentials prior to but not after the addition of d-amphetamine. Amphetamine (0.27 mM) elicited bursting of action potentials 20 min after perfusion. Subsequent addition of 10 mM H8 in the amphetamine-containing perfusion medium did not alter the effect of amphetamine even after 3 h of further incubation with H8 and amphetamine. However, in the preparations pretreated with H8 perfusion medium containing H8 (10 mM) for 40 min, subsequent concomitant treatment of neurons with d-amphetamine (0.27 mM) and H8 (10 mM) did not induce bursting activity for more than 60 min. The d-amphetamine elicited bursting firing of action potentials returned if H8 was washed off. The effects of pre-administration of H8 on the amphetamine elicited bursting firing of action potentials were shown in Fig. 2. Similar results were found in 10 other preparations. It appears that H8 significantly blocked the bursting firing of action potentials elicited by amphetamine if H8 was pre-administered to the preparation.
Fig. 2. Effects of H8 (10 mM) on the d-amphetamine elicited bursting firing of action potentials. A, B, C, D and E were from the same neuron. (A) Control, showing the spontaneous action potentials of RP-4 neuron. (B) the potentials after 40 min of H8 (10 mM) administration. (C) and (D) were potentials 25 and 60 min, respectively, after further incubation with d-amphetamine (0.27 mM) and H8 (10 mM). (E) 60 min after washing off H8 (10 mM) from (D). The horizontal bar on the top left side was the membrane potential at 0 mV. Note that amphetamine elicited bursting firing of action potentials which were blocked with H8 pre-treatment.
3.4. Effect of intracellular injection of 3 %-deoxyadenosine on the d-amphetamine elicited bursting of potentials To test the role of adenylyl cyclase in the generation of the bursting activity of potentials elicited by amphetamine, 3%-deoxyadenosine, an inhibitor of adenylyl cyclase was studied. The results were shown in Fig. 3. Injection micropipettes contained 10 mM 3%-deoxyadenosine and 100 mM KCl. 3%-Deoxyadenosine reversibly depolarized the resting membrane potentials of the RP4 neuron. Immediately after 3%-deoxyadenosine injection, the resting membrane potential of the RP4 neuron was depolarized from −60.190.6 mV (n= 3) to −44.29 0.7 mV (n=3, PB0.001). The frequency of the spontaneously generated action potentials were increased from 439 3 (n= 5) to 130910 pulse/min (n= 5, PB0.001) 30 s after intracellular injection (Fig. 3). Twenty min after intracellular injection, the resting membrane potentials returned to the control value of −59.09 1.6 mV (n= 4), although the frequency of the spontaneously generated action potentials was still significantly elevated (62.0 92.5 pulses/ min, n= 4, P B0.001). In our system, the volume injected was about 2.6910 − 10 l, resulting in the concentration of 3%-deoxyadenosine inside the neuron of around 80 mM if we assumed the volume of the RP4 neuron was 3.26× 10 − 8 l (Tsai and Chen, 1995). If the 3%-deoxyadenosine injected RP4 neuron was further in-
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cubated with amphetamine (0.27 mM) for 60 min, an intermittent firing pattern of the potentials was observed. However, the oscillation of the membrane potential of bursting activity elicited by amphetamine was not observed. The resting membrane potential was not altered following amphetamine treatment. Similar results were found in 3 other preparations. An example of the effect of 3%-deoxyadenosine on the bursting activity is shown in Fig. 3.
3.5. Effects of forskolin on the d-amphetamine elicited bursting of action potentials To test whether adenylyl cyclase was involved in the generation of the bursting firing of action potentials elicited by amphetamine, the effects of forskolin, an activator of adenylyl cyclase were evaluated. Forskoline did not affect the resting membrane potentials and the frequency of the spontaneously generated action potentials of the neuron. The resting membrane potential of the identified RP4 neuron was −60.0 91.3 mV (n = 10, mean9 S.E.M.). Sixty min after application of forskolin (100 mM), the resting membrane potential was − 59.790.9 mV (n =3, P =0.67). The spontaneous firing of potentials in control and in preparation pre-treated with forskolin (100 mM) for 60 min were 39.59 1.8 pulses/min (n = 10) and 42.3 92.3 pulses/min (n=3, P =0.5), respectively. No bursting firing of action potentials was observed in forskolin treated preparations (Fig. 4). It seemed that forskoline alone did not elicit the bursting of firing action potentials in the neuron.
Fig. 3. Effects of intracellular injection of 3%-deoxyadenosine on the d-amphetamine elicited bursting firing of action potentials. A, B and C were from the same neuron. (A) the spontaneous action potentials of RP 4 neuron. At the dot, 3%-deoxyadenosine (10 mM) was injected into the RP4 neuron. (B) the potentials 20 min after intracellular injection of 3%-deoxyadenosine (10 mM). C: the potentials 60 min after incubation with d-amphetamine (0.27 mM) from B. The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that d-amphetamine elicited normal bursting firing of action potentials in 3%-deoxyadenosine injected neuron.
Fig. 4. A continuous recording showing the effects of forskolin (100 mM) on the d-amphetamine elicited bursting firing of action potenitals in RP4 neuron. A, B, C, D, E and F were from the same RP4 neuron. (A) Control, the spontaneous action potentials. (B) the potentials 60 min after incubation with d-amphetamine (80 mM). (C) after 40 min incubation with forskolin (100 mM) following d-amphetamine (80 mM) from B. (D) 60 min after washing off forskolin (100 mM) and d-amphetamine (80 mM). (E) the potentials 40 min after perfusion of forskolin (100 mM) from (D). (F) 60 min after further incubation with d-amphetamine (80 mM) and forskolin (100 mM) from (E). The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that neither d-amphetamine (80 mM) nor forskolin (100 mM) alone elicited bursting firing of action potentials in RP4 neuron. However, d-amphetamine (80 mM) elicited bursting firing of action potentials after forskolin (100 mM) pretreatment.
The facilitatory effects of forskoline on the bursting of potentials elicited by d-amphetamine were tested in RP4 neurons pretreated with low concentrations of amphetamine. d-Amphetamine at 0.08 mM did not elicit bursting of potentials in the RP4 neuron even after 4 h of incubation. However, if forskolin (100 mM) was added to the preparation pre-treated with lower concentration of d-amphetamine (0.08 mM), bursting firing of action potentials were found 60 min after perfusion. The bursting firing of action potentials ceased if both forskoline and amphetamine were washed out (Fig. 4). It appeared that neither d-amphetamine (at lower concentration, 80 mM) nor forskolin (100 mM) alone elicited bursting firing of action potentials of the RP4 neuron, while forskolin (100 mM) reversibly facilitated the generation of the
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bursting firing of action potentials elicited by d-amphetamine (80 mM). Similar results were found in 4 other preparations.
3.6. Effects of 8 -bromo-cAMP on d-amphetamine elicited bursting acti6ity To evaluate the effect of the cAMP analog on the generation of the bursting firing of action potentials induced by amphetamine, the effects of 8-bromocAMP, a nonhydrolyzable analog of cAMP, were studied. 8-Bromo-cAMP (3 mM) did not alter the resting membrane potential and the frequency of the spontaneously generated action potentials of the RP4 neuron (Fig. 5). The resting membrane potential of the RP4 neuron in control and 40 min after 8-bromo-cAMP (3 mM) treatment were −60.0 9 1.3 mV (n =10) and − 57.79 1.2 mV (n =3, P =0.38, mean 9S.E.M.), respectively. The frequency of the spontaneously generated action potentials in control preparation and 40 min after 8-bromo-cAMP (3 mM) treatment were 39.591.8 pulses/min (n =10) and 37.7 91.5 pulses/min (n=3, P=0.60), respectively (Fig. 5). d-Amphetamine, at concentration 0.08 mM, did not elicit any bursting activity on the RP4 neuron. However, it did elicit bursting firing of action potentials if 8-bromo-cAMP (3 mM) was added to the preparation for an additional 40 min (Fig. 6). Similar results were found in 3 other preparations. It appeared that neither d-amphetamine (0.08 mM) nor 8-bromo-cAMP (3 mM) alone elicited bursting firing of action potentials in the RP4 neuron. However, 8-bromo-cAMP (3 mM) facilitated the generation of the bursting firing of potentials elicited by d-amphetamine.
3.7. Effects of extracellular application of H7 on the bursting firing of action potentials elicited by d-amphetamine The PKC and inositol 1,4,5-triphosphate (InsP3)
Fig. 6. Effects of 8-bromo-cyclic AMP (3 mM) on d-amphetamine elicited bursting firing of action potentials in RP4 neuron. A, B and C were from the same neuron. (A) Control, the spontaneous action potentials. (B) the potentials 60 min after incubation with d-amphetamine (80 mM). (C) additional 40 min incubation with 8-bromocyclic AMP (3 mM) +d-amphetamine (80 mM) from B. The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that d-amphetamine (80 mM) elicited bursting firing of action potentials only after addition of 8-bromo-cyclic AMP (3 mM).
pathway were two branches of the PI pathway. To examine whether PKC was involved in the generation the bursting firing of action potentials elicited by amphetamine, the effects of H7, an inhibitor of PKC, on the amphetamine-elicited bursting activity of potentials were tested. The resting membrane potential of the RP4 neuron in control and 40 min after H7 (10 mM) application were −60.091.3 mV (n=10, mean9 S.E.M.) and − 57.79 1.2 mV (n=3, P= 0.38), respectively. The frequency of spontaneously generated action potential in control and 40 min after H7 (10 mM) application were 39.59 1.8 pulses/min (n= 10) and 42.09 5.7 pulses/min (n=3, P=0.58), respectively. Thus, H7 did not alter the resting membrane potential and the frequency of spontaneously generated action potentials of the RP4 neuron. Furthermore, H7 did not affect d-amphetamine-mediated bursting firing of action potentials in the RP4 neuron. Similar results were found in 3 other preparations. An example on the effects of H7 on the bursting firing of action potentials elicited by d-amphetamine is shown in Fig. 7.
3.8. Effects of anisomycin on d-amphetamine elicited bursting acti6ity of the RP4 neuron
Fig. 5. Effects of 8-bromo-cyclic AMP (3 mM) on the spontaneously generated potentials of RP4 neuron. (A) Control., the spontaneous action potentials. (B) the potentials 60 min after incubation with 8-bromo-cyclic AMP (3 mM). The horizontal bar on the top left side was the membrane potential at 0 mV. Note that 8-bromo-cyclic AMP (3 mM) did not alter the pattern of the spontaneously generated action potentials of RP 4 neuron.
To elucidate whether new protein synthesis is involved in the generation of the bursting firing of action potentials elicited by d-amphetamine, the effects of anisomycin, an inhibitor of protein synthesis, on the bursting of potentials elicited by d-amphetamine was tested and an example was shown in Fig. 8. Anisomycin (1 mM) did not alter the resting membrane potentials of
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Fig. 7. Effects of H7 (10 mM) on the d-amphetamine elicited bursting firing of action potentials in RP4 neuron. A, B and C were from the same neuron. (A) Control, the spontaneous action potentials. (B) the potentials 40 min after perfusion with H7 (10 mM). (C) 40 min after further incubation with d-amphetamine (0.27 mM)+ H7 (10 mM) to the neuron from (B). The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that d-amphetamine elicited bursting firing of action potentials in the presence of H7.
the neuron. The resting membrane potential of the identified RP4 neuron in control and 3 h after anisomycin (1 mM) treatment were −60.0 9 1.3 mV (n= 10, mean9 S.E.M.) and −58.7 90.3 mV (n= 3, P =0.6), respectively. However, anisomycin significantly decreased the frequency of the spontaneously generated action potentials of the neuron. The frequency of spontaneously generated action potenitials in control and 3 h after anisomycin (1 mM) treatment were 39.59 1.8 pulses/min (n =10) and 18.79 1.3 pulses/min (n=3), (P B0.0001) respectively. No bursting of potentials was found in anisomycin treated preparations. However, anisomycin (1 mM) pretreat-
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Fig. 9. Anisomycin (10 mM) decreased the d-amphetamine elicited bursting firing of action potentials in RP4 neuron. (A), (B) and (C) were from the same neuron. (A) Control, the spontaneous action potentials. (B) the bursting firing of action potentials elicited by d-amphetamine (0.27 mM). (C) 90 min after further incubation with anisomycin (10 mM)+d-amphetamine (0.27 mM) from (B). The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that the bursting firing of action potentials elicited by d-amphetamine (0.27 mM) was blocked by anisomycin (10 mM) treatment.
ment prevented the bursting firing of action potentials elicited by d-amphetamine. In the preparation pretreated with anisomycin (1 mM) for 3 h, subsequent treatment with d-amphetamine (0.27 mM) for additional 90 min did not elicit bursting firing of action potentials. Similar results was found in 3 other preparations. It appeared that anisomycin pre-treatment abolished the generation of bursting firing of action potentials elicited by d-amphetamine. Addition of anisomycin (10 mM) after administration of d-amphetamine also blocked the bursting firing of action potentials elicited by amphetamine (0.27 mM) (Fig. 9).
3.9. Effects of intracellular injection of GDPbs on the d-amphetamine elicited bursting acti6ity
Fig. 8. Effects of anisomycin (10 mM) on d-amphetamine elicited bursting firing of action potentials in RP4 neuron. (A), (B) and (C) were from the same neuron. (A) Control., the spontaneous action potentials. (B) the potentials 3 h after incubation with anisomysin (10 mM). (C) 90 min after further incubation with d-amphetamine (0.27 mM) and anisomycin (10 mM). The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that d-amphetamine (0.27 mM) did not elicite the bursting firing of action potentials in anisomycin pretreated RP4 neuron.
The effect of G protein on the bursting firing of action potentials elicited by amphetamine was tested by intracellular injection of GDPbS, an inhibitor of G protein and the result is shown in Fig. 10. GDPbS was applied by the micropipettes containing 10 mM GDPbS and 100 mM KCl. The resting membrane potentials of the neuron in the control and 20 min after injection testing compound were − 60.09 1.3 mV (n= 10, mean9 S.E.M.) and 60.390.3 mV (n=3, P= 0.60), respectively. The frequency of the spontaneously generated action potentials of the neuron in the control and 20 min after injection of GDPbS were 39.5 91.8 pulses/min (n=10) and 26.093.1 pulse/min (n=3, P B0.001), respectively. The system we used for intracellular injection will provide injection of about 2.6× 10 − 10 l of the solution into the RP4 neuron. The concentrations of GDPbS inside the neuron was
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around 80 mM if we assumed the volume of the RP4 neuron was 3.26 ×10 − 8 l. Amphetamine (0.27 mM) elicited the bursting firing of action potentials in the GDPbS injected neuron. As shown in Fig. 10, 60 min after the addition of d-amphetamine (0.27 mM) in GDPbS injected neuron, bursting of potentials were observed. It appeared that intracellular injection of GDPbS did not alter the d-amphetamine elicited bursting firing of the action potentials. Similar results were found in 3 other preparations.
4. Discussion Amphetamine elicited bursting firing of action potentials in the central snail neuron in a concentration dependent manner. At 80 mM, amphetamine did not elicit bursting firing of action potentials in the RP4 neuron even after 4 h of incubation, while at 0.27 mM, it elicited bursting firing of action potentials 20 min after amphetamine treatment and the bursting of potentials continued for more than 3 h (Tsai and Chen, 1995). Second messengers are intimately related to the functions of the neuron. Evidence suggests the involvement of cAMP in alterations of cellular membrane properties (Hausdorff et al., 1990). For example, cAMP elicited biphasic current in the snail neuron whose activation was mediated through protein phosphorylation (Watanabe and Funase, 1991). It has been shown in many cells that calcium channels may be regulated by cAMP-dependent protein kinase (Penner et al., 1988). Interestingly, forskolin, an activator of adenylyl cyclase (Seamon and Daly, 1981), has been shown to activate
Fig. 10. Effects of intracellular injection of GDPbS on the d-amphetamine elicited bursting activity. (A), (B) and (C) were from the same RP4 neuron. (A) Control, the spontaneous action potentials. (B) the potentials 20 min after injection of GDPbS (10 mM). (C) 40 min after further incubation with d-amphetamine (0.27 mM) to the preparation from (B). The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that d-amphetamine (0.27 mM) elicited normal bursting firing of action potentials in GDPbS injected neuron.
the calcium current of the motor nerve terminal (Tsai et al., 1992). In the present study we found that activation of adenylyl cyclase may facilitate the generation of the bursting firing of action potentials elicited by d-amphetamine. At lower concentrations, e.g. 0.08 mM, d-amphetamine did not elicit bursting firing of action potentials in the snail neuron. However, at this concentration, d-amphetamine did elicit bursting of action potentials if forskolin was added prior to the administration of d-amphetamine. These results suggest that the activity of adenylyl cyclase may be involved in the generation of the bursting firing of action potentials elicited by d-amphetamine. This view was further supported by the observation that 8-bromo-cAMP, an analog of cAMP, also facilitated the generation of the bursting firing of action potentials in the RP4 neuron pretreated with subthreshold concentration of d-amphetamine (0.08 mM). However, the precise relationship between adenylyl cyclase and the bursting firing of action potentials elicited by d-amphetamine needs further study. Though intracellular injection of 3%-deoxyadenosine, an adenylyl cyclase inhibitor (Hidaka et al., 1984), did not abolish the bursting firing of action potentials elicited by d-amphetamine. The time course suggested that it exhibited a transient effect, lasting about 20–30 min. Thus, the inability of 3%-deoxyadenosine to alter the effect of d-amphetamine may be due to its rapid metabolic inactivation, which suggest that 3%-deoxyadenosine, is not an appropriate inhibitor for in vivo experiments. The cAMP-dependent protein phosphorylation, probably plays an essential role on the d-amphetamine elicited bursting firing of action potentials in the RP4 neuron. Pretreatment with H8, an inhibitor of PKA (Funase et al., 1993), reversibly blocked the d-amphetamine-elicited bursting firing of action potentials. However, once the bursting firing of action potentials was elicited by d-amphetamine, H8 could no longer block the effect of d-amphetamine. It is interesting to note that cAMP-dependent protein phosphorylation appears to play an important role on the bursting pacemaker activity in the serotonin activated neuron because H8 significantly decreased the serotonin elicited ionic current in the PON neuron of the snail (Funase et al., 1993). PKC is a family of plasma-membrane protein kinases. In the absence of hormone stimulation, PKC is present as a soluble cytosolic protein that is catalytically inactive. A rise in the cytosolic Ca2 + level causes PKC to bind to the cytoplasmic leaflet of the plasma membrane, where it can be activated by membrane-associated 1,2-diacylglycerol (DAG). Thus activation of PKC depends on both the Ca2 + ion and DAG, suggesting an interaction between the two branches of the inositol lipid signaling pathway (Nishizuka, 1992). In the present study, we found that H7, a PKC inhibitor
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(Singh et al., 1994; Duraj et al., 1995), did not block the bursting firing of action potentials induced by d-amphetamine. These results suggested that the bursting firing of action potentials elicited by d-amphetamine probably was not associated with the inositol lipid signaling pathway in the neuron. G-protein plays an important role on the functions of the gastropod. The potassium channels were opened through activation of the pertussis toxin-sensitive G protein, Gi or Go (Takahashi et al., 1989) and voltagedependent sodium channels were opened through activation of the cholera toxin-sensitive G protein (Kudo et al., 1991). However, in the present study, we found that intracellular injection of GDPbS, a G protein inhibitor (Chiba et al., 1992), did not affect the bursting firing of action potentials elicited by d-amphetamine. The data suggested that the d-amphetamine elicited bursting firing of action potentials may not involve G protein in the neuron. Anisomycin, a protein synthesis inhibitor (Schacher et al., 1988), alone did not elicit bursting firing of action potential in the RP4 neuron. However, anisomycin did block the bursting firing of action potentials mediated by d-amphetamine. The mechanism of the blocking effects remained to be determined. Anisomycin blocked the protein phosphorylation in sensory neurons of Aplysia (Homayouni et al., 1995) and the regulatory subunits of the Aplysia cAMP-dependent protein kinase (Bergold et al., 1990). It is interesting to note that P70, a specific protein found in the cobalt-induced epileptogenic focus of the rat cerebral cortex, induced bursting activity of action potentials when intracellularly injected into the identified neuron of the snail Euhadra peliomphala (Onozuka et al., 1991b). cAMP-dependent protein kinases possess multiple signal transduction pathways (Walsh and Van Patten, 1994). The role of the phosphorylation of the regulatory subunits of cAMP-dependent protein kinase on the d-amphetamine elicited bursting firing of action potentials remain an interesting subject for further study. Together, our data indicated that d-amphetamine elicited bursting firing of action potentials in the snail RP4 neuron. The bursting of action potentials may be associated with the cAMP dependent kinase system. On the contrary, the effect may not be associated with PKC and G protein activity in the neuron.
Acknowledgements We are grateful to Professor Ted H Chiu (Department of Pharmacology, Medical College of Ohio, USA) for his geneous comments and suggestions of the manuscript and to Dr RL Walsh (Research Technology Branch, National Institute of Drug Abuse, USA) for the generous supply of d-amphetamine. This work was
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supported by grants, NSC-85-2331-B-002-289 and NSC-86-2341-B-002-329, from the National Science Council, Taipei, Taiwan.
References Arvanov, V.L., Chen, R.C., Chen, Y.H., Liou, H.H., Chan, Y.L., Arvanov, V.A., Tsai, M.C. (1994) Modulation of pentylenetetrazol induced bursting activity by electrogenic Na pump in Achatina fulica neurons. Asia Pacific J. Pharmacol., 9: 37–42. Bergold, P.J., Sweatt, J.D., Winicov, I., Weiss, K.R., Kandel, E.R. and Schwartz, J.H. (1990) Protein synthesis during acquisition of long-term facilitation is needed for the persistent loss of regulatory subunits of the Aplysia cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA, 87: 3788 – 3791. Chen, Y.H. and Tsai, M.C. (1996) Bursting firing of action potential in central snail neuron elicited by d-amphetamine: role of calcium ions. Comp. Physiol. Pharmacol., 115: 195 – 205. Chiba, O., Sasaki, K., Higuchi, H. and Takashima, K. (1992) G Protein mediating the slow depolarization induced by FMRFamide in the ganglion cells of Aplysia. Neurosci. Res., 15: 255 – 254. Duraj, J., Kovacikova, M., Sedlak, J., Koppel, J., Sobel, A. and Chorvath, B. (1995) The protein kinase C inhibitor H7 blocks phosphorylation of stathmin during TPA-induced growth inhibition of human pre-B leukemia REH6 cells. Leukemia Res., 19: 457 – 461. Ferrendelli, J.A. and Kinscherf, D.A. (1977) Cyclic nucleotides in epileptic brain: effects of pentylenetetrazol on regional cyclic AMP levels in vivo. Epilepsia, 18: 525 – 531. Funase, K., Watanabe, K. and Onozuka, M. (1993) Augmentation of bursting pacemaker activity by serotonin in an identified Achatina fulica neurone: an increase in sodium- and calcium-activated negative slope resistance via cyclic-AMP dependent protein phosphorylation. J. Exp. Biol., 175: 33 – 44. Hausdorff, W.P., Caron, M.G. and Lefkowitz, R.J. (1990) Turning off the signal: desensitization of b-adrenergic receptor function. FASEB J., 4: 2881 – 2889. Hidaka, H., Inagaki, M., Kawamoto, S., Sasaki, Y. (1984) Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry, 23: 5036 – 5041. Homayouni, R., Byrne, J.H. and Eskin, A. (1995) Dynamic of protein phosphorylation in sensory neurons of Aplysia. J. Neurosci., 15: 429 – 438. Kerkut, G.A., Lambert, J.D.C., Gayton, R.J., Loker, J.E. and Walker, R.J. (1975) Mapping of nerve cells in the subesophageal ganglia of Helix aspersa. Comp. Biochem. Physiol., 50: 1–25. Kudo, A., Sasaki, K., Tamazawa, Y. and Matsumoto, M. (1991) A slow voltage-dependent Na + -current induced by 5-hydroxytryptamine and the G-protein-coupled activation mechanism in the ganglion cells of Aplysia. Jpn. J. Physiol., 41: 259-275. Kurahashi, T. and Kaneko, A. (1993) Gating properties of the cAMP- gated channel in toad olfactory receptor cells. J. Physiol., 466: 287 – 302. Levistre, R., Berguerand,M., Bereziat, G. and Masliah, J. (1995) The cross-regulation of Gi-protein by cholera toxin involves a phosphorylation by protein kinase A. Biochem. J., 306: 765–769. Levitan, E.S. and Levitan, I.B. (1988) Serotonin acting via cyclic AMP enhances both the hyperpolarizating and depolarizing phase of bursting pacemaker activity in the Aplysia neuron R 15. J. Neuroscience, 8: 1152 – 1161. Magoski, N.S., Bauce, L.G., Syed, N.I. and Bulloch, A.G.M. (1995) Dopaminergic transmission between identified neurons from the mollusk, Lymnaea stagnalis. J. Neurophysiol., 74: 1287–1300.
Y.H. Chen, M.C. Tsai / Neuroscience Research 27 (1997) 295–304
304
Nishizuka, Y. (1992) Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science, 258: 607 – 614. Onozuka, M., Kishii, K., Furuchi, H. and Sugaya, E. (1983) Behavior of intracellular cyclic nucleotide and calcium in pentylenetetrazole-induced bursting activity in snail neurons. Brain Res. 269: 277 – 286. Onozuka, M., Kubo, K.A. and Ozono, S. (1991a) The molecular mechanism underlying pentylenetetrazole-induced bursting activity in Euhadra neurons: involvement of protein phosphorylation. Comp. Biochem. Physiol., 100: 423–432. Onozuka, M., Imai, S., Furuichi, H. and Ozono, S. (1991b) A specific 70K protein found in epileptic rat cortex: induction of bursting activity and negative resistance by its intracellular application in Euhadra neurons. J. Neurobiol., 22: 287–297. Onozuka, M., Imai, S. and Sugaya, E. (1986) Pentylenetetrazol induced bursting activity and cellular protein phosphorylation in snail neurons. Brain Res., 362: 33–39. Penner, R., Mathews, G. and Neher, E. (1988) Regulation of calcium influx by second messengers in rat mast cells. Nature, 334: 499 – 504. Price, C.J. and Goldberg, J.I. (1993) Serotonin activation of a cyclic AMP-dependent sodium current in an identified neuron from Helisoma trivolvis. J. Neurosci. 13: 4979–4987. Rees, W.J. (1950) The giant African snail. Proc. Zool. Soc. London, 120: 577 – 599. Rhee, S.G. and Choi, K.D. (1992) Mutiple forms of phospholipase C isozymes and their activation mechanisms. In: J.W. Putney Jr (Ed.), Advances in Second Messenger and Phsophoprotein Research, Vol. 26, Raven Press, New York, 1992, pp. 35 – 61. Schacher, S., Castellucci, V.F. and Kandel, E.R. (1988) cAMP evokes long-term facilitation in Aplysia sensory neurons that requires new protein synthesis. Science, 24: 1667–1669. Seamon, K.B. and Daly, J.W. (1981) Forskolin: a unique diterpene activator of cyclic AMP-generating systems. J. Cyclic Nucleotide Res., 7: 201 – 224. Singh, V.K., Cheng, J.F. and Leu, S.J.C. (1994) Effect of substance P and protein kinase inhibitors on b-amyloid peptide-induced proliferation of cultured brain cells. Brain Res., 660: 353 – 356. Slater, N.T., Hall, A.F. and Carpenter, D.O. (1984) Kinetic properties of cholinergic desensitization in Aplysia neurons. Proc. R. Soc. London B. Biol. Sci., 223: 63–78.
Sugaya, E., Furuichi, H., Takagi, T., Kajiwara, K. and Komatsubara, J. (1987) Intracellular calcium concentration during pentylenetetrazol-induced bursting activity in snail neurons. Brain Res., 416: 183 – 186. Sugaya, E., Goldring, S. and O’Leary, J.L. (1964) Intracellular potentials discharge in cat. Electroencephalogr. Clin. Neurophysiol., 17: 661 – 669. Sugaya, E. and Onozuka, M. (1978a) Intracellular calcium: its movement during pentylenetetrazol-induced bursting activity. Science, 200: 797 – 799. Sugaya, E. and Onozuka, M. (1978b) Intracellular calcium: its release from granules during bursting activity in snail neurons. Science, 202: 1195 – 1197. Takahashi, J., Sasaki, K. and Matsumoto, M. (1989) The gating mechanism of K + -channels coupled to the FMRFamide receptor in the ganglion cells of Aplysia. Nippon Serigaku Zasshi (J. Physiol. Soc. Jpn.), 51: 363 – 378. Takeuchi, H., Araki, Y., Emaduddin, M. et al. (1996) Identifiable Achatina giant neurones: their localizations in ganglia, axonal pathways and pharmacological features. Gen. Pharmacol., 27: 3 – 32. Taussig, R. and Gilman, A.G. (1995) Mammalian membrane-bound adenylyl cyclases. J. Biol. Chem., 270: 1 – 4. Tsai, M.C. and Chen, M.L. (1989) A new method for screening anticonvulsants: 1. effects of anticonvulsants on pentylenetetrazolinduced neuronal activity of the giant African snail, Achatina fulica Ferussac. Asia Pacific J. Pharmacol., 4: 203 –207. Tsai, M.C., Chen, M.L., Su, J.L., Hsieh, W.H. and Fan, S.Z. (1992) Effects of a phorbol ester and forskolin on nerve terminal currents in mouse motor nerves. Asia Pacific J. Pharmacol., 7: 27 – 31. Tsai, M.C. and Chen, Y.H. (1995) Bursting firing of action potential in central snail neuron elicited by d-amphetamine: role of electrogenic sodium pump. Comp. Physiol. Pharmacol., 111: 131– 141. Walsh, D.A. and Van Patten, S.M. (1994) Multiple pathway signal transduction by the cAMP-dependent protein kinase. FASEB J., 8: 1227 – 1236. Watanabe, K. and Funase, K. (1991) Cyclic AMP elicits biphasic current whose activation is mediated through protein phosphorylation in snail neurons. Neurosci. Res., 10: 64 – 70.
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Bursting firing of action potentials in central snail neurons elicited by d-amphetamine: role of cytoplasmic second messengers Yi Hung Chen, Ming Cheng Tsai * Department of Pharmacology, College of Medicine, National Taiwan Uni6ersity, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan Received 15 October 1996; accepted 27 December 1996
Abstract The role of the intracellular second messengers on the bursting firing of action potentials in central snail neurons elicited by d-amphetamine was studied in the identified RP4 neuron of the African snail Achatina fulica Ferussac. Oscillation of membrane potential and bursting firing of action potentials were elicited by d-amphetamine in a concentration dependent manner. The bursting firing of action potentials was 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-isoquinolinesulphonyl)-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 intracellular injection of 3%-deoxyadenosine, an adenylyl-cyclase inhibitor. These results suggested that the bursting firing of action potentials elicited by d-amphetamine in snail neurons may be associated with the cyclic adenosine monophosphate (cAMP) second messenger system; on the other hand it may not be associated with the G protein and protein kinase C activity. © 1997 Elsevier Science Ireland Ltd. Keywords: Amphetamine; Second messenger; Drug abuse; Snail; Central neuron; Anisomycin; Action potential; cAMP
1. Introduction The central ganglia of the snail (Rees, 1950) contains many identifiable neurotransmitters and receptors (Kerkut et al., 1975; Takeuchi et al., 1996). Convulsants such as pentylenetetrazole-induced bursting activity of action potentials in the central neurons of the snail (Ferrendelli and Kinscherf, 1977; Sugaya and Onozuka, 1978a,b; Sugaya et al., 1987; Onozuka et al., 1983, 1986; 1991a; Tsai and Chen, 1989; Arvanov et al., 1994). The response strongly resembled the pentylenetrazole-induced seizure changes in cerebral cortical neurons of mammals (Sugaya et al., 1964). We have shown that * Corresponding author. Tel.: +886 2 3966786; fax: + 886 2 3915297; e-mail: [email protected]
amphetamine elicited bursting firing of action potentials in the central RP4 neuron of the African snail Achatina fulica Ferussac (Tsai and Chen, 1995). This bursting activity was not blocked in high magnesium medium or after continuous perfusion of propranolol, prazosin, haloperidol, phenobarbital, hexamethonium, d-tubocurarine, atropine or calcium free solution containing EDTA or verapamil. These results suggested that the bursting activity of potentials elicited by d-amphetamine was not due to (1) the synaptic effects of neurotransmitters or (2) the activity of cholinergic or adrenergic receptors of the excitable membrane (Tsai and Chen, 1995). However, it was associated with intracellular calcium ions because the bursting firing of action potentials elicited by amphetamine was reduced following intracellular injection with EGTA (Chen and Tsai, 1996).
0168-0102/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 8 - 0 1 0 2 ( 9 7 ) 0 1 1 5 9 - 0
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The cytoplasmic concentration of Ca2 + is regulated by regulation of several distinct Ca2 + -specific channels in the plasma membrane and its release from intracellular storage sites. Ca2 + channels may be opened by electrical depolarization, phosphorylation, cyclic adenosine monophosphate (cAMP)-dependent protein kinase, protein kinase C (PKC), G protein, etc. Since cytoplasmic second messengers play important roles in the functions of the neuron, the aim of the present study was to elucidate the interaction between the amphetamine-elicited bursting firing of action potentials and the second messengers in the snail central neuron. Evidence suggests that cAMP may alter membrane properties (Hausdorff et al., 1990). For example, cAMP elicited biphasic current in snail neurons whose activation was mediated through protein phosphorylation (Watanabe and Funase, 1991); cAMP elicited gating currents in toad olefactory cells (Kurahashi and Kaneko, 1993). In addition, cAMP-dependent protein kinase and PKC have been shown to modulate membrane Ca2 + and K + channels (Penner et al., 1988). Forskolin, a known activator of adenylate cyclase (Seamon and Daly, 1981; Levitan and Levitan, 1988), was found to activate the Ca2 + current of the motor nerve terminal (Tsai et al., 1992). To elucidate the role of adenylate cyclase and cAMP dependent protein kinase in the generation of the bursting activity induced by amphetamine, the effects of adenylyl cyclase activator, e.g. forskolin and inhibitor, e.g. 3%-deoxyadenosine on the bursting potentials were examined. The effects of a specific protein kinase A (PKA) inhibitor, N-(2-methylamino) ethyl-3-isoquinoline sulphonamide dihydrochloride (H8) (Levistre et al., 1995) and 8-bromo-cyclic AMP, a nonhydrolyzable analog of cAMP (Price and Goldberg, 1993) on the amphetamine-elicited bursting potentials were studied. Release of Ca2 + from intracellular stores may be modulated by inositol 1,4,5-triphosphate (IP3) which is produced from the membrane lipid, catalyzed by phospholipase C (Rhee and Choi, 1992). Both the PKC and IP3 pathways are branches of the PI pathway. To test whether PKC was involved in the generation of the bursting firing of action potentials mediated by amphetamine, the effects of a specific PKC inhibitor, 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride (H7) (Duraj et al., 1995), on the amphetamine elicited bursting activity of potentials were tested. G-proteins has been demonstrated to modulate the activity of adenylyl cyclase (Taussig and Gilman, 1995). Therefore, the roles of G protein and newly synthesized proteins on the bursting of action potentials induced by amphetamine were also examined with guanosine 5%-O2-thiodiphohsphate (GDPbS), an inhibitor of G protein (Chiba et al., 1992; Magoski et al., 1995) and
with anisomycin, an inhibitor of protein synthesis (Schacher et al., 1988). Our results indicated that the d-amphetamine elicited bursting of action potentials in the snail neuron may be associated with intracellular activity of cAMP and newly synthesized protein(s). In contrast, they may not be associated with G protein or PKC.
2. Methods
2.1. Electrophysiological recordings Experiments were performed on identified RP4 neuron from the subesophageal ganglia of the African snail A. fulica. The ganglia were pinned to the bottom of a 0.7 ml sylgard-coated perfusion chamber and carefully freed from the connective tissue sheath to allow easy identification and penetration by microelectrodes. For measuring the potentials, two microelectrodes penetrated into the neuron. The recording electrode (5 MV) was filled with 3 M potassium acetate (KAc) and the other electrode was filled with injection solutions for intracellular injection. The experimental chamber was perfused with control saline i.e. NaCl (mM) 85, KCl 4.0, CaCl2 8, MgCl2 7, Tris–HCl 10 (pH, 7.5) at room temperature of 23–24°C. The potentials were amplified using Axoclamp 2A amplifier and monitored on Tektronic oscilloscopes (Type 5441 and 5110). Neurons were studied only if they exhibited resting membrane potentials more negative than − 50 mV (Tsai and Chen, 1989; 1995), the time constant at around 5–8 ms and the rate of rise of the action potentials at around 5–8 DV/s. For the voltage clamp study, the neurons were clamped by means of an Axoclamp 2A amplifier. For the recording of membrane currents, the neurons were maintained at a standard potential of −75 mV. Current and voltage electrodes (5 MV) were filled with 3 M KAc. In order to eliminate the chloride effects from the electrodes during recording, acetate was used for the intracellular recording of the whole cell preparation (Tsai and Chen, 1995). Microperfusion was used for the rapid application of agonists. The perfusion tube, 0.9 mm inside diameter, was placed on the top of the neuron under microscopic control. This system allows rapid exchange of the bathing medium without apparent mechanical disturbance of the neuron under study (Slater et al., 1984). All potentials were recorded on tape via a digitalizing unit (Digidata 1200) and analyzed using a pCLAMP system. The mean amplitude of the potentials and the resting membrane potentials after various treatments were compared with the pre-drug control by means of Student’s two-tailed t-test. Differences were considered significant at PB 0.05.
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2.2. Intracellular injection For intracellular injections, 3%-deoxyladenosine or guanosine 5%-O-2-thiodiphohsphate (GDPbS) was dissolved in 100 mM KCl (Watanabe and Funase, 1991), and the intracellular injection solutions were filled into the micropipettes with a tip diameter of about 1 mm adjusted with a microforge (Narishige MF-83). Drugs were buffered to pH 7.5 with KOH or HCl. Injections were made with a pulse under manual control over the course of the experiment. The duration of each injection pulse is 2 s with a pressure of 200 mmHg. The diameter of the neuron was measured with a micrometer, assuming the shape of the neuron was spherical. The volumes of solutions injected into the neuron were measured by the application of similar pressure and duration to the pipettes containing solutions before impalement. The droplets on the edge of the micropipettes was measured with a micrometer, assuming the shapes of droplets were spherical. According to our previous work, the diameters of the RP4 neuron and the droplets were about 400 and 80 mm, respectively. The volume of the neuron and the volume of solution injected into the neuron were around 3.26 × 10 − 8 and 2.6 × 10 − 10 l, respectively (Tsai and Chen, 1989).
3. Results
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3.2. Effect of extracellular d-amphetamine on RP4 neuron The identified neuron in the right parietal ganglion, RP4 neuron had a prominent response to d-amphetamine. Twenty min after extracellular perfusion of d-amphetamine (0.027 mM), the frequency of spontaneously firing action potentials was reduced by 28% (from 39.591.8 pulses/min, n= 10) to 28.391.2 pulses/min, n=3, P= 0.003). Higher concentration of d-amphetamine, e.g. 0.08 mM, further decreased the frequency of the action potentials by 45% to 21.6 91.8 pulses/min, n =3, PB 0.001). An intermittent firing pattern was also observed. No bursting firing of action potentials was observed even after 4 h of incubation. However, 20 min after increasing the extracellular damphetamine concentration to 0.27 mM, the firing pattern changed from regularly spaced single spikes to one in which bursts of 2–20 action potentials were separated by large hyperpolarization of membrane potential (up to 8 mV) lasting 1–40 s (Fig. 1). Oscillation of membrane potentials with a phasic depolarization was followed by a sustained depolarization. The resting membrane potential was phasically depolarized from − 61.49 0.9 mV (n=11) to − 57.69 1.0 mV (n=11, PB 0.05). The effect of d-amphetamine continued throughout its application (up to 3 h). The membrane potential also showed a phasic depolarization followed by a sustained depolarization. The sustained depolar-
3.1. Some electrical characteristics of the identifiable RP4 neuron The resting membrane potential (RMP) of the identified RP4 neuron was −60.0 91.3 mV (n= 10, mean9 S.E.M.) and it showed a spontaneous firing frequency of about 39.591.8 pulses/min (n= 10) (Tsai and Chen, 1995). The mean amplitude of the spontaneously generated action potentials was 87.3 9 1.8 mV (n=10). The time constant of the action potential was 6.690.7 ms (n =5) and the rate of rise of the action potentials was 5.7 9 0.7 DV/s (n = 5). The neuron was sensitive to several neurotransmitters. Acetylcholine and g-aminobutyric acid (GABA) induced hyperpolarization of the membrane potential and decreased the frequencies of the spontaneous firing of action potentials (results not shown). GABA and ACh also elicited inward currents in the voltage clamped neuron. Glutamic acid and dopamine increased the frequency of the spontaneous action potentials. Serotonin induced biphasic responses, i.e. an initial hyperpolarization of membrane potential followed by an increase in the frequency of action potentials.
Fig. 1. Effects of d-amphetamine on a central RP 4 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 RP-4 neuron after 40 min of d-amphetamine (80 mM) administration. (C) bursting firing of action potentials of the neuron after 40 min of d-amphetamine (0.27 mM) administration. (D) the potentials after 75 min washing off d-amphetamine (0.27 mM). The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that d-amphetamine at 80 mM did not, while d-amphetamine at 0.27 mM did, elicit bursting firing of action potentials of RP4 neuron.
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ization reached −24.8 92.3 mV (n =9) 3 h after 0.27 mM d-amphetamine application. A sudden depolarization with a train of spike changes on its rising phase was observed. This was followed by a long fall to a hyperpolarization level ( − 69.4 9 1.4 mV, n = 11, PB 0.001). The train of spike changes on the rising phase was reduced after 2 or 3 h of d-amphetamine perfusion. The effect of d-amphetamine on the neuronal activities was reversible. After 120 min, of continuous washing, the spontaneously generated spikes of the central neuron almost returned to the control level albeit with a slower frequency of spontaneous firing. An example of the effects of d-amphetamine on the action potentials of the snail neuron are shown in Fig. 1.
3.3. Effects of H8 on d-amphetamine elicited bursting acti6ity of the RP4 neuron To examine the role of cAMP-dependent protein kinase in the generation of the bursting activity elicited by d-amphetamine, the effects of H8, an inhibitor of PKA, were studied. H8 did not alter the resting membrane potentials of the RP4 neuron. The resting membrane potentials of the RP4 neuron in saline was − 60.0 9 1.3 mV (n= 10, mean 9S.E.M.). Sixty min after 10 mM H8 treatment, the RMP of the neuron was −59.0 90.6 mV (n= 10, P= 0.5). However, H8 decreased the frequency of the spontaneously generated action potentials of the RP4 neuron from normal saline control of 39.59 1.8 (n= 10) to 8.79 0.7 pulses/min (n =10, P B0.001) 60 min after 10 mM H8 treatment. The frequency returned to the control level after 40 min of washing. It should be pointed out that H8 did not alter the amplitudes of the action potentials. An example of the effects of H8 on the spontaneously generated action potentials of the RP4 neuron are shown in Fig. 2. H8 blocked the bursting firing of action potentials prior to but not after the addition of d-amphetamine. Amphetamine (0.27 mM) elicited bursting of action potentials 20 min after perfusion. Subsequent addition of 10 mM H8 in the amphetamine-containing perfusion medium did not alter the effect of amphetamine even after 3 h of further incubation with H8 and amphetamine. However, in the preparations pretreated with H8 perfusion medium containing H8 (10 mM) for 40 min, subsequent concomitant treatment of neurons with d-amphetamine (0.27 mM) and H8 (10 mM) did not induce bursting activity for more than 60 min. The d-amphetamine elicited bursting firing of action potentials returned if H8 was washed off. The effects of pre-administration of H8 on the amphetamine elicited bursting firing of action potentials were shown in Fig. 2. Similar results were found in 10 other preparations. It appears that H8 significantly blocked the bursting firing of action potentials elicited by amphetamine if H8 was pre-administered to the preparation.
Fig. 2. Effects of H8 (10 mM) on the d-amphetamine elicited bursting firing of action potentials. A, B, C, D and E were from the same neuron. (A) Control, showing the spontaneous action potentials of RP-4 neuron. (B) the potentials after 40 min of H8 (10 mM) administration. (C) and (D) were potentials 25 and 60 min, respectively, after further incubation with d-amphetamine (0.27 mM) and H8 (10 mM). (E) 60 min after washing off H8 (10 mM) from (D). The horizontal bar on the top left side was the membrane potential at 0 mV. Note that amphetamine elicited bursting firing of action potentials which were blocked with H8 pre-treatment.
3.4. Effect of intracellular injection of 3 %-deoxyadenosine on the d-amphetamine elicited bursting of potentials To test the role of adenylyl cyclase in the generation of the bursting activity of potentials elicited by amphetamine, 3%-deoxyadenosine, an inhibitor of adenylyl cyclase was studied. The results were shown in Fig. 3. Injection micropipettes contained 10 mM 3%-deoxyadenosine and 100 mM KCl. 3%-Deoxyadenosine reversibly depolarized the resting membrane potentials of the RP4 neuron. Immediately after 3%-deoxyadenosine injection, the resting membrane potential of the RP4 neuron was depolarized from −60.190.6 mV (n= 3) to −44.29 0.7 mV (n=3, PB0.001). The frequency of the spontaneously generated action potentials were increased from 439 3 (n= 5) to 130910 pulse/min (n= 5, PB0.001) 30 s after intracellular injection (Fig. 3). Twenty min after intracellular injection, the resting membrane potentials returned to the control value of −59.09 1.6 mV (n= 4), although the frequency of the spontaneously generated action potentials was still significantly elevated (62.0 92.5 pulses/ min, n= 4, P B0.001). In our system, the volume injected was about 2.6910 − 10 l, resulting in the concentration of 3%-deoxyadenosine inside the neuron of around 80 mM if we assumed the volume of the RP4 neuron was 3.26× 10 − 8 l (Tsai and Chen, 1995). If the 3%-deoxyadenosine injected RP4 neuron was further in-
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cubated with amphetamine (0.27 mM) for 60 min, an intermittent firing pattern of the potentials was observed. However, the oscillation of the membrane potential of bursting activity elicited by amphetamine was not observed. The resting membrane potential was not altered following amphetamine treatment. Similar results were found in 3 other preparations. An example of the effect of 3%-deoxyadenosine on the bursting activity is shown in Fig. 3.
3.5. Effects of forskolin on the d-amphetamine elicited bursting of action potentials To test whether adenylyl cyclase was involved in the generation of the bursting firing of action potentials elicited by amphetamine, the effects of forskolin, an activator of adenylyl cyclase were evaluated. Forskoline did not affect the resting membrane potentials and the frequency of the spontaneously generated action potentials of the neuron. The resting membrane potential of the identified RP4 neuron was −60.0 91.3 mV (n = 10, mean9 S.E.M.). Sixty min after application of forskolin (100 mM), the resting membrane potential was − 59.790.9 mV (n =3, P =0.67). The spontaneous firing of potentials in control and in preparation pre-treated with forskolin (100 mM) for 60 min were 39.59 1.8 pulses/min (n = 10) and 42.3 92.3 pulses/min (n=3, P =0.5), respectively. No bursting firing of action potentials was observed in forskolin treated preparations (Fig. 4). It seemed that forskoline alone did not elicit the bursting of firing action potentials in the neuron.
Fig. 3. Effects of intracellular injection of 3%-deoxyadenosine on the d-amphetamine elicited bursting firing of action potentials. A, B and C were from the same neuron. (A) the spontaneous action potentials of RP 4 neuron. At the dot, 3%-deoxyadenosine (10 mM) was injected into the RP4 neuron. (B) the potentials 20 min after intracellular injection of 3%-deoxyadenosine (10 mM). C: the potentials 60 min after incubation with d-amphetamine (0.27 mM) from B. The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that d-amphetamine elicited normal bursting firing of action potentials in 3%-deoxyadenosine injected neuron.
Fig. 4. A continuous recording showing the effects of forskolin (100 mM) on the d-amphetamine elicited bursting firing of action potenitals in RP4 neuron. A, B, C, D, E and F were from the same RP4 neuron. (A) Control, the spontaneous action potentials. (B) the potentials 60 min after incubation with d-amphetamine (80 mM). (C) after 40 min incubation with forskolin (100 mM) following d-amphetamine (80 mM) from B. (D) 60 min after washing off forskolin (100 mM) and d-amphetamine (80 mM). (E) the potentials 40 min after perfusion of forskolin (100 mM) from (D). (F) 60 min after further incubation with d-amphetamine (80 mM) and forskolin (100 mM) from (E). The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that neither d-amphetamine (80 mM) nor forskolin (100 mM) alone elicited bursting firing of action potentials in RP4 neuron. However, d-amphetamine (80 mM) elicited bursting firing of action potentials after forskolin (100 mM) pretreatment.
The facilitatory effects of forskoline on the bursting of potentials elicited by d-amphetamine were tested in RP4 neurons pretreated with low concentrations of amphetamine. d-Amphetamine at 0.08 mM did not elicit bursting of potentials in the RP4 neuron even after 4 h of incubation. However, if forskolin (100 mM) was added to the preparation pre-treated with lower concentration of d-amphetamine (0.08 mM), bursting firing of action potentials were found 60 min after perfusion. The bursting firing of action potentials ceased if both forskoline and amphetamine were washed out (Fig. 4). It appeared that neither d-amphetamine (at lower concentration, 80 mM) nor forskolin (100 mM) alone elicited bursting firing of action potentials of the RP4 neuron, while forskolin (100 mM) reversibly facilitated the generation of the
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bursting firing of action potentials elicited by d-amphetamine (80 mM). Similar results were found in 4 other preparations.
3.6. Effects of 8 -bromo-cAMP on d-amphetamine elicited bursting acti6ity To evaluate the effect of the cAMP analog on the generation of the bursting firing of action potentials induced by amphetamine, the effects of 8-bromocAMP, a nonhydrolyzable analog of cAMP, were studied. 8-Bromo-cAMP (3 mM) did not alter the resting membrane potential and the frequency of the spontaneously generated action potentials of the RP4 neuron (Fig. 5). The resting membrane potential of the RP4 neuron in control and 40 min after 8-bromo-cAMP (3 mM) treatment were −60.0 9 1.3 mV (n =10) and − 57.79 1.2 mV (n =3, P =0.38, mean 9S.E.M.), respectively. The frequency of the spontaneously generated action potentials in control preparation and 40 min after 8-bromo-cAMP (3 mM) treatment were 39.591.8 pulses/min (n =10) and 37.7 91.5 pulses/min (n=3, P=0.60), respectively (Fig. 5). d-Amphetamine, at concentration 0.08 mM, did not elicit any bursting activity on the RP4 neuron. However, it did elicit bursting firing of action potentials if 8-bromo-cAMP (3 mM) was added to the preparation for an additional 40 min (Fig. 6). Similar results were found in 3 other preparations. It appeared that neither d-amphetamine (0.08 mM) nor 8-bromo-cAMP (3 mM) alone elicited bursting firing of action potentials in the RP4 neuron. However, 8-bromo-cAMP (3 mM) facilitated the generation of the bursting firing of potentials elicited by d-amphetamine.
3.7. Effects of extracellular application of H7 on the bursting firing of action potentials elicited by d-amphetamine The PKC and inositol 1,4,5-triphosphate (InsP3)
Fig. 6. Effects of 8-bromo-cyclic AMP (3 mM) on d-amphetamine elicited bursting firing of action potentials in RP4 neuron. A, B and C were from the same neuron. (A) Control, the spontaneous action potentials. (B) the potentials 60 min after incubation with d-amphetamine (80 mM). (C) additional 40 min incubation with 8-bromocyclic AMP (3 mM) +d-amphetamine (80 mM) from B. The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that d-amphetamine (80 mM) elicited bursting firing of action potentials only after addition of 8-bromo-cyclic AMP (3 mM).
pathway were two branches of the PI pathway. To examine whether PKC was involved in the generation the bursting firing of action potentials elicited by amphetamine, the effects of H7, an inhibitor of PKC, on the amphetamine-elicited bursting activity of potentials were tested. The resting membrane potential of the RP4 neuron in control and 40 min after H7 (10 mM) application were −60.091.3 mV (n=10, mean9 S.E.M.) and − 57.79 1.2 mV (n=3, P= 0.38), respectively. The frequency of spontaneously generated action potential in control and 40 min after H7 (10 mM) application were 39.59 1.8 pulses/min (n= 10) and 42.09 5.7 pulses/min (n=3, P=0.58), respectively. Thus, H7 did not alter the resting membrane potential and the frequency of spontaneously generated action potentials of the RP4 neuron. Furthermore, H7 did not affect d-amphetamine-mediated bursting firing of action potentials in the RP4 neuron. Similar results were found in 3 other preparations. An example on the effects of H7 on the bursting firing of action potentials elicited by d-amphetamine is shown in Fig. 7.
3.8. Effects of anisomycin on d-amphetamine elicited bursting acti6ity of the RP4 neuron
Fig. 5. Effects of 8-bromo-cyclic AMP (3 mM) on the spontaneously generated potentials of RP4 neuron. (A) Control., the spontaneous action potentials. (B) the potentials 60 min after incubation with 8-bromo-cyclic AMP (3 mM). The horizontal bar on the top left side was the membrane potential at 0 mV. Note that 8-bromo-cyclic AMP (3 mM) did not alter the pattern of the spontaneously generated action potentials of RP 4 neuron.
To elucidate whether new protein synthesis is involved in the generation of the bursting firing of action potentials elicited by d-amphetamine, the effects of anisomycin, an inhibitor of protein synthesis, on the bursting of potentials elicited by d-amphetamine was tested and an example was shown in Fig. 8. Anisomycin (1 mM) did not alter the resting membrane potentials of
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Fig. 7. Effects of H7 (10 mM) on the d-amphetamine elicited bursting firing of action potentials in RP4 neuron. A, B and C were from the same neuron. (A) Control, the spontaneous action potentials. (B) the potentials 40 min after perfusion with H7 (10 mM). (C) 40 min after further incubation with d-amphetamine (0.27 mM)+ H7 (10 mM) to the neuron from (B). The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that d-amphetamine elicited bursting firing of action potentials in the presence of H7.
the neuron. The resting membrane potential of the identified RP4 neuron in control and 3 h after anisomycin (1 mM) treatment were −60.0 9 1.3 mV (n= 10, mean9 S.E.M.) and −58.7 90.3 mV (n= 3, P =0.6), respectively. However, anisomycin significantly decreased the frequency of the spontaneously generated action potentials of the neuron. The frequency of spontaneously generated action potenitials in control and 3 h after anisomycin (1 mM) treatment were 39.59 1.8 pulses/min (n =10) and 18.79 1.3 pulses/min (n=3), (P B0.0001) respectively. No bursting of potentials was found in anisomycin treated preparations. However, anisomycin (1 mM) pretreat-
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Fig. 9. Anisomycin (10 mM) decreased the d-amphetamine elicited bursting firing of action potentials in RP4 neuron. (A), (B) and (C) were from the same neuron. (A) Control, the spontaneous action potentials. (B) the bursting firing of action potentials elicited by d-amphetamine (0.27 mM). (C) 90 min after further incubation with anisomycin (10 mM)+d-amphetamine (0.27 mM) from (B). The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that the bursting firing of action potentials elicited by d-amphetamine (0.27 mM) was blocked by anisomycin (10 mM) treatment.
ment prevented the bursting firing of action potentials elicited by d-amphetamine. In the preparation pretreated with anisomycin (1 mM) for 3 h, subsequent treatment with d-amphetamine (0.27 mM) for additional 90 min did not elicit bursting firing of action potentials. Similar results was found in 3 other preparations. It appeared that anisomycin pre-treatment abolished the generation of bursting firing of action potentials elicited by d-amphetamine. Addition of anisomycin (10 mM) after administration of d-amphetamine also blocked the bursting firing of action potentials elicited by amphetamine (0.27 mM) (Fig. 9).
3.9. Effects of intracellular injection of GDPbs on the d-amphetamine elicited bursting acti6ity
Fig. 8. Effects of anisomycin (10 mM) on d-amphetamine elicited bursting firing of action potentials in RP4 neuron. (A), (B) and (C) were from the same neuron. (A) Control., the spontaneous action potentials. (B) the potentials 3 h after incubation with anisomysin (10 mM). (C) 90 min after further incubation with d-amphetamine (0.27 mM) and anisomycin (10 mM). The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that d-amphetamine (0.27 mM) did not elicite the bursting firing of action potentials in anisomycin pretreated RP4 neuron.
The effect of G protein on the bursting firing of action potentials elicited by amphetamine was tested by intracellular injection of GDPbS, an inhibitor of G protein and the result is shown in Fig. 10. GDPbS was applied by the micropipettes containing 10 mM GDPbS and 100 mM KCl. The resting membrane potentials of the neuron in the control and 20 min after injection testing compound were − 60.09 1.3 mV (n= 10, mean9 S.E.M.) and 60.390.3 mV (n=3, P= 0.60), respectively. The frequency of the spontaneously generated action potentials of the neuron in the control and 20 min after injection of GDPbS were 39.5 91.8 pulses/min (n=10) and 26.093.1 pulse/min (n=3, P B0.001), respectively. The system we used for intracellular injection will provide injection of about 2.6× 10 − 10 l of the solution into the RP4 neuron. The concentrations of GDPbS inside the neuron was
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around 80 mM if we assumed the volume of the RP4 neuron was 3.26 ×10 − 8 l. Amphetamine (0.27 mM) elicited the bursting firing of action potentials in the GDPbS injected neuron. As shown in Fig. 10, 60 min after the addition of d-amphetamine (0.27 mM) in GDPbS injected neuron, bursting of potentials were observed. It appeared that intracellular injection of GDPbS did not alter the d-amphetamine elicited bursting firing of the action potentials. Similar results were found in 3 other preparations.
4. Discussion Amphetamine elicited bursting firing of action potentials in the central snail neuron in a concentration dependent manner. At 80 mM, amphetamine did not elicit bursting firing of action potentials in the RP4 neuron even after 4 h of incubation, while at 0.27 mM, it elicited bursting firing of action potentials 20 min after amphetamine treatment and the bursting of potentials continued for more than 3 h (Tsai and Chen, 1995). Second messengers are intimately related to the functions of the neuron. Evidence suggests the involvement of cAMP in alterations of cellular membrane properties (Hausdorff et al., 1990). For example, cAMP elicited biphasic current in the snail neuron whose activation was mediated through protein phosphorylation (Watanabe and Funase, 1991). It has been shown in many cells that calcium channels may be regulated by cAMP-dependent protein kinase (Penner et al., 1988). Interestingly, forskolin, an activator of adenylyl cyclase (Seamon and Daly, 1981), has been shown to activate
Fig. 10. Effects of intracellular injection of GDPbS on the d-amphetamine elicited bursting activity. (A), (B) and (C) were from the same RP4 neuron. (A) Control, the spontaneous action potentials. (B) the potentials 20 min after injection of GDPbS (10 mM). (C) 40 min after further incubation with d-amphetamine (0.27 mM) to the preparation from (B). The horizontal bar on the top left side indicated the membrane potential at 0 mV. Note that d-amphetamine (0.27 mM) elicited normal bursting firing of action potentials in GDPbS injected neuron.
the calcium current of the motor nerve terminal (Tsai et al., 1992). In the present study we found that activation of adenylyl cyclase may facilitate the generation of the bursting firing of action potentials elicited by d-amphetamine. At lower concentrations, e.g. 0.08 mM, d-amphetamine did not elicit bursting firing of action potentials in the snail neuron. However, at this concentration, d-amphetamine did elicit bursting of action potentials if forskolin was added prior to the administration of d-amphetamine. These results suggest that the activity of adenylyl cyclase may be involved in the generation of the bursting firing of action potentials elicited by d-amphetamine. This view was further supported by the observation that 8-bromo-cAMP, an analog of cAMP, also facilitated the generation of the bursting firing of action potentials in the RP4 neuron pretreated with subthreshold concentration of d-amphetamine (0.08 mM). However, the precise relationship between adenylyl cyclase and the bursting firing of action potentials elicited by d-amphetamine needs further study. Though intracellular injection of 3%-deoxyadenosine, an adenylyl cyclase inhibitor (Hidaka et al., 1984), did not abolish the bursting firing of action potentials elicited by d-amphetamine. The time course suggested that it exhibited a transient effect, lasting about 20–30 min. Thus, the inability of 3%-deoxyadenosine to alter the effect of d-amphetamine may be due to its rapid metabolic inactivation, which suggest that 3%-deoxyadenosine, is not an appropriate inhibitor for in vivo experiments. The cAMP-dependent protein phosphorylation, probably plays an essential role on the d-amphetamine elicited bursting firing of action potentials in the RP4 neuron. Pretreatment with H8, an inhibitor of PKA (Funase et al., 1993), reversibly blocked the d-amphetamine-elicited bursting firing of action potentials. However, once the bursting firing of action potentials was elicited by d-amphetamine, H8 could no longer block the effect of d-amphetamine. It is interesting to note that cAMP-dependent protein phosphorylation appears to play an important role on the bursting pacemaker activity in the serotonin activated neuron because H8 significantly decreased the serotonin elicited ionic current in the PON neuron of the snail (Funase et al., 1993). PKC is a family of plasma-membrane protein kinases. In the absence of hormone stimulation, PKC is present as a soluble cytosolic protein that is catalytically inactive. A rise in the cytosolic Ca2 + level causes PKC to bind to the cytoplasmic leaflet of the plasma membrane, where it can be activated by membrane-associated 1,2-diacylglycerol (DAG). Thus activation of PKC depends on both the Ca2 + ion and DAG, suggesting an interaction between the two branches of the inositol lipid signaling pathway (Nishizuka, 1992). In the present study, we found that H7, a PKC inhibitor
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(Singh et al., 1994; Duraj et al., 1995), did not block the bursting firing of action potentials induced by d-amphetamine. These results suggested that the bursting firing of action potentials elicited by d-amphetamine probably was not associated with the inositol lipid signaling pathway in the neuron. G-protein plays an important role on the functions of the gastropod. The potassium channels were opened through activation of the pertussis toxin-sensitive G protein, Gi or Go (Takahashi et al., 1989) and voltagedependent sodium channels were opened through activation of the cholera toxin-sensitive G protein (Kudo et al., 1991). However, in the present study, we found that intracellular injection of GDPbS, a G protein inhibitor (Chiba et al., 1992), did not affect the bursting firing of action potentials elicited by d-amphetamine. The data suggested that the d-amphetamine elicited bursting firing of action potentials may not involve G protein in the neuron. Anisomycin, a protein synthesis inhibitor (Schacher et al., 1988), alone did not elicit bursting firing of action potential in the RP4 neuron. However, anisomycin did block the bursting firing of action potentials mediated by d-amphetamine. The mechanism of the blocking effects remained to be determined. Anisomycin blocked the protein phosphorylation in sensory neurons of Aplysia (Homayouni et al., 1995) and the regulatory subunits of the Aplysia cAMP-dependent protein kinase (Bergold et al., 1990). It is interesting to note that P70, a specific protein found in the cobalt-induced epileptogenic focus of the rat cerebral cortex, induced bursting activity of action potentials when intracellularly injected into the identified neuron of the snail Euhadra peliomphala (Onozuka et al., 1991b). cAMP-dependent protein kinases possess multiple signal transduction pathways (Walsh and Van Patten, 1994). The role of the phosphorylation of the regulatory subunits of cAMP-dependent protein kinase on the d-amphetamine elicited bursting firing of action potentials remain an interesting subject for further study. Together, our data indicated that d-amphetamine elicited bursting firing of action potentials in the snail RP4 neuron. The bursting of action potentials may be associated with the cAMP dependent kinase system. On the contrary, the effect may not be associated with PKC and G protein activity in the neuron.
Acknowledgements We are grateful to Professor Ted H Chiu (Department of Pharmacology, Medical College of Ohio, USA) for his geneous comments and suggestions of the manuscript and to Dr RL Walsh (Research Technology Branch, National Institute of Drug Abuse, USA) for the generous supply of d-amphetamine. This work was
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supported by grants, NSC-85-2331-B-002-289 and NSC-86-2341-B-002-329, from the National Science Council, Taipei, Taiwan.
References Arvanov, V.L., Chen, R.C., Chen, Y.H., Liou, H.H., Chan, Y.L., Arvanov, V.A., Tsai, M.C. (1994) Modulation of pentylenetetrazol induced bursting activity by electrogenic Na pump in Achatina fulica neurons. Asia Pacific J. Pharmacol., 9: 37–42. Bergold, P.J., Sweatt, J.D., Winicov, I., Weiss, K.R., Kandel, E.R. and Schwartz, J.H. (1990) Protein synthesis during acquisition of long-term facilitation is needed for the persistent loss of regulatory subunits of the Aplysia cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA, 87: 3788 – 3791. Chen, Y.H. and Tsai, M.C. (1996) Bursting firing of action potential in central snail neuron elicited by d-amphetamine: role of calcium ions. Comp. Physiol. Pharmacol., 115: 195 – 205. Chiba, O., Sasaki, K., Higuchi, H. and Takashima, K. (1992) G Protein mediating the slow depolarization induced by FMRFamide in the ganglion cells of Aplysia. Neurosci. Res., 15: 255 – 254. Duraj, J., Kovacikova, M., Sedlak, J., Koppel, J., Sobel, A. and Chorvath, B. (1995) The protein kinase C inhibitor H7 blocks phosphorylation of stathmin during TPA-induced growth inhibition of human pre-B leukemia REH6 cells. Leukemia Res., 19: 457 – 461. Ferrendelli, J.A. and Kinscherf, D.A. (1977) Cyclic nucleotides in epileptic brain: effects of pentylenetetrazol on regional cyclic AMP levels in vivo. Epilepsia, 18: 525 – 531. Funase, K., Watanabe, K. and Onozuka, M. (1993) Augmentation of bursting pacemaker activity by serotonin in an identified Achatina fulica neurone: an increase in sodium- and calcium-activated negative slope resistance via cyclic-AMP dependent protein phosphorylation. J. Exp. Biol., 175: 33 – 44. Hausdorff, W.P., Caron, M.G. and Lefkowitz, R.J. (1990) Turning off the signal: desensitization of b-adrenergic receptor function. FASEB J., 4: 2881 – 2889. Hidaka, H., Inagaki, M., Kawamoto, S., Sasaki, Y. (1984) Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry, 23: 5036 – 5041. Homayouni, R., Byrne, J.H. and Eskin, A. (1995) Dynamic of protein phosphorylation in sensory neurons of Aplysia. J. Neurosci., 15: 429 – 438. Kerkut, G.A., Lambert, J.D.C., Gayton, R.J., Loker, J.E. and Walker, R.J. (1975) Mapping of nerve cells in the subesophageal ganglia of Helix aspersa. Comp. Biochem. Physiol., 50: 1–25. Kudo, A., Sasaki, K., Tamazawa, Y. and Matsumoto, M. (1991) A slow voltage-dependent Na + -current induced by 5-hydroxytryptamine and the G-protein-coupled activation mechanism in the ganglion cells of Aplysia. Jpn. J. Physiol., 41: 259-275. Kurahashi, T. and Kaneko, A. (1993) Gating properties of the cAMP- gated channel in toad olfactory receptor cells. J. Physiol., 466: 287 – 302. Levistre, R., Berguerand,M., Bereziat, G. and Masliah, J. (1995) The cross-regulation of Gi-protein by cholera toxin involves a phosphorylation by protein kinase A. Biochem. J., 306: 765–769. Levitan, E.S. and Levitan, I.B. (1988) Serotonin acting via cyclic AMP enhances both the hyperpolarizating and depolarizing phase of bursting pacemaker activity in the Aplysia neuron R 15. J. Neuroscience, 8: 1152 – 1161. Magoski, N.S., Bauce, L.G., Syed, N.I. and Bulloch, A.G.M. (1995) Dopaminergic transmission between identified neurons from the mollusk, Lymnaea stagnalis. J. Neurophysiol., 74: 1287–1300.
Y.H. Chen, M.C. Tsai / Neuroscience Research 27 (1997) 295–304
304
Nishizuka, Y. (1992) Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science, 258: 607 – 614. Onozuka, M., Kishii, K., Furuchi, H. and Sugaya, E. (1983) Behavior of intracellular cyclic nucleotide and calcium in pentylenetetrazole-induced bursting activity in snail neurons. Brain Res. 269: 277 – 286. Onozuka, M., Kubo, K.A. and Ozono, S. (1991a) The molecular mechanism underlying pentylenetetrazole-induced bursting activity in Euhadra neurons: involvement of protein phosphorylation. Comp. Biochem. Physiol., 100: 423–432. Onozuka, M., Imai, S., Furuichi, H. and Ozono, S. (1991b) A specific 70K protein found in epileptic rat cortex: induction of bursting activity and negative resistance by its intracellular application in Euhadra neurons. J. Neurobiol., 22: 287–297. Onozuka, M., Imai, S. and Sugaya, E. (1986) Pentylenetetrazol induced bursting activity and cellular protein phosphorylation in snail neurons. Brain Res., 362: 33–39. Penner, R., Mathews, G. and Neher, E. (1988) Regulation of calcium influx by second messengers in rat mast cells. Nature, 334: 499 – 504. Price, C.J. and Goldberg, J.I. (1993) Serotonin activation of a cyclic AMP-dependent sodium current in an identified neuron from Helisoma trivolvis. J. Neurosci. 13: 4979–4987. Rees, W.J. (1950) The giant African snail. Proc. Zool. Soc. London, 120: 577 – 599. Rhee, S.G. and Choi, K.D. (1992) Mutiple forms of phospholipase C isozymes and their activation mechanisms. In: J.W. Putney Jr (Ed.), Advances in Second Messenger and Phsophoprotein Research, Vol. 26, Raven Press, New York, 1992, pp. 35 – 61. Schacher, S., Castellucci, V.F. and Kandel, E.R. (1988) cAMP evokes long-term facilitation in Aplysia sensory neurons that requires new protein synthesis. Science, 24: 1667–1669. Seamon, K.B. and Daly, J.W. (1981) Forskolin: a unique diterpene activator of cyclic AMP-generating systems. J. Cyclic Nucleotide Res., 7: 201 – 224. Singh, V.K., Cheng, J.F. and Leu, S.J.C. (1994) Effect of substance P and protein kinase inhibitors on b-amyloid peptide-induced proliferation of cultured brain cells. Brain Res., 660: 353 – 356. Slater, N.T., Hall, A.F. and Carpenter, D.O. (1984) Kinetic properties of cholinergic desensitization in Aplysia neurons. Proc. R. Soc. London B. Biol. Sci., 223: 63–78.
Sugaya, E., Furuichi, H., Takagi, T., Kajiwara, K. and Komatsubara, J. (1987) Intracellular calcium concentration during pentylenetetrazol-induced bursting activity in snail neurons. Brain Res., 416: 183 – 186. Sugaya, E., Goldring, S. and O’Leary, J.L. (1964) Intracellular potentials discharge in cat. Electroencephalogr. Clin. Neurophysiol., 17: 661 – 669. Sugaya, E. and Onozuka, M. (1978a) Intracellular calcium: its movement during pentylenetetrazol-induced bursting activity. Science, 200: 797 – 799. Sugaya, E. and Onozuka, M. (1978b) Intracellular calcium: its release from granules during bursting activity in snail neurons. Science, 202: 1195 – 1197. Takahashi, J., Sasaki, K. and Matsumoto, M. (1989) The gating mechanism of K + -channels coupled to the FMRFamide receptor in the ganglion cells of Aplysia. Nippon Serigaku Zasshi (J. Physiol. Soc. Jpn.), 51: 363 – 378. Takeuchi, H., Araki, Y., Emaduddin, M. et al. (1996) Identifiable Achatina giant neurones: their localizations in ganglia, axonal pathways and pharmacological features. Gen. Pharmacol., 27: 3 – 32. Taussig, R. and Gilman, A.G. (1995) Mammalian membrane-bound adenylyl cyclases. J. Biol. Chem., 270: 1 – 4. Tsai, M.C. and Chen, M.L. (1989) A new method for screening anticonvulsants: 1. effects of anticonvulsants on pentylenetetrazolinduced neuronal activity of the giant African snail, Achatina fulica Ferussac. Asia Pacific J. Pharmacol., 4: 203 –207. Tsai, M.C., Chen, M.L., Su, J.L., Hsieh, W.H. and Fan, S.Z. (1992) Effects of a phorbol ester and forskolin on nerve terminal currents in mouse motor nerves. Asia Pacific J. Pharmacol., 7: 27 – 31. Tsai, M.C. and Chen, Y.H. (1995) Bursting firing of action potential in central snail neuron elicited by d-amphetamine: role of electrogenic sodium pump. Comp. Physiol. Pharmacol., 111: 131– 141. Walsh, D.A. and Van Patten, S.M. (1994) Multiple pathway signal transduction by the cAMP-dependent protein kinase. FASEB J., 8: 1227 – 1236. Watanabe, K. and Funase, K. (1991) Cyclic AMP elicits biphasic current whose activation is mediated through protein phosphorylation in snail neurons. Neurosci. Res., 10: 64 – 70.
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