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The modulation effects of d-amphetamine and procaine on the spontaneously generated action potentials in the central neuron of snail, Achatina fulica Ferussac
Comparative Biochemistry and Physiology, Part C 141 (2005) 58 – 68 www.elsevier.com/locate/cbpc
The modulation effects of d-amphetamine and procaine on the spontaneously generated action potentials in the central neuron of snail, Achatina fulica Ferussac Chia-Hsien Lin, Ming-Cheng Tsai* Department of Pharmacology, College of Medicine, National Taiwan University, No.1, Sec.1, Jen-Ai Road, Taipei, Taiwan Received 22 February 2005; received in revised form 5 May 2005; accepted 5 May 2005 Available online 9 June 2005
Abstract The modulation effects of d-amphetamine and procaine on the spontaneously generated action potentials were studied on the RP1 central neuron of giant African snails (Achatina fulica Ferussac). Extra-cellular application of d-amphetamine or procaine reversibly elicited bursts of potential (BoP). Prazosin, propranolol, atropine or d-tubocurarine did not alter the BoP elicited by either d-amphetamine or procaine. KT5720 or H89 (protein kinase A inhibitors) blocked d-amphetamine-elicited BoP, whereas they did not block the procaine-elicited BoP. U73122, neomycin (phospholipase C inhibitors) blocked the procaine-elicited BoP, whereas they did not block the d-amphetamine-elicited BoP in the same neuron. These results suggest that BoP elicited by d-amphetamine or procaine were associated with protein kinase A and phospholipase C activity in the neuron. D 2005 Elsevier Inc. All rights reserved. Keywords: Phospholipase; cAMP; d-amphetamine; Procaine; Neuron; Bursts of potential; Epilepsy; Synaptic transmission; Second messengers; Potential modulation
1. Introduction Physiological signals are integrated within the cell as a result of interactions between second messenger pathways. The cyclic adenosine 3V, 5V-monophosphate (cAMP) and the phosphoinositide (PI) signal-transduction pathways are the intracellular targets that mediate the cellular functions. cAMP-dependent phosphorylation modulated the dihydropyridine-sensitive calcium channels in cardiac myocytes (McDonald et al., 1994). Phospholipase C (PLC) hydrolyzed phosphatidylinositol 4, 5- bisphosphate to generate the second messengers, inositol 1, 4, 5- trisphosphate (IP3) and diacylglycerol (DAG). IP3 induced a transient increase in intracellular free Ca2+. The enzymes known to regulate cAMP levels, adenyl –cyclase and phosphodiesterase, and the functions of phospholipase C have been studied in
* Corresponding author. Tel.: +886 2 23966786; fax: +886 2 23915297. E-mail address: [email protected] (M.-C. Tsai). 1532-0456/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2005.05.003
detail. However, whether these messengers modulate potentials in the same central neuron previously remained unclear. In the present study, we found that both damphetamine and procaine elicited BoP on the same central neuron of giant African snails. The effects were altered after administration of protein kinase A (PKA) or phospholipase C (PLC) inhibitors. These results suggested that PKA and PLC in the neuron were closely associated to the modulation of potential changes in the RP1 neuron.
2. Materials and methods Experiments were performed on identified central RP1 neurons from the sub-esophageal ganglia of the African snail (Achatina fulica Ferussac). The ganglia were pinned to the bottom of a 2 mL Sylgard-coated perfusion chamber and carefully freed from the connective tissue sheath to allow easy identification and penetration by microelectrodes (Chen and Tsai, 2000). For intracellular recording, a Gene
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Clamp 500 amplifier (Axon Instruments, Inc., USA) was used. Micro-electrodes (5 – 6 MV) for recording membrane potentials were filled with 3 M KCl. The experimental chamber was perfused with control saline which had following composition (mM): NaCl, 85; KCl, 4; CaCl2, 8; MgCl2, 7; Tris –HCl, 10 (pH 7.6), at room temperature of 23 –24 -C with perfusion speed of 8 mL/min. The Ca2+-free solution was substituted for the calcium ion with equimolar amounts (8 mM) of CoCl2 (Kim et al., 1991). In the lithium substitute solution (sodium-free solution), NaCl (85 mM) was substituted with LiCl. For testing the effect of procaine or d-amphetamine on resting membrane potential (RMP), amplitudes of action potentials and the frequency of single spikes of action potentials of the RP1, RMP, amplitudes of action potentials and the frequency of single spikes of action potentials were recorded 60 min after procaine or d-amphetamine administration or recorded at the temporary steady-state level. To test the effects of prazosin, propranolol, atropine, dtubocurarine, KT-5720, H89, chelerythrine chloride, Ro 318220 or U73122 on d-amphetamine-or procaine-elicited potential changes, the RP1 neuron was first treated with damphetamine (270 AM) or procaine (10 mM) for 60 min, and then d-amphetamine or procaine were washed off as a pre-drug control. After the BoP elicited by d-amphetamine or procaine had recovered to the control level, prazosin, propranolol, atropine, d-tubocurarine, KT-5720, H89, chelerythrine chloride, Ro 31-8220 were added for 40 min, or U73122 were added for 60 min, and then d-amphetamine (270 AM) or procaine (10 mM) was further added for 60 min, and then washed off with normal saline for 120 min. To test the effects of neomycin on procaine-or damphetamine-elicited potential changes, the RP1 neuron was first treated with d-amphetamine or procaine for 60 min until BoP were observed, then, neomycin was further added for 120 min. To test the effects of calcium-free solution on d-amphetamine-or procaine-elicited potentials changes in RP1 neuron, the neuron was first treated with calcium-free solution for 30 min, then d-amphetamine or procaine were further added for 60 min. To test the effects of the high-magnesium solution or lithium substitute solution (sodium-free solution) on damphetamine-or procaine-elicited potential changes, the RP1 neuron was first treated with d-amphetamine (270 AM) or procaine (10 mM) for 60 min or until BoP were observed. Then a high-magnesium solution was further added for 30 min or lithium substitute solution (sodium-free solution) was further added for 40 min. Topical microperfusion was used for the rapid application of drugs, as described previously (Chen and Tsai, 1997, 2000). For intracellular injections of d-amphetamine or procaine, CaCl2, MgCl2, KCl into the RP1 neuron; d-amphetamine (13.5 mM), procaine (1 M), CaCl2 (250 mM), MgCl2 (500 mM) (Brown and McCrohan, 1991) were dissolved in 100 mM KCl (Watanabe and Funase, 1991) and the
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intracellular injection solutions were filled into micropipettes with tip diameter of about 1 Am adjusted by microforge (Narishige MF-83). Injections were made by a pulse under manual control over the course of the experiment. The duration of each injection pulse was 2 s with a pressure of 200 mmHg and the calculation of injected intracellular drug concentration was as described previously (Chen and Tsai, 1996). All potentials were recorded on tape by a digitalizing unit (Digidata 1200) and analyzed. The mean amplitude of the potentials after various treatments was compared with the pre-drug control by means of Student’s two-tailed t-test. Differences were considered significant at p < 0.05 (Chen and Tsai, 2000). Procaine, neomycin, H89, Ro 31-8220, U73122 (1-[6[((17h)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]1H-pyrrole-2,5-dione), prazosin, propranolol, atropine and d-tubocurarine were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). KT -5720 was purchased from Calbiochem Ltd. (USA). Chelerythrine chloride was purchased from Tocris (Bristol, UK). LiCl were purchased from Riedel-deHae¨n\ (USA). All drug stocks were made with double-distilled water except for prazosin, U73122 and KT-5720, which were prepared in dimethyl sulfoxide (DMSO). The presence of DMSO ( 0.1%) alone did not affect the RMP, amplitude and frequency of the spontaneous firing of action potential in the RP1 neuron.
3. Results 3.1. Identifiable RP1 neuron of Achatina fulica ferussac The central right parietal neuron 1 (RP1 neuron), the identified neuron in the sub-esophageal ganglia of the African snail A. fulica Ferussac (Chen and Tsai, 1996), was sensitive to several neurotransmitters. Glutamic acid (50 AM) induced hyperpolarization of the membrane potential. Serotonin (5-HT) (50 AM), g-aminobutyric acid (GABA) (50 AM), dopamine (50 AM) or acetylcholine (ACh) (50 AM) increased the frequency of the spontaneous action potential of the neuron. 3.2. Effects of d-amphetamine or procaine on the RP1 neuron The RP1 neuron had a RMP of 60.3 T 0.6 mV (n = 20, mean T S.E.M.), and it showed a spontaneous firing of action potential at a frequency of 58.7 T 1.2 pulses/min (n = 20). The action potentials showed regularly spaced single spikes, and no BoP was observed in the control RP1 neuron. The mean amplitude of the spontaneously generated action potential was 83.7 T 1.6 mV (n = 20). The effects of various concentrations of d-amphetamine on the RP1 neurons were tested. After 60 min of extracellular incubation of d-
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amphetamine (67.5 AM), the RMP, amplitude and frequency of the spontaneous firing action potential were 60.4 T 1.1, 85.6 T 1.1 and 57.5 T 1.2 pulses/min (n = 10), respectively. Higher concentrations of d-amphetamine (135 AM) increased the frequency of the action potential to 104.6 T 6.4 pulses/min (n = 10) ( p < 0.05) 60 min after damphetamine (135 AM) treatment. The RMP, amplitude of action potential and frequency of single spikes remained at the same level, even after 2h of d-amphetamine (135 AM) treatment. However, some phasic depolarization followed by a hyper-polarization phase was occasionally found 30 min after d-amphetamine (135 AM) treatment. Sixty min after d-amphetamine (270 AM) treatment, the firing patterns of the neuron had changed from regularly spaced single spikes to ones in which there were bursts of 8– 20 action potentials. Oscillation of membrane potentials with a phasic depolarization followed by a sustained depolarization with burst of action potential was found in d-amphetamine (270 AM)-treated preparations. At the end of the potential bursts, the neuron also elicited a hyper-polarization phase. The large hyper-polarization phase (hyperpolarization of membrane potential up to 8 mV), lasted 1 – 25 s after damphetamine administration. The frequency of bursts was 9.8 T 0.4 bursts/min (n = 10). The RMP of the neurons changed from 60.3 T 0.6 mV (n = 20) to –72.2 T 1.4 mV (n = 10) during the hyper-polarization phase. After the phasic depolarization and hyper-polarization phase, the RMP reached a temporary steady state level. There was no single-spike action potential 40 – 60 min after damphetamine (270 AM) treatment. The effect of d-amphetamine on the neuronal activities was reversible. After 120 min of continuous washing, the BoP of the central neuron returned to control levels, albeit with a slower frequency of
spontaneous firing. d-Amphetamine (270 AM) increased the RMP and amplitude of action potential and elicited BoP on the RP1 neuron. An example of the effects of different concentrations of d-amphetamine (67.5, 135, 270 AM) on the RP1 neuron is shown in Fig. 1A. The effect of intracellular injection of d-amphetamine on the spontaneously generated action potentials of the RP1 neuron was tested. Here, d-amphetamine was applied by micropipettes containing 13.5 mM d-amphetamine and 100 mM KCl. Immediately after d-amphetamine injection, BoP was observed. The RMP of the RP1 neuron were depolarized from 61.5 T 2.3 to 52.2 T 1.4 mV (n = 6). The system we used for intracellular injection will provide injection with about 2.6 10 10 L of the solution into the RP1 neuron. The concentration of d-amphetamine inside the neuron was approximately 108 AM, assuming the volume of the RP1 neuron was 3.26 10 8 L. The RMP and BoP elicited by intracellular injection of damphetamine were reversible. Ten minutes after intracellular injection, the RMPs were recovered to 60.9 T 0.8 mV (n = 6), indicating that intracellular injection of d-amphetamine elicited BoP in the RP1 neuron. Sixty min after procaine (1, 3 mM) administration, the frequency of the spontaneous firing of action potential had decreased from 58.7 T 1.2 pulses/min (control, n = 20) to 35.6 T 2.5 pulses/min ( p < 0.05, n = 10) and 26.5 T 1.6 pulses/ min, (n = 10) ( p < 0.05), respectively. Increasing procaine concentration to 10 mM elicited BoP 40 min after procaine administration, and these BoP lasted for more than 2 h. The firing pattern changed from regularly spaced single spikes to ones in which there were bursts of 2 –10 action potentials. The frequency of bursts was 5.8 T 0.5 bursts/min. The BoP elicited by procaine was recovered to control level after 120
Fig. 1. Effects of various concentrations of d-amphetamine or procaine on the central RP1 neuron of snails. A1 and B1 were control, showing spontaneous firing of action potential. A2, A3, A4 were the potentials 60 min after application of d-amphetamine (67.5, 135, or 270 AM), respectively. B2, B3, B4 were the potentials 60 min after application of procaine (1, 3 or 10 mM), respectively. A5 and B5 were the potentials 120 min after washing off from A4 and B4 with normal physiological solution, respectively. The horizontal bar at the top left side indicates the membrane potential at 0 mV. Noted that d-amphetamine (270 AM) and procaine (10 mM) elicited bursts of potential (BoP) on the RP1 neurons.
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Table 1 Effect of KT-5720, H89, chelerythrine chloride, Ro 31-8220, U73122, neomycin, lithium substitute solution (sodium-free solution), calcium-free solution and high-magnesium solution on d-amphetamine or procaine-elicited changes of the RMPs (mV) on the RP1 neuron Variable Control KT-5720 (10 AM) H89 (10 AM) Chelerythrine chloride (10 AM) Ro 31-8220 (10 AM) U73122 (10 AM) Neomycin (3.5 mM) LiCl Calcium-free solution High-magnesium solution (30 mM)
Physiological solution 60.3 T 0.6 (n = 20) 63.8 T 0.6 (n = 10) 63.7 T 1.8 (n = 9) 61.4 T 0.7 (n = 11) 62.7 T 1.9 (n = 8) 63.1 T1.3 (n = 10) 42.7 T 2.3a (n = 8) 60.7 T 0.9 (n = 11) 52.0 T 0.8a (n = 10) 58.7 T 2.7 (n = 8)
d-amphetamine (270 AM) 72.2 T 1.4a (n = 10) 63.9 T 1.6b (n = 5) 69.3 T 1.4b,d (n = 5) 69.5 T 0.9e (n = 4) 72.0 T 1.7f (n = 4) 61.0 T 3.2b (n = 4) 46.3 T 1.2b (n = 3) 51.3 T 1.5b,g (n = 5) 49.6 T 1.5b (n = 5) 69.0 T 2.6b,i (n = 4)
Procaine (10 mM) 58.3 T 0.9 (n = 10) 65.5 T 1.1c (n = 5) 64.7 T 1.7c (n = 4) 58.8 T 0.6 (n = 7) 60.1 T1.7 (n = 4) 59.5 T 0.9 (n = 6) 47.7 T 3.7c (n = 5) 47.8 T 0.7c,g (n = 6) 34.8 T 1.3c,h (n = 5) 47.3 T 0.7c,i (n = 4)
a
: Statistically significant compared with the data in physiological solution (control), p < 0.05. b : Statistically significant compared with d-amphetamine (270 AM) in physiological solution, p < 0.05. c : Statistically significant compared with procaine (10 mM) in physiological solution, p < 0.05. d : Statistically significant compared with H89 (10 AM) in physiological solution, p < 0.05. e : Statistically significant compared with chelerythrine chloride (10 AM) in physiological solution, p < 0.05. f : Statistically significant compared with Ro 31-8220 (10 AM) in physiological solution, p < 0.05. g : Statistically significant compared with LiCl in physiological solution, p < 0.05. h : Statistically significant compared with calcium-free solution in physiological solution, p < 0.05. i : Statistically significant compared with high Mg2+ (30 mM) in physiological solution, p < 0.05.
min of continuously washing off the procaine (Fig. 1B). Procaine did not alter the RMP and amplitude of the RP1 neuron at all concentrations tested (1 –10 mM), but procaine (1, 3 mM) did decrease the frequency of spontaneous action potential of the RP1 neuron. It appears that higher concentration of procaine (10 mM) reversibly elicited BoP on the RP1 neuron. An example of the effects of procaine (1– 10 mM) on the RP1 neuron is shown in Fig. 1B. The effects of intracellular injection of procaine on the spontaneously generated action potentials of the RP1 neuron were tested. Procaine was applied by micropipettes containing 1 M procaine and 100 mM KCl. Immediately after procaine injection, BoP was observed. The RMP of the RP1 neuron were depolarized from 61.5 T 2.3 to 54.2 T 1.6 mV (n = 6). The concentration of procaine inside the neuron was approximately 8 mM, assuming the volume of the RP1 neuron was 3.26 10 8 L (Chen and Tsai, 1996). The RMP and BoP elicited by intracellular injection of procaine were
reversible. Ten minutes after intracellular injection, the RMP had recovered to 60.9 T 0.8 mV (n = 6). The RP1 neuron was depolarized if injected for longer period of procaine, indicating that intracellular injection of procaine elicited BoP in the RP1 neuron. 3.3. Effects of protein kinase A inhibitors on d-amphetamine-or procaine-elicited burst activity of the RP1 neuron To examine the role of cAMP-dependent protein kinase in the generation of burst activity elicited by d-amphetamine or procaine, the effects of KT-5720 or H89, inhibitors of protein kinase A, were studied. The RMP, amplitude and frequency of the spontaneous firing action potential of the RP1 neuron with d-amphetamine (270 AM) or procaine (10 mM) and KT-5720 (10 AM) are shown in Tables 1– 3, respectively. KT-5720 reversibly decreased the frequency of the spontaneous action potential
Table 2 Effect of KT-5720, H89, chelerythrine chloride, Ro 31-8220, U73122, neomycin, lithium substitute solution (sodium-free solution), calcium-free solution and high-magnesium solution on d-amphetamine or procaine-elicited changes of the amplitude of action potential (mV) on the RP1 neuron Variable
Physiological solution
d-amphetamine (270 AM)
Procaine (10 mM)
Control KT-5720 (10 AM) H89 (10 AM) Chelerythrine chloride (10 AM) Ro 31-8220 (10 AM) U73122 (10 AM) Neomycin (3.5 mM) LiCl Calcium-free solution High-magnesium solution (30 mM)
83.7 T 1.6 (n = 20) 87.2 T 0.7 (n = 10) 83.4 T 2.3 (n = 9) 85.7 T 0.6 (n = 11) 83.8 T 1.1 (n = 8) 84.9 T 2.7 (n = 10) 51.1 T 2.6a (n = 8) 86.0 T 1.0 (n = 11) 66.0 T 1.0a (n = 10) 85.2 T 0.4 (n = 8)
97.5 T 1.6a (n = 10) 88.9 T 1.8b (n = 5) 89.3 T 1.9b,d (n = 5) 94.5 T 0.7e (n = 4) 94.0 T 2.2f (n = 4) 80.0 T 5.7b (n = 4) 59.5 T 3.0b (n = 3) 59.8 T 3.2b,g (n = 5) 63.0 T 1.9b (n = 5) 92.0 T 1.2i (n = 4)
79.5 T 1.2 (n = 10) 89.3 T 1.3c (n = 5) 85.4 T 1.9c (n = 4) 80.6 T 0.6e (n = 7) 84.7 T 1.5 (n = 4) 79.1 T1.3 (n = 6) 54.2 T 2.4c (n = 5) 64.4 T 0.9c,g (n = 6) 43.6 T 1.1c,h (n = 5) 64.6 T 0.9c,i (n = 4)
a : Statistically significant compared with the data in physiological solution (control), p < 0.05. b : Statistically significant compared with d-amphetamine (270 AM) in physiological solution, p < 0.05. c : Statistically significant compared with procaine (10 mM) in physiological solution, p < 0.05. d : Statistically significant compared with H89 (10 AM) in physiological solution, p < 0.05. e : Statistically significant compared with chelerythrine chloride (10 AM) in physiological solution, p < 0.05. f : Statistically significant compared with Ro 31-8220 (10 AM) in physiological solution, p < 0.05. g : Statistically significant compared with LiCl in physiological solution, p < 0.05. h : Statistically significant compared with calcium-free solution in physiological solution, p < 0.05. i : Statistically significant compared with high Mg2+ (30 mM) in physiological solution, p < 0.05.
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Table 3 Effect of KT-5720, H89, chelerythrine chloride, Ro 31-8220, U73122, neomycin, lithium substitute solution (sodium-free solution), calcium-free solution and high-magnesium solution on d-amphetamine or procaine-elicited changes on frequency of spikes of the RP1 neuron Variable
Physiological solution
d-amphetamine (270 AM)
Procaine (10 mM)
Control KT-5720 (10 AM) H89 (10 AM) Chelerythrine chloride (10 AM) Ro 31-8220 (10 AM) U73122 (10 AM) Neomycin (3.5 mM) LiCl Calcium-free solution High-magnesium solution (30 mM)
58.7 T 1.2 pulses/min (n = 20) 34.8 T 1.5a pulses/min (n = 10) 39.0 T 2.4a pulses/min (n = 8) 55.0 T 1.4 pulses/min (n = 11) 55.3 T 2.6 pulses/min (n = 8) 57.1 T 2.2 pulses/min (n = 10) 74.0 T 5.2a pulses/min (n = 8) 31.0 T 1.7a pulses/min (n = 11) 41.7 T 2.1a pulses/min (n = 10) 51.5 T 5.5 pulses/min (n = 8)
9.8 T 0.4 bursts/min (n = 10) 52.9 T 3.5d pulses/min (n = 5) 53.5 T 2.5e pulses /min (n = 5) 13.4 T 0.2b bursts/min (n = 4) 11.6 T 0.3b bursts/min (n = 4) 5.0 T 0.6b bursts/min (n = 4) 5.3 T 1.5b bursts/min(n = 3) 68.0 T 3.3h pulses /min(n = 5) 2.3 T 0.3b bursts/min (n = 5) 7.3 T 1.8 bursts/min (n = 4)
5.8 T 0.5 bursts/min (n = 10) 5.1 T1.3 bursts/min (n = 5) 5.1 T 2.2 bursts/min (n = 4) 7.1 T1.4c bursts/min (n = 7) 6.3 T 1.9 bursts/min (n = 4) 32.3 T 1.4f pulses/min (n = 6) 9.7 T 1.5g pulses/min (n = 5) 43.0 T 3.2h pulses/min (n = 6) 3.4 T 0.4c bursts/min (n = 5) 8.0 T 1.0i pulses/min (n = 4)
a
: Statistically significant compared with the data in physiological solution (control), p < 0.05. b : Statistically significant compared with d-amphetamine (270 AM) in physiological solution, p < 0.05. c : Statistically significant compared with procaine (10 mM) in physiological solution, p < 0.05. d : Statistically significant compared with KT-5720 (10 AM) in physiological solution, p < 0.05. e : Statistically significant compared with H89 (10 AM) in physiological solution, p < 0.05. f : Statistically significant compared with U73122 (10 AM) in physiological solution, p < 0.05. g : Statistically significant compared with neomycin (3.5 mM) in physiological solution, p < 0.05. h : Statistically significant compared with LiCl in physiological solution, p < 0.05. i : Statistically significant compared with high Mg2+ (30 mM) in physiological solution, p < 0.05.
of the neuron ( p < 0.05), whereas it did not alter the RMP and amplitude of the action potential of the RP1 neuron ( p > 0.05). No BoP was found in the KT-5720-and d-amphetaminetreated preparations tested. Compared with the neurons treated with KT-5720 (10 AM), the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were not altered in d-amphetamine-(270 AM) and KT-5720-(10 AM) treated preparations ( p > 0.05), and an example of the effects of KT-5720 on the d-amphetamineelicited potential changes is shown in Fig. 2A.
The RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were not altered in procaine-(10 mM) and KT-5720-(10 AM) treated preparations ( p > 0.05) compared with the neurons treated with KT5720 (10 AM). BoP was found in KT-5720-and procainetreated preparations. Procaine (10 mM) still elicited BoP even after increasing KT-5720 concentration to 100 AM. An example of the effects of KT-5720 on the procaine-elicited potential changes is shown in Fig. 2B. In order to test the role of PKA on the BoP elicited by damphetamine and procaine, the RP1 neuron was first treated
Fig. 2. Effects of KT-5720 (10 AM) on the d-amphetamine-or procaine-elicited BoP on the central RP1 neurons. A1 and B1 were control, showing spontaneous firing of action potential. A2 and B2 were the potentials of the neuron 40 min after application with KT-5720 (10 AM). A3 and B3 were the potentials 60 min after further incubation with d-amphetamine (270 AM) or procaine (10 mM) from A2, B2, respectively. A4 and B4 were the potentials 120 min after washing off from A3 and B3 with normal physiological solution, respectively. The horizontal bar at the top left side indicates the membrane potential at 0 mV. KT-5720 blocked the d-amphetamine-elicited BoP, while KT-5720 did not block the procaine-elicited BoP on the RP1 neuron.
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with d-amphetamine until BoP appeared. Then KT-5720 was added until BoP elicited by d-amphetamine had shifted to single spikes, and then procaine was further added. BoP was found in 4 of the preparations tested, and an example is shown in Fig. 3A. KT-5720 blocked the potentials elicited by d-amphetamine, whereas it did not block the BoP elicited by procaine. Effects of H89 (10 AM) on d-amphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP (mV), amplitude of action potential (mV), and frequency of action potential on the RP1 neuron are shown in Tables 1 –3, respectively. H89 reversibly decreased the frequency of spontaneous action potential of the neuron ( p < 0.05), whereas it did not alter the RMP and amplitudes of the action potential of the RP1 neuron ( p > 0.05). The RMP, amplitude and frequency of BoP in the RP1 neuron after sixty minutes incubation in d-amphetamine (270 AM) and H89 (10 AM) are shown in Tables 1 –3, respectively. Compared with the neurons treated with H89 (10 AM), the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron increased in damphetamine-(270 AM) and H89-(10 AM) treated preparations ( p < 0.05). Furthermore, H89 significantly decreased the BoP elicited by d-amphetamine. The RMP, amplitude and frequency of BoP in the RP1 neuron after sixty minutes incubation in procaine (10 mM) and H89 (10 AM) are shown in Tables 1 –3, respectively.
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Compared with the neurons treated with H89 (10 AM), the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were not altered in procaine-(10 mM) and H89-(10 AM) treated preparations ( p > 0.05). BoP was found in H89-and procaine-treated preparations. H89 did not alter the BoP elicited by procaine. 3.4. Effects of protein kinase C inhibitors on d-amphetamine-or procaine-elicited bursting activity of the RP1 neuron To examine whether protein kinase C was involved in the generation of burst firing of the frequency of spontaneously generated action potentials of the RP1 neuron, the effects of chelerythrine chloride or Ro 31-8220, inhibitors of protein kinase C, on the d-amphetamine-or procaine-elicited bursting activity of potentials were tested. Effects of chelerythrine chloride (10 AM) on d-amphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP (mV), amplitude of action potential (mV), and frequency of action potential on the RP1 neuron are shown in Tables 1 –3, respectively. Chelerythrine chloride did not alter the RMP, amplitude and frequency of the action potential of the RP1 neuron ( p > 0.05). Compared with the neurons treated with chelerythrine chloride (10 AM), the RMP and amplitude of the sponta-
Fig. 3. Effects of KT-5720 (10 AM) and U73122 (10 AM) on d-amphetamine-or procaine-elicited BoP on the central RP1 neuron of snails. A1 and B1 were control, showing spontaneous firing of action potential. A2 and B2 were the potentials 60 min after addition of d-amphetamine (270 AM) or procaine (10 mM) from A1 and B1, respectively. A3 was the potentials after addition KT-5720 (10 AM) for 60 min from A2. A4 was the potentials after further addition of procaine (10 mM) for 60 min from A3. B3 was the potentials after addition U73122 (10 AM) for 60 min from B2. B4 was the potentials after further addition of d-amphetamine (270 AM) for 60 min from B3. A5 and B5 were the potentials after washing off with normal physiological solution for 120 min from A4 and B4, respectively. The horizontal bar at the top left side indicates the membrane potential at 0 mV. Notes that procaine-elicited BoP in the preparation treated with both d-amphetamine and KT-5720 (A4) and d-amphetamine-elicited BoP in the preparation treated with procaine and U73122 (B4).
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neously generated action potential of the RP1 neuron were increased in d-amphetamine-(270 AM) and chelerythrine chloride-(10 AM) treated preparations ( p < 0.05). d-Amphetamine (270 AM) still elicited BoP in a preparation pretreated with chelerythrine chloride (10 AM). Compared to the neurons treated with chelerythrine chloride (10 AM), the RMP were not altered ( p > 0.05), however the amplitude of the spontaneously generated action potential of the RP1 neuron treated preparations were decreased in procaine-(10 mM) and chelerythrine chloride-(10 AM) treated preparations ( p < 0.05). Procaine still elicited BoP in preparations pretreated with chelerythrine chloride (10 AM). Effects of Ro 31-8220 (10 AM) on d-amphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP (mV), amplitude of action potential (mV), and frequency of action potential on the RP1 neuron are shown in Tables 1– 3, respectively. Ro 31-8220 did not alter the RMP, amplitude or frequency of the action potential of the RP1 neuron ( p > 0.05). The RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were increased in damphetamine-(270 AM) and Ro 31-8220-(10 AM) treated preparations compared with the neurons treated with Ro 318220 (10 AM). BoP was found in Ro 31-8220-and damphetamine-treated preparations. d-Amphetamine (270 AM) still elicited BoP in Ro 31-8220 (10 AM) pre-treated preparation. The RMP and amplitude of the spontaneously generated action potential of the RP1 neuron treated preparations were not altered in procaine-(10 mM) and Ro 31-8220-(10 AM)
treated preparations ( p > 0.05) compared with the neurons treated with Ro 31-8220 (10 AM). BoP was found in Ro 318220-and procaine-treated preparations. Procaine still elicited BoP in preparations pretreated with Ro 31-8220 (10 AM). 3.5. Effects of phospholipase C inhibitors on d-amphetamine-or procaine-elicited potential changes in the RP1 neuron To examine whether phospholipase C was involved in the generation of burst firing of the frequency of spontaneously generated action potentials of the RP1 neuron, the effects of U73122 or neomycin, inhibitors of phospholipase C, on the d-amphetamine-or procaine-elicited bursting activity of potentials were tested. Effects of U73122 (10 AM) on d-amphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP (mV), amplitude of action potential (mV), and frequency of action potential on the RP1 neuron are shown in Tables 1– 3, respectively. U73122 did not alter the RMP, amplitude and frequency of the action potential of the RP1 neuron ( p > 0.05). The RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were not altered in damphetamine-(270 AM) and U73122-(10 AM) treated preparations ( p > 0.05) compared with the neurons treated with U73122 (10 AM). BoP was found in U73122-and d-amphetamine-treated preparations, and an example of the effects of U73122 on the d-amphetamine-elicited potential changes is shown in Fig. 4A. d-Amphetamine (270 AM) still elicited BoP in preparation pre-treated with U73122 (10 AM).
Fig. 4. Effects of U73122 (10 AM) (PLC inhibitor) on d-amphetamine-or procaine-elicited BoP on the central RP1 neuron of snails. A1 and B1 were control, showing spontaneous firing of action potential. A2 and B2 were the potentials of the neuron 60 min after application with U73122 (10 AM). A3 and A4 were the potentials 30, 60 min after addition d-amphetamine (270 AM) from A2, respectively. B3 and B4 were the potentials after addition procaine (10 mM) 30, 60 min, respectively from B2. A5 and B5 were the potentials 120 min after washing off from A4 and B4 with normal physiological solution, respectively. The horizontal bar at the top left side indicates the membrane potential at 0 mV. Pretreatment with U73122 blocked the procaine, but it did not block the damphetamine-elicited BoP.
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No BoP was found in U73122-and procaine-treated preparations tested. The RMP and amplitude of the spontaneously generated action potential of the RP1 neuron treated preparations were not altered in procaine (10 mM) and U73122 (10 AM) treated preparations ( p > 0.05) compared with the neurons treated with U73122 (10 AM), and an example of the effects of U73122 on the procaineelicited potential changes is shown in Fig. 4B. In order to test the role of PLC (phospholipase C) on the BoP elicited by d-amphetamine and procaine, the RP1 neuron was first treated with procaine until BoP appeared. Then U73122 (phospholipase C inhibitor) was added until BoP elicited by procaine had shifted to single spikes, and then d-amphetamine was further added. BoP was found in 4 of the preparations tested, and an example is shown in Fig. 3B. U73122 blocked the potentials elicited by procaine, whereas it did not block the BoP elicited by d-amphetamine. Effects of neomycin (3.5 mM) on d-amphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP (mV), amplitude of action potential (mV), and frequency of action potential on the RP1 neuron are shown in Tables 1 – 3, respectively. At 120 min after incubation with neomycin (3.5 mM), neomycin decreased the RMP and amplitudes of the action potentials of the neuron, whereas it increased the frequency of spontaneous action potentials of the RP1 neuron ( p < 0.05). The spontaneous action potentials of the RP1 neuron recovered to control level after 120 min of continuous washing with normal physiological solution. Compared with the neurons treated with neomycin (3.5 mM), the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were not altered in d-amphetamine-(270 AM) and neomycin-(3.5 mM) treated preparations ( p > 0.05). BoP was found in neomycin-and d-amphetamine-treated preparations. Neomycin did not alter the BoP elicited by d-amphetamine. Compared with the neurons treated with neomycin (3.5 mM), the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were not altered in procaine-(10 mM) and neomycin-(3.5 mM) treated preparations ( p > 0.05). No BoP was found in the neomycin-and procaine-treated preparations tested, indicating that neomycin significantly altered the BoP elicited by procaine. 3.6. Effects of sodium ion composition and extra-or intracellular calcium, magnesium ions on d-amphetamine-or procaine-elicited potential changes in the RP1 neuron 3.6.1. Lithium substituted for sodium solution Effects of lithium substitute solution (sodium-free solution) on d-amphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP, amplitude of action potential, and frequency of action potential on the RP1 neuron are shown in Tables 1 –3, respectively. Forty min after perfusing with lithium substituted for sodium solution,
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the RMP and amplitudes of the RP1 neurons were not significantly altered ( p > 0.05). However, the frequency of the action potential of the RP1 neurons was decreased ( p < 0.05). Sixty min after substituting lithium ion for sodium solution, the RMP, amplitude and frequency of action potential of the RP1 neurons were 53.3 T 2.9 mV, 69.3 T 1.7 mV and 105.8 T 3.7 pulses/min (n = 11), respectively. Thus the RMP and amplitude of the RP1 neurons were significantly decreased ( p < 0.05). The frequency of the action potentials of the RP1 neurons was increased ( p < 0.05). Eighty min after perfusion with the solution having lithium substituted for sodium, the RMP remained in the same depolarized state ( 52.4 T 1.3 mV), while the action potentials of the RP1 neurons were eliminated. The depolarized RMP and the action potentials of the neurons recovered to control level after 120 min of continuous washing with normal physiological solution. Compared with the neurons treated with lithium substituted (sodium-free) solution, the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were decreased in d-amphetamine (270 AM) and lithium-substituted (sodium-free) solution-treated preparations ( p < 0.05). No BoP was found in the lithium-substituted (sodium-free) solution and d-amphetamine-treated preparations tested. It appears that the lithium-substituted (sodiumfree) solution significantly decreased the BoP elicited by damphetamine (270 AM). Compared with the neurons treated with lithium-substituted (sodium-free) solution, the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were decreased in procaine (10 mM) and lithium substituted (sodium-free) solution treated preparations ( p < 0.05). No BoP was found in the lithium-substituted (sodium-free) solution and the procaine-treated preparations tested, indicating that the lithium-substituted (sodium-free) solution significantly decreased the BoP elicited by procaine (10 mM). 3.6.2. Ca2+-free solution Effects of Ca2+-free solution on d-amphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP, amplitude of action potential, and frequency of action potential on the RP1 neuron are shown in Tables 1– 3, respectively. Compared with the RMP, amplitudes and frequency of the action potentials of the RP1 neuron in normal saline, the RMP, amplitudes and frequency of the action potentials of the RP1 neuron had decreased ( p < 0.05) after 30 min of incubation in Ca2+-free solution. The effects were recovered to control level after 120 min of continuous washing with normal physiological solution. Compared with the neurons treated with Ca2+-free solution, the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were not altered in d-amphetamine (270 AM) and Ca2+-free solution treated preparations ( p > 0.05). BoP was found in Ca2+-free
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solution and d-amphetamine-treated preparations. Ca2+-free solution did not alter the BoP elicited by d-amphetamine. Compared with the neurons treated with Ca2+-free solution, the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were decreased in procaine (10 mM) and Ca2+-free solution treated preparations ( p < 0.05). BoP was found in Ca2+-free solution and procaine-treated preparations. Procaine still elicited BoP in preparations pretreated with Ca2+-free solution. The effects of intracellular injection of calcium ions on the potentials of the RP1 neuron were tested. The micropipettes containing calcium chloride (250 mM) were pressure injected using 200 mmHg for 2 s. Assuming that the volume of the RP1 neuron and the volume of calcium solution injected were 3.26 10 8 and 2.6 10 10 L, respectively, the injection increased the intracellular calcium concentration by 2 mM. The RMP was depolarized by 15.1 T1.3 mV (n = 7) immediately after intracellular injection, and BoP was also observed immediately after injection. The BoP elicited by intracellular injection of calcium ions were recovered to preinjection level 5 s after injection. The RP1 neuron was depolarized if injected for longer period of calcium ions. 3.6.3. High-magnesium solution Effects of high-magnesium solution (30 mM) on damphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP, amplitude of action potential, and frequency of action potential on the RP1 neuron are shown in Tables 1– 3, respectively. High-magnesium solution (30 mM) did not alter the RMP, amplitude and frequency of the action potential of the RP1 neuron ( p > 0.05). Compared with the neurons treated with high-magnesium solution (30 mM), the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were increased in d-amphetamine (270 AM) and high-magnesium solution (30 mM) treated preparations ( p < 0.05). BoP was found in the high-magnesium solution and d-amphetamine-treated preparations. Highmagnesium solution did not alter the pattern of BoP elicited by d-amphetamine. Compared with the neurons treated with high-magnesium solution (30 mM), the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were decreased in procaine-(10 mM) and high-magnesium solution (30 mM) treated preparations ( p < 0.05). No BoP was found in the high-magnesium solution and the procainetreated preparations tested. For intracellular injection of magnesium ion, magnesium chloride was injected using 500 mM MgCl2 (200 mmHg for 2 s). The injection increased the intracellular magnesium concentration by 4 mM, assuming the volume of the RP1 neuron and the volume of magnesium ion injected were 3.26 10 8 and 2.6 10 10 L, respectively. The mean RMP of control neurons was 61.5 T 1.4 mV (n = 6). One and thirty minutes after intracellular injection of magnesium
ion, the RMP were 58.3 T 2.4 mV (n = 6) and 57.3 T 1.6 mV (n = 6), respectively. It appears that the RMP was not altered 1 and 30 min after intracellular magnesium injection. The frequency of the action potential of the RP1 neuron in 1 and 30 min after intracellular injection of magnesium ion were 68.7 T 3.4 pulses/min (n = 6) and 50.4 T 2.6 pulses/min (n = 6), respectively. It appears that intracellular injection of magnesium ion increased the frequency of spontaneously generated action potentials for short periods. The BoP elicited by procaine (10 mM) perfusion decreased immediately after intracellular magnesium ion injection. Then, BoP was resumed 30 min after injection. The BoP elicited by d-amphetamine perfusion decreased immediately after intracellular magnesium ion injection. Then, BoP was resumed 10 min after injection. This indicates that intra-neuronal injection of magnesium ion blocked the BoP elicited by either by procaine or by d-amphetamine. 3.7. Effects of adrenergic and cholinergic blocking agents on d-amphetamine-or procaine-elicited potential changes on the RP1 neuron To test whether adrenergic or cholinergic receptors were involved in the d-amphetamine or procaine-elicited burst activity, the effects of prazosin and propranolol, the adrenergic blocking agents or atropine and d-tubocurarine, the cholinergic blocking agents on the d-amphetamine-or procaine-treated RP1 neuron were tested. The RMP, amplitudes of action potential and the frequency of the spontaneously generated action potential of the RP1 neurons were not altered after 2h of incubation with prazosin (100 AM), propranolol (100 AM), atropine (1 mM), d-tubocurarine (100 AM). However, BoP elicited by procaine (10 mM) or d-amphetamine (270 AM), still existed in prazosin (100 AM), propranolol (100 AM), atropine (1 mM), or d-tubocurarine (100 AM) treated preparations. Similar results were found in 5 of the preparations tested. This indicates that procaine (10 mM) or d-amphetamine (270 AM) still elicited BoP on the RP1 neuron in the presence of prazosin (100 AM), propranolol (100 AM), atropine (1 mM), or d-tubocurarine (100 AM).
4. Discussion The present study presents, the modulation effects of damphetamine and procaine on the spontaneously generated action potentials in the central neuron of the giant African snail, A. fulica Ferussac. Both d-amphetamine (Tsai and Chen, 1995) and procaine reversibly elicited BoP on the same central neuron, whether d-amphetamine or procaine was applied by extra-neuronal perfusion or intra-neuronal injection. However, intracellular injection of procaine and damphetamine produced short term hyper-excitability which is not identical to bath application, which results in longlasting effects for tens of minutes. The delay of the effects
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after extra-cellular application of both drugs suggested that the sites of action of both drugs are inside of the neuron. Our previous study revealed that forskolin (adenyl – cyclase activator), rolipram (phosphodiesterase inhibitor) (Lin et al., 2004) and m-3M3FBS (PLC activator) (unpublished results) elicited BoP on central snail neuron. Those results suggested that cAMP and PLC activity in the neuron was closely associated with the BoP. The pattern of the BoP elicited by d-amphetamine and procaine were not altered after chelerythrine chloride, Ro 31-8220 treatments. Chelerythrine is commonly used as a protein kinase C (PKC) inhibitor (Gainer, 1972), and Ro 318220 is a synthetic PKC inhibitor (Hurd et al., 2002). These results suggested that the BoP elicited by d-amphetamine or procaine were not associated with the activation of protein kinase C. However, the pattern of the BoP elicited by damphetamine and procaine were decreased after replacing the sodium ion with lithium ion in physiological saline. These results may be due to the effect of the lithium ion on the intracellular calcium ion. Intracellular accumulation of lithium displaced intracellular sodium, which in turn, decreased intracellular calcium (Hussain et al., 2002). Intra-neuronal injection of magnesium ion also abolished the BoP elicited by d-amphetamine or procaine. Intracellular injection of EGTA abolished the BoP, and intracellular injection of calcium ion elicited BoP in the central snail neuron (Chen and Tsai, 1996; Jakab and Bowyer, 2002). Ca2+ and cAMP signaling pathways are tightly interconnected and they reciprocally modulate each other (Derlet et al., 1990). The reciprocal modulation of messengers in the central snail neuron remains an interesting subject for further study. The lithium ion has also been shown to inhibit turnover of arachidonic acid (AA) by phospholipase A2 (PLA2) (Kavalali et al., 1997). Pretreatment with KT-5720 or H89 blocked d-amphetamine-elicited BoP, whereas KT-5720 or H89 did not alter the pattern of BoP elicited by procaine. Pretreatment of H8 or anisomycin also blocked the BoP elicited by d-amphetamine (Chen and Tsai, 1997). Furthermore, KT-5720, H89 and H8 were commonly used as protein kinase A inhibitors (Kim et al., 2000). It appeared that the protein kinase A activity in the neuron was closely associated with the generation of BoP elicited by d-amphetamine (Chen and Tsai, 1996, 1997, 2000), whereas the PKA activity was not associated with the generation of BoP elicited by procaine. This view was further supported by the finding that (a) in the preparation treated with d-amphetamine and KT-5720, procaine still elicited BoP on the same neuron; and (b) in the preparation pretreated with procaine followed by U73122, neomycin or high-magnesium solution which blocked the BoP elicited by procaine and BoP was further observed if damphetamine was further added. Neomycin and U73122 are commonly used as phospholipase C inhibitors (Kodama et al., 2001). A high-magnesium solution (30 mM) decreased the transmitter-releasing process of the nerve terminal (Lee and Linstedt, 2000). Magnesium sulfate also inhibited the
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phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activity (McDonald et al., 1994). U73122, neomycin or high-magnesium solution decreased the BoP elicited by procaine, whereas these compounds did not alter the BoP elicited by d-amphetamine. These results suggest that the phospholipase activity in the neuron was closely associated with the generation of BoP elicited by procaine, and this effect was not directly associated with the protein kinase A activity. It is concluded that PKA and PLC in the same neuron were closely associated to the modulation of potential changes elicited by d-amphetamine or procaine. Acknowledgements We are grateful to Dr. R.L. Walsh (Research Technology Branch, National Institute of Drug Abuse, U.S.A.) for the generous supply of d-amphetamine and to KT Lee foundation for the development of science and technology, Taiwan for the generous suggestions of the manuscript. This work was supported by grant, NSC-93-2320-B-002-130 from National Science Council, Taipei, Taiwan. References Brown, A.M., McCrohan, C.R., 1991. Differential responses of two identified neurons of the pond snail Lymnaea stagnalis to the convulsant drug pentylenetetrazol. Brain Res. 565, 247 – 253. Chen, Y.H., Tsai, M.C., 1996. Bursting firing of action potentials in central snail neurons elicited by d-amphetamine: role of the intracellular calcium ions. Comp. Biochem. Physiol., A 115, 195 – 205. Chen, Y.H., Tsai, M.C., 1997. Bursting firing of action potentials in central snail neurons elicited by d-amphetamine: role of cytoplasmic second messengers. Neurosci. Res. 27, 295 – 304. Chen, Y.H., Tsai, M.C., 2000. Action potential bursts in central snail neurons elicited by d-amphetamine: role of ionic currents. Neuroscience 96, 237 – 248. Derlet, R.W., Albertson, T.E., Rice, P., 1990. The effect of SCH 23390 against toxic doses of cocaine, d-amphetamine and methamphetamine. Life Sci. 47, 821 – 827. Gainer, H., 1972. Electrophysiological behavior of an endogenously active neurosecretory cell. Brain Res. 39, 403 – 418. Hurd, W.W., Natarajan, V., Fischer, J.R., Singh, D.M., Gibbs, S.G., Fomin, V.P., 2002. Magnesium sulfate inhibits the oxytocin-induced production of inositol 1,4,5-trisphosphate in cultured human myometrial cells. Am. J. Obstet. Gynecol. 187, 419 – 424. Hussain, S., Assender, J.W., Bond, M., Wong, L.F., Murphy, D., Newby, A.C., 2002. Activation of protein kinase Czeta is essential for cytokine-induced metalloproteinase-1,-3, and-9 secretion from rabbit smooth muscle cells and inhibits proliferation. J. Biol. Chem. 277, 27345 – 27352. Jakab, R.L., Bowyer, J.F., 2002. Parvalbumin neuron circuits and microglia in three dopamine-poor cortical regions remain sensitive to amphetamine exposure in the absence of hyperthermia, seizure and stroke. Brain Res. 958, 52 – 69. Kavalali, E.T., Hwang, K.S., Plummer, M.R., 1997. cAMP-dependent enhancement of dihydropyridine-sensitive calcium channel availability in hippocampal neurons. J. Neurosci. 17, 5334 – 5348. Kim, K.H., Takeuchi, H., Kamatani, Y., Minakata, H., Nomoto, K., 1991. Slow inward current induced by achatin-I, an endogenous peptide with a D-Phe residue. Eur. J. Pharmacol. 194, 99 – 106.
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Kim, M.J., Kim, E., Ryu, S.H., Suh, P.G., 2000. The mechanism of phospholipase C-gamma1 regulation. Exp. Mol. Med. 32, 101 – 109. Kodama, M., Yamada, N., Sato, K., Sato, T., Morimoto, K., Kuroda, S., 2001. The insular but not the perirhinal cortex is involved in the expression of fully-kindled amygdaloid seizures in rats. Epilepsy Res. 46, 69 – 78. Lee, T.H., Linstedt, A.D., 2000. Potential role for protein kinases in regulation of bidirectional endoplasmic reticulum-to-Golgi transport revealed by protein kinase inhibitor H89. Mol. Biol. Cell. 11, 2577 – 2590. Lin, P.J., Lin, C.H., Lin, P.L., Chen, I.M., Tsai, M.C., 2004. Regulation of action potential bursting by phosphodiesterase inhibitors in snail RP4 neurons. Changhua J. Med. 10, 162 – 171.
McDonald, T.F., Pelzer, S., Trautwein, W., Pelzer, D., 1994. Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. Physiol. Rev. 74, 365 – 507. Tsai, M.C., Chen, Y.H., 1995. Bursting firing of action potentials in central snail neurons elicited by d-amphetamine: role of the electrogenic sodium pump. Comp. Biochem. Physiol. C 111, 131 – 141. Watanabe, K., Funase, K., 1991. Cyclic AMP elicits biphasic current whose activation is mediated through protein phosphorylation in snail neurons. Neurosci. Res. 10, 64 – 70.
The modulation effects of d-amphetamine and procaine on the spontaneously generated action potentials in the central neuron of snail, Achatina fulica Ferussac Chia-Hsien Lin, Ming-Cheng Tsai* Department of Pharmacology, College of Medicine, National Taiwan University, No.1, Sec.1, Jen-Ai Road, Taipei, Taiwan Received 22 February 2005; received in revised form 5 May 2005; accepted 5 May 2005 Available online 9 June 2005
Abstract The modulation effects of d-amphetamine and procaine on the spontaneously generated action potentials were studied on the RP1 central neuron of giant African snails (Achatina fulica Ferussac). Extra-cellular application of d-amphetamine or procaine reversibly elicited bursts of potential (BoP). Prazosin, propranolol, atropine or d-tubocurarine did not alter the BoP elicited by either d-amphetamine or procaine. KT5720 or H89 (protein kinase A inhibitors) blocked d-amphetamine-elicited BoP, whereas they did not block the procaine-elicited BoP. U73122, neomycin (phospholipase C inhibitors) blocked the procaine-elicited BoP, whereas they did not block the d-amphetamine-elicited BoP in the same neuron. These results suggest that BoP elicited by d-amphetamine or procaine were associated with protein kinase A and phospholipase C activity in the neuron. D 2005 Elsevier Inc. All rights reserved. Keywords: Phospholipase; cAMP; d-amphetamine; Procaine; Neuron; Bursts of potential; Epilepsy; Synaptic transmission; Second messengers; Potential modulation
1. Introduction Physiological signals are integrated within the cell as a result of interactions between second messenger pathways. The cyclic adenosine 3V, 5V-monophosphate (cAMP) and the phosphoinositide (PI) signal-transduction pathways are the intracellular targets that mediate the cellular functions. cAMP-dependent phosphorylation modulated the dihydropyridine-sensitive calcium channels in cardiac myocytes (McDonald et al., 1994). Phospholipase C (PLC) hydrolyzed phosphatidylinositol 4, 5- bisphosphate to generate the second messengers, inositol 1, 4, 5- trisphosphate (IP3) and diacylglycerol (DAG). IP3 induced a transient increase in intracellular free Ca2+. The enzymes known to regulate cAMP levels, adenyl –cyclase and phosphodiesterase, and the functions of phospholipase C have been studied in
* Corresponding author. Tel.: +886 2 23966786; fax: +886 2 23915297. E-mail address: [email protected] (M.-C. Tsai). 1532-0456/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2005.05.003
detail. However, whether these messengers modulate potentials in the same central neuron previously remained unclear. In the present study, we found that both damphetamine and procaine elicited BoP on the same central neuron of giant African snails. The effects were altered after administration of protein kinase A (PKA) or phospholipase C (PLC) inhibitors. These results suggested that PKA and PLC in the neuron were closely associated to the modulation of potential changes in the RP1 neuron.
2. Materials and methods Experiments were performed on identified central RP1 neurons from the sub-esophageal ganglia of the African snail (Achatina fulica Ferussac). The ganglia were pinned to the bottom of a 2 mL Sylgard-coated perfusion chamber and carefully freed from the connective tissue sheath to allow easy identification and penetration by microelectrodes (Chen and Tsai, 2000). For intracellular recording, a Gene
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Clamp 500 amplifier (Axon Instruments, Inc., USA) was used. Micro-electrodes (5 – 6 MV) for recording membrane potentials were filled with 3 M KCl. The experimental chamber was perfused with control saline which had following composition (mM): NaCl, 85; KCl, 4; CaCl2, 8; MgCl2, 7; Tris –HCl, 10 (pH 7.6), at room temperature of 23 –24 -C with perfusion speed of 8 mL/min. The Ca2+-free solution was substituted for the calcium ion with equimolar amounts (8 mM) of CoCl2 (Kim et al., 1991). In the lithium substitute solution (sodium-free solution), NaCl (85 mM) was substituted with LiCl. For testing the effect of procaine or d-amphetamine on resting membrane potential (RMP), amplitudes of action potentials and the frequency of single spikes of action potentials of the RP1, RMP, amplitudes of action potentials and the frequency of single spikes of action potentials were recorded 60 min after procaine or d-amphetamine administration or recorded at the temporary steady-state level. To test the effects of prazosin, propranolol, atropine, dtubocurarine, KT-5720, H89, chelerythrine chloride, Ro 318220 or U73122 on d-amphetamine-or procaine-elicited potential changes, the RP1 neuron was first treated with damphetamine (270 AM) or procaine (10 mM) for 60 min, and then d-amphetamine or procaine were washed off as a pre-drug control. After the BoP elicited by d-amphetamine or procaine had recovered to the control level, prazosin, propranolol, atropine, d-tubocurarine, KT-5720, H89, chelerythrine chloride, Ro 31-8220 were added for 40 min, or U73122 were added for 60 min, and then d-amphetamine (270 AM) or procaine (10 mM) was further added for 60 min, and then washed off with normal saline for 120 min. To test the effects of neomycin on procaine-or damphetamine-elicited potential changes, the RP1 neuron was first treated with d-amphetamine or procaine for 60 min until BoP were observed, then, neomycin was further added for 120 min. To test the effects of calcium-free solution on d-amphetamine-or procaine-elicited potentials changes in RP1 neuron, the neuron was first treated with calcium-free solution for 30 min, then d-amphetamine or procaine were further added for 60 min. To test the effects of the high-magnesium solution or lithium substitute solution (sodium-free solution) on damphetamine-or procaine-elicited potential changes, the RP1 neuron was first treated with d-amphetamine (270 AM) or procaine (10 mM) for 60 min or until BoP were observed. Then a high-magnesium solution was further added for 30 min or lithium substitute solution (sodium-free solution) was further added for 40 min. Topical microperfusion was used for the rapid application of drugs, as described previously (Chen and Tsai, 1997, 2000). For intracellular injections of d-amphetamine or procaine, CaCl2, MgCl2, KCl into the RP1 neuron; d-amphetamine (13.5 mM), procaine (1 M), CaCl2 (250 mM), MgCl2 (500 mM) (Brown and McCrohan, 1991) were dissolved in 100 mM KCl (Watanabe and Funase, 1991) and the
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intracellular injection solutions were filled into micropipettes with tip diameter of about 1 Am adjusted by microforge (Narishige MF-83). Injections were made by a pulse under manual control over the course of the experiment. The duration of each injection pulse was 2 s with a pressure of 200 mmHg and the calculation of injected intracellular drug concentration was as described previously (Chen and Tsai, 1996). All potentials were recorded on tape by a digitalizing unit (Digidata 1200) and analyzed. The mean amplitude of the potentials after various treatments was compared with the pre-drug control by means of Student’s two-tailed t-test. Differences were considered significant at p < 0.05 (Chen and Tsai, 2000). Procaine, neomycin, H89, Ro 31-8220, U73122 (1-[6[((17h)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]1H-pyrrole-2,5-dione), prazosin, propranolol, atropine and d-tubocurarine were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). KT -5720 was purchased from Calbiochem Ltd. (USA). Chelerythrine chloride was purchased from Tocris (Bristol, UK). LiCl were purchased from Riedel-deHae¨n\ (USA). All drug stocks were made with double-distilled water except for prazosin, U73122 and KT-5720, which were prepared in dimethyl sulfoxide (DMSO). The presence of DMSO ( 0.1%) alone did not affect the RMP, amplitude and frequency of the spontaneous firing of action potential in the RP1 neuron.
3. Results 3.1. Identifiable RP1 neuron of Achatina fulica ferussac The central right parietal neuron 1 (RP1 neuron), the identified neuron in the sub-esophageal ganglia of the African snail A. fulica Ferussac (Chen and Tsai, 1996), was sensitive to several neurotransmitters. Glutamic acid (50 AM) induced hyperpolarization of the membrane potential. Serotonin (5-HT) (50 AM), g-aminobutyric acid (GABA) (50 AM), dopamine (50 AM) or acetylcholine (ACh) (50 AM) increased the frequency of the spontaneous action potential of the neuron. 3.2. Effects of d-amphetamine or procaine on the RP1 neuron The RP1 neuron had a RMP of 60.3 T 0.6 mV (n = 20, mean T S.E.M.), and it showed a spontaneous firing of action potential at a frequency of 58.7 T 1.2 pulses/min (n = 20). The action potentials showed regularly spaced single spikes, and no BoP was observed in the control RP1 neuron. The mean amplitude of the spontaneously generated action potential was 83.7 T 1.6 mV (n = 20). The effects of various concentrations of d-amphetamine on the RP1 neurons were tested. After 60 min of extracellular incubation of d-
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amphetamine (67.5 AM), the RMP, amplitude and frequency of the spontaneous firing action potential were 60.4 T 1.1, 85.6 T 1.1 and 57.5 T 1.2 pulses/min (n = 10), respectively. Higher concentrations of d-amphetamine (135 AM) increased the frequency of the action potential to 104.6 T 6.4 pulses/min (n = 10) ( p < 0.05) 60 min after damphetamine (135 AM) treatment. The RMP, amplitude of action potential and frequency of single spikes remained at the same level, even after 2h of d-amphetamine (135 AM) treatment. However, some phasic depolarization followed by a hyper-polarization phase was occasionally found 30 min after d-amphetamine (135 AM) treatment. Sixty min after d-amphetamine (270 AM) treatment, the firing patterns of the neuron had changed from regularly spaced single spikes to ones in which there were bursts of 8– 20 action potentials. Oscillation of membrane potentials with a phasic depolarization followed by a sustained depolarization with burst of action potential was found in d-amphetamine (270 AM)-treated preparations. At the end of the potential bursts, the neuron also elicited a hyper-polarization phase. The large hyper-polarization phase (hyperpolarization of membrane potential up to 8 mV), lasted 1 – 25 s after damphetamine administration. The frequency of bursts was 9.8 T 0.4 bursts/min (n = 10). The RMP of the neurons changed from 60.3 T 0.6 mV (n = 20) to –72.2 T 1.4 mV (n = 10) during the hyper-polarization phase. After the phasic depolarization and hyper-polarization phase, the RMP reached a temporary steady state level. There was no single-spike action potential 40 – 60 min after damphetamine (270 AM) treatment. The effect of d-amphetamine on the neuronal activities was reversible. After 120 min of continuous washing, the BoP of the central neuron returned to control levels, albeit with a slower frequency of
spontaneous firing. d-Amphetamine (270 AM) increased the RMP and amplitude of action potential and elicited BoP on the RP1 neuron. An example of the effects of different concentrations of d-amphetamine (67.5, 135, 270 AM) on the RP1 neuron is shown in Fig. 1A. The effect of intracellular injection of d-amphetamine on the spontaneously generated action potentials of the RP1 neuron was tested. Here, d-amphetamine was applied by micropipettes containing 13.5 mM d-amphetamine and 100 mM KCl. Immediately after d-amphetamine injection, BoP was observed. The RMP of the RP1 neuron were depolarized from 61.5 T 2.3 to 52.2 T 1.4 mV (n = 6). The system we used for intracellular injection will provide injection with about 2.6 10 10 L of the solution into the RP1 neuron. The concentration of d-amphetamine inside the neuron was approximately 108 AM, assuming the volume of the RP1 neuron was 3.26 10 8 L. The RMP and BoP elicited by intracellular injection of damphetamine were reversible. Ten minutes after intracellular injection, the RMPs were recovered to 60.9 T 0.8 mV (n = 6), indicating that intracellular injection of d-amphetamine elicited BoP in the RP1 neuron. Sixty min after procaine (1, 3 mM) administration, the frequency of the spontaneous firing of action potential had decreased from 58.7 T 1.2 pulses/min (control, n = 20) to 35.6 T 2.5 pulses/min ( p < 0.05, n = 10) and 26.5 T 1.6 pulses/ min, (n = 10) ( p < 0.05), respectively. Increasing procaine concentration to 10 mM elicited BoP 40 min after procaine administration, and these BoP lasted for more than 2 h. The firing pattern changed from regularly spaced single spikes to ones in which there were bursts of 2 –10 action potentials. The frequency of bursts was 5.8 T 0.5 bursts/min. The BoP elicited by procaine was recovered to control level after 120
Fig. 1. Effects of various concentrations of d-amphetamine or procaine on the central RP1 neuron of snails. A1 and B1 were control, showing spontaneous firing of action potential. A2, A3, A4 were the potentials 60 min after application of d-amphetamine (67.5, 135, or 270 AM), respectively. B2, B3, B4 were the potentials 60 min after application of procaine (1, 3 or 10 mM), respectively. A5 and B5 were the potentials 120 min after washing off from A4 and B4 with normal physiological solution, respectively. The horizontal bar at the top left side indicates the membrane potential at 0 mV. Noted that d-amphetamine (270 AM) and procaine (10 mM) elicited bursts of potential (BoP) on the RP1 neurons.
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Table 1 Effect of KT-5720, H89, chelerythrine chloride, Ro 31-8220, U73122, neomycin, lithium substitute solution (sodium-free solution), calcium-free solution and high-magnesium solution on d-amphetamine or procaine-elicited changes of the RMPs (mV) on the RP1 neuron Variable Control KT-5720 (10 AM) H89 (10 AM) Chelerythrine chloride (10 AM) Ro 31-8220 (10 AM) U73122 (10 AM) Neomycin (3.5 mM) LiCl Calcium-free solution High-magnesium solution (30 mM)
Physiological solution 60.3 T 0.6 (n = 20) 63.8 T 0.6 (n = 10) 63.7 T 1.8 (n = 9) 61.4 T 0.7 (n = 11) 62.7 T 1.9 (n = 8) 63.1 T1.3 (n = 10) 42.7 T 2.3a (n = 8) 60.7 T 0.9 (n = 11) 52.0 T 0.8a (n = 10) 58.7 T 2.7 (n = 8)
d-amphetamine (270 AM) 72.2 T 1.4a (n = 10) 63.9 T 1.6b (n = 5) 69.3 T 1.4b,d (n = 5) 69.5 T 0.9e (n = 4) 72.0 T 1.7f (n = 4) 61.0 T 3.2b (n = 4) 46.3 T 1.2b (n = 3) 51.3 T 1.5b,g (n = 5) 49.6 T 1.5b (n = 5) 69.0 T 2.6b,i (n = 4)
Procaine (10 mM) 58.3 T 0.9 (n = 10) 65.5 T 1.1c (n = 5) 64.7 T 1.7c (n = 4) 58.8 T 0.6 (n = 7) 60.1 T1.7 (n = 4) 59.5 T 0.9 (n = 6) 47.7 T 3.7c (n = 5) 47.8 T 0.7c,g (n = 6) 34.8 T 1.3c,h (n = 5) 47.3 T 0.7c,i (n = 4)
a
: Statistically significant compared with the data in physiological solution (control), p < 0.05. b : Statistically significant compared with d-amphetamine (270 AM) in physiological solution, p < 0.05. c : Statistically significant compared with procaine (10 mM) in physiological solution, p < 0.05. d : Statistically significant compared with H89 (10 AM) in physiological solution, p < 0.05. e : Statistically significant compared with chelerythrine chloride (10 AM) in physiological solution, p < 0.05. f : Statistically significant compared with Ro 31-8220 (10 AM) in physiological solution, p < 0.05. g : Statistically significant compared with LiCl in physiological solution, p < 0.05. h : Statistically significant compared with calcium-free solution in physiological solution, p < 0.05. i : Statistically significant compared with high Mg2+ (30 mM) in physiological solution, p < 0.05.
min of continuously washing off the procaine (Fig. 1B). Procaine did not alter the RMP and amplitude of the RP1 neuron at all concentrations tested (1 –10 mM), but procaine (1, 3 mM) did decrease the frequency of spontaneous action potential of the RP1 neuron. It appears that higher concentration of procaine (10 mM) reversibly elicited BoP on the RP1 neuron. An example of the effects of procaine (1– 10 mM) on the RP1 neuron is shown in Fig. 1B. The effects of intracellular injection of procaine on the spontaneously generated action potentials of the RP1 neuron were tested. Procaine was applied by micropipettes containing 1 M procaine and 100 mM KCl. Immediately after procaine injection, BoP was observed. The RMP of the RP1 neuron were depolarized from 61.5 T 2.3 to 54.2 T 1.6 mV (n = 6). The concentration of procaine inside the neuron was approximately 8 mM, assuming the volume of the RP1 neuron was 3.26 10 8 L (Chen and Tsai, 1996). The RMP and BoP elicited by intracellular injection of procaine were
reversible. Ten minutes after intracellular injection, the RMP had recovered to 60.9 T 0.8 mV (n = 6). The RP1 neuron was depolarized if injected for longer period of procaine, indicating that intracellular injection of procaine elicited BoP in the RP1 neuron. 3.3. Effects of protein kinase A inhibitors on d-amphetamine-or procaine-elicited burst activity of the RP1 neuron To examine the role of cAMP-dependent protein kinase in the generation of burst activity elicited by d-amphetamine or procaine, the effects of KT-5720 or H89, inhibitors of protein kinase A, were studied. The RMP, amplitude and frequency of the spontaneous firing action potential of the RP1 neuron with d-amphetamine (270 AM) or procaine (10 mM) and KT-5720 (10 AM) are shown in Tables 1– 3, respectively. KT-5720 reversibly decreased the frequency of the spontaneous action potential
Table 2 Effect of KT-5720, H89, chelerythrine chloride, Ro 31-8220, U73122, neomycin, lithium substitute solution (sodium-free solution), calcium-free solution and high-magnesium solution on d-amphetamine or procaine-elicited changes of the amplitude of action potential (mV) on the RP1 neuron Variable
Physiological solution
d-amphetamine (270 AM)
Procaine (10 mM)
Control KT-5720 (10 AM) H89 (10 AM) Chelerythrine chloride (10 AM) Ro 31-8220 (10 AM) U73122 (10 AM) Neomycin (3.5 mM) LiCl Calcium-free solution High-magnesium solution (30 mM)
83.7 T 1.6 (n = 20) 87.2 T 0.7 (n = 10) 83.4 T 2.3 (n = 9) 85.7 T 0.6 (n = 11) 83.8 T 1.1 (n = 8) 84.9 T 2.7 (n = 10) 51.1 T 2.6a (n = 8) 86.0 T 1.0 (n = 11) 66.0 T 1.0a (n = 10) 85.2 T 0.4 (n = 8)
97.5 T 1.6a (n = 10) 88.9 T 1.8b (n = 5) 89.3 T 1.9b,d (n = 5) 94.5 T 0.7e (n = 4) 94.0 T 2.2f (n = 4) 80.0 T 5.7b (n = 4) 59.5 T 3.0b (n = 3) 59.8 T 3.2b,g (n = 5) 63.0 T 1.9b (n = 5) 92.0 T 1.2i (n = 4)
79.5 T 1.2 (n = 10) 89.3 T 1.3c (n = 5) 85.4 T 1.9c (n = 4) 80.6 T 0.6e (n = 7) 84.7 T 1.5 (n = 4) 79.1 T1.3 (n = 6) 54.2 T 2.4c (n = 5) 64.4 T 0.9c,g (n = 6) 43.6 T 1.1c,h (n = 5) 64.6 T 0.9c,i (n = 4)
a : Statistically significant compared with the data in physiological solution (control), p < 0.05. b : Statistically significant compared with d-amphetamine (270 AM) in physiological solution, p < 0.05. c : Statistically significant compared with procaine (10 mM) in physiological solution, p < 0.05. d : Statistically significant compared with H89 (10 AM) in physiological solution, p < 0.05. e : Statistically significant compared with chelerythrine chloride (10 AM) in physiological solution, p < 0.05. f : Statistically significant compared with Ro 31-8220 (10 AM) in physiological solution, p < 0.05. g : Statistically significant compared with LiCl in physiological solution, p < 0.05. h : Statistically significant compared with calcium-free solution in physiological solution, p < 0.05. i : Statistically significant compared with high Mg2+ (30 mM) in physiological solution, p < 0.05.
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Table 3 Effect of KT-5720, H89, chelerythrine chloride, Ro 31-8220, U73122, neomycin, lithium substitute solution (sodium-free solution), calcium-free solution and high-magnesium solution on d-amphetamine or procaine-elicited changes on frequency of spikes of the RP1 neuron Variable
Physiological solution
d-amphetamine (270 AM)
Procaine (10 mM)
Control KT-5720 (10 AM) H89 (10 AM) Chelerythrine chloride (10 AM) Ro 31-8220 (10 AM) U73122 (10 AM) Neomycin (3.5 mM) LiCl Calcium-free solution High-magnesium solution (30 mM)
58.7 T 1.2 pulses/min (n = 20) 34.8 T 1.5a pulses/min (n = 10) 39.0 T 2.4a pulses/min (n = 8) 55.0 T 1.4 pulses/min (n = 11) 55.3 T 2.6 pulses/min (n = 8) 57.1 T 2.2 pulses/min (n = 10) 74.0 T 5.2a pulses/min (n = 8) 31.0 T 1.7a pulses/min (n = 11) 41.7 T 2.1a pulses/min (n = 10) 51.5 T 5.5 pulses/min (n = 8)
9.8 T 0.4 bursts/min (n = 10) 52.9 T 3.5d pulses/min (n = 5) 53.5 T 2.5e pulses /min (n = 5) 13.4 T 0.2b bursts/min (n = 4) 11.6 T 0.3b bursts/min (n = 4) 5.0 T 0.6b bursts/min (n = 4) 5.3 T 1.5b bursts/min(n = 3) 68.0 T 3.3h pulses /min(n = 5) 2.3 T 0.3b bursts/min (n = 5) 7.3 T 1.8 bursts/min (n = 4)
5.8 T 0.5 bursts/min (n = 10) 5.1 T1.3 bursts/min (n = 5) 5.1 T 2.2 bursts/min (n = 4) 7.1 T1.4c bursts/min (n = 7) 6.3 T 1.9 bursts/min (n = 4) 32.3 T 1.4f pulses/min (n = 6) 9.7 T 1.5g pulses/min (n = 5) 43.0 T 3.2h pulses/min (n = 6) 3.4 T 0.4c bursts/min (n = 5) 8.0 T 1.0i pulses/min (n = 4)
a
: Statistically significant compared with the data in physiological solution (control), p < 0.05. b : Statistically significant compared with d-amphetamine (270 AM) in physiological solution, p < 0.05. c : Statistically significant compared with procaine (10 mM) in physiological solution, p < 0.05. d : Statistically significant compared with KT-5720 (10 AM) in physiological solution, p < 0.05. e : Statistically significant compared with H89 (10 AM) in physiological solution, p < 0.05. f : Statistically significant compared with U73122 (10 AM) in physiological solution, p < 0.05. g : Statistically significant compared with neomycin (3.5 mM) in physiological solution, p < 0.05. h : Statistically significant compared with LiCl in physiological solution, p < 0.05. i : Statistically significant compared with high Mg2+ (30 mM) in physiological solution, p < 0.05.
of the neuron ( p < 0.05), whereas it did not alter the RMP and amplitude of the action potential of the RP1 neuron ( p > 0.05). No BoP was found in the KT-5720-and d-amphetaminetreated preparations tested. Compared with the neurons treated with KT-5720 (10 AM), the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were not altered in d-amphetamine-(270 AM) and KT-5720-(10 AM) treated preparations ( p > 0.05), and an example of the effects of KT-5720 on the d-amphetamineelicited potential changes is shown in Fig. 2A.
The RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were not altered in procaine-(10 mM) and KT-5720-(10 AM) treated preparations ( p > 0.05) compared with the neurons treated with KT5720 (10 AM). BoP was found in KT-5720-and procainetreated preparations. Procaine (10 mM) still elicited BoP even after increasing KT-5720 concentration to 100 AM. An example of the effects of KT-5720 on the procaine-elicited potential changes is shown in Fig. 2B. In order to test the role of PKA on the BoP elicited by damphetamine and procaine, the RP1 neuron was first treated
Fig. 2. Effects of KT-5720 (10 AM) on the d-amphetamine-or procaine-elicited BoP on the central RP1 neurons. A1 and B1 were control, showing spontaneous firing of action potential. A2 and B2 were the potentials of the neuron 40 min after application with KT-5720 (10 AM). A3 and B3 were the potentials 60 min after further incubation with d-amphetamine (270 AM) or procaine (10 mM) from A2, B2, respectively. A4 and B4 were the potentials 120 min after washing off from A3 and B3 with normal physiological solution, respectively. The horizontal bar at the top left side indicates the membrane potential at 0 mV. KT-5720 blocked the d-amphetamine-elicited BoP, while KT-5720 did not block the procaine-elicited BoP on the RP1 neuron.
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with d-amphetamine until BoP appeared. Then KT-5720 was added until BoP elicited by d-amphetamine had shifted to single spikes, and then procaine was further added. BoP was found in 4 of the preparations tested, and an example is shown in Fig. 3A. KT-5720 blocked the potentials elicited by d-amphetamine, whereas it did not block the BoP elicited by procaine. Effects of H89 (10 AM) on d-amphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP (mV), amplitude of action potential (mV), and frequency of action potential on the RP1 neuron are shown in Tables 1 –3, respectively. H89 reversibly decreased the frequency of spontaneous action potential of the neuron ( p < 0.05), whereas it did not alter the RMP and amplitudes of the action potential of the RP1 neuron ( p > 0.05). The RMP, amplitude and frequency of BoP in the RP1 neuron after sixty minutes incubation in d-amphetamine (270 AM) and H89 (10 AM) are shown in Tables 1 –3, respectively. Compared with the neurons treated with H89 (10 AM), the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron increased in damphetamine-(270 AM) and H89-(10 AM) treated preparations ( p < 0.05). Furthermore, H89 significantly decreased the BoP elicited by d-amphetamine. The RMP, amplitude and frequency of BoP in the RP1 neuron after sixty minutes incubation in procaine (10 mM) and H89 (10 AM) are shown in Tables 1 –3, respectively.
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Compared with the neurons treated with H89 (10 AM), the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were not altered in procaine-(10 mM) and H89-(10 AM) treated preparations ( p > 0.05). BoP was found in H89-and procaine-treated preparations. H89 did not alter the BoP elicited by procaine. 3.4. Effects of protein kinase C inhibitors on d-amphetamine-or procaine-elicited bursting activity of the RP1 neuron To examine whether protein kinase C was involved in the generation of burst firing of the frequency of spontaneously generated action potentials of the RP1 neuron, the effects of chelerythrine chloride or Ro 31-8220, inhibitors of protein kinase C, on the d-amphetamine-or procaine-elicited bursting activity of potentials were tested. Effects of chelerythrine chloride (10 AM) on d-amphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP (mV), amplitude of action potential (mV), and frequency of action potential on the RP1 neuron are shown in Tables 1 –3, respectively. Chelerythrine chloride did not alter the RMP, amplitude and frequency of the action potential of the RP1 neuron ( p > 0.05). Compared with the neurons treated with chelerythrine chloride (10 AM), the RMP and amplitude of the sponta-
Fig. 3. Effects of KT-5720 (10 AM) and U73122 (10 AM) on d-amphetamine-or procaine-elicited BoP on the central RP1 neuron of snails. A1 and B1 were control, showing spontaneous firing of action potential. A2 and B2 were the potentials 60 min after addition of d-amphetamine (270 AM) or procaine (10 mM) from A1 and B1, respectively. A3 was the potentials after addition KT-5720 (10 AM) for 60 min from A2. A4 was the potentials after further addition of procaine (10 mM) for 60 min from A3. B3 was the potentials after addition U73122 (10 AM) for 60 min from B2. B4 was the potentials after further addition of d-amphetamine (270 AM) for 60 min from B3. A5 and B5 were the potentials after washing off with normal physiological solution for 120 min from A4 and B4, respectively. The horizontal bar at the top left side indicates the membrane potential at 0 mV. Notes that procaine-elicited BoP in the preparation treated with both d-amphetamine and KT-5720 (A4) and d-amphetamine-elicited BoP in the preparation treated with procaine and U73122 (B4).
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neously generated action potential of the RP1 neuron were increased in d-amphetamine-(270 AM) and chelerythrine chloride-(10 AM) treated preparations ( p < 0.05). d-Amphetamine (270 AM) still elicited BoP in a preparation pretreated with chelerythrine chloride (10 AM). Compared to the neurons treated with chelerythrine chloride (10 AM), the RMP were not altered ( p > 0.05), however the amplitude of the spontaneously generated action potential of the RP1 neuron treated preparations were decreased in procaine-(10 mM) and chelerythrine chloride-(10 AM) treated preparations ( p < 0.05). Procaine still elicited BoP in preparations pretreated with chelerythrine chloride (10 AM). Effects of Ro 31-8220 (10 AM) on d-amphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP (mV), amplitude of action potential (mV), and frequency of action potential on the RP1 neuron are shown in Tables 1– 3, respectively. Ro 31-8220 did not alter the RMP, amplitude or frequency of the action potential of the RP1 neuron ( p > 0.05). The RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were increased in damphetamine-(270 AM) and Ro 31-8220-(10 AM) treated preparations compared with the neurons treated with Ro 318220 (10 AM). BoP was found in Ro 31-8220-and damphetamine-treated preparations. d-Amphetamine (270 AM) still elicited BoP in Ro 31-8220 (10 AM) pre-treated preparation. The RMP and amplitude of the spontaneously generated action potential of the RP1 neuron treated preparations were not altered in procaine-(10 mM) and Ro 31-8220-(10 AM)
treated preparations ( p > 0.05) compared with the neurons treated with Ro 31-8220 (10 AM). BoP was found in Ro 318220-and procaine-treated preparations. Procaine still elicited BoP in preparations pretreated with Ro 31-8220 (10 AM). 3.5. Effects of phospholipase C inhibitors on d-amphetamine-or procaine-elicited potential changes in the RP1 neuron To examine whether phospholipase C was involved in the generation of burst firing of the frequency of spontaneously generated action potentials of the RP1 neuron, the effects of U73122 or neomycin, inhibitors of phospholipase C, on the d-amphetamine-or procaine-elicited bursting activity of potentials were tested. Effects of U73122 (10 AM) on d-amphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP (mV), amplitude of action potential (mV), and frequency of action potential on the RP1 neuron are shown in Tables 1– 3, respectively. U73122 did not alter the RMP, amplitude and frequency of the action potential of the RP1 neuron ( p > 0.05). The RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were not altered in damphetamine-(270 AM) and U73122-(10 AM) treated preparations ( p > 0.05) compared with the neurons treated with U73122 (10 AM). BoP was found in U73122-and d-amphetamine-treated preparations, and an example of the effects of U73122 on the d-amphetamine-elicited potential changes is shown in Fig. 4A. d-Amphetamine (270 AM) still elicited BoP in preparation pre-treated with U73122 (10 AM).
Fig. 4. Effects of U73122 (10 AM) (PLC inhibitor) on d-amphetamine-or procaine-elicited BoP on the central RP1 neuron of snails. A1 and B1 were control, showing spontaneous firing of action potential. A2 and B2 were the potentials of the neuron 60 min after application with U73122 (10 AM). A3 and A4 were the potentials 30, 60 min after addition d-amphetamine (270 AM) from A2, respectively. B3 and B4 were the potentials after addition procaine (10 mM) 30, 60 min, respectively from B2. A5 and B5 were the potentials 120 min after washing off from A4 and B4 with normal physiological solution, respectively. The horizontal bar at the top left side indicates the membrane potential at 0 mV. Pretreatment with U73122 blocked the procaine, but it did not block the damphetamine-elicited BoP.
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No BoP was found in U73122-and procaine-treated preparations tested. The RMP and amplitude of the spontaneously generated action potential of the RP1 neuron treated preparations were not altered in procaine (10 mM) and U73122 (10 AM) treated preparations ( p > 0.05) compared with the neurons treated with U73122 (10 AM), and an example of the effects of U73122 on the procaineelicited potential changes is shown in Fig. 4B. In order to test the role of PLC (phospholipase C) on the BoP elicited by d-amphetamine and procaine, the RP1 neuron was first treated with procaine until BoP appeared. Then U73122 (phospholipase C inhibitor) was added until BoP elicited by procaine had shifted to single spikes, and then d-amphetamine was further added. BoP was found in 4 of the preparations tested, and an example is shown in Fig. 3B. U73122 blocked the potentials elicited by procaine, whereas it did not block the BoP elicited by d-amphetamine. Effects of neomycin (3.5 mM) on d-amphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP (mV), amplitude of action potential (mV), and frequency of action potential on the RP1 neuron are shown in Tables 1 – 3, respectively. At 120 min after incubation with neomycin (3.5 mM), neomycin decreased the RMP and amplitudes of the action potentials of the neuron, whereas it increased the frequency of spontaneous action potentials of the RP1 neuron ( p < 0.05). The spontaneous action potentials of the RP1 neuron recovered to control level after 120 min of continuous washing with normal physiological solution. Compared with the neurons treated with neomycin (3.5 mM), the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were not altered in d-amphetamine-(270 AM) and neomycin-(3.5 mM) treated preparations ( p > 0.05). BoP was found in neomycin-and d-amphetamine-treated preparations. Neomycin did not alter the BoP elicited by d-amphetamine. Compared with the neurons treated with neomycin (3.5 mM), the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were not altered in procaine-(10 mM) and neomycin-(3.5 mM) treated preparations ( p > 0.05). No BoP was found in the neomycin-and procaine-treated preparations tested, indicating that neomycin significantly altered the BoP elicited by procaine. 3.6. Effects of sodium ion composition and extra-or intracellular calcium, magnesium ions on d-amphetamine-or procaine-elicited potential changes in the RP1 neuron 3.6.1. Lithium substituted for sodium solution Effects of lithium substitute solution (sodium-free solution) on d-amphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP, amplitude of action potential, and frequency of action potential on the RP1 neuron are shown in Tables 1 –3, respectively. Forty min after perfusing with lithium substituted for sodium solution,
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the RMP and amplitudes of the RP1 neurons were not significantly altered ( p > 0.05). However, the frequency of the action potential of the RP1 neurons was decreased ( p < 0.05). Sixty min after substituting lithium ion for sodium solution, the RMP, amplitude and frequency of action potential of the RP1 neurons were 53.3 T 2.9 mV, 69.3 T 1.7 mV and 105.8 T 3.7 pulses/min (n = 11), respectively. Thus the RMP and amplitude of the RP1 neurons were significantly decreased ( p < 0.05). The frequency of the action potentials of the RP1 neurons was increased ( p < 0.05). Eighty min after perfusion with the solution having lithium substituted for sodium, the RMP remained in the same depolarized state ( 52.4 T 1.3 mV), while the action potentials of the RP1 neurons were eliminated. The depolarized RMP and the action potentials of the neurons recovered to control level after 120 min of continuous washing with normal physiological solution. Compared with the neurons treated with lithium substituted (sodium-free) solution, the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were decreased in d-amphetamine (270 AM) and lithium-substituted (sodium-free) solution-treated preparations ( p < 0.05). No BoP was found in the lithium-substituted (sodium-free) solution and d-amphetamine-treated preparations tested. It appears that the lithium-substituted (sodiumfree) solution significantly decreased the BoP elicited by damphetamine (270 AM). Compared with the neurons treated with lithium-substituted (sodium-free) solution, the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were decreased in procaine (10 mM) and lithium substituted (sodium-free) solution treated preparations ( p < 0.05). No BoP was found in the lithium-substituted (sodium-free) solution and the procaine-treated preparations tested, indicating that the lithium-substituted (sodium-free) solution significantly decreased the BoP elicited by procaine (10 mM). 3.6.2. Ca2+-free solution Effects of Ca2+-free solution on d-amphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP, amplitude of action potential, and frequency of action potential on the RP1 neuron are shown in Tables 1– 3, respectively. Compared with the RMP, amplitudes and frequency of the action potentials of the RP1 neuron in normal saline, the RMP, amplitudes and frequency of the action potentials of the RP1 neuron had decreased ( p < 0.05) after 30 min of incubation in Ca2+-free solution. The effects were recovered to control level after 120 min of continuous washing with normal physiological solution. Compared with the neurons treated with Ca2+-free solution, the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were not altered in d-amphetamine (270 AM) and Ca2+-free solution treated preparations ( p > 0.05). BoP was found in Ca2+-free
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solution and d-amphetamine-treated preparations. Ca2+-free solution did not alter the BoP elicited by d-amphetamine. Compared with the neurons treated with Ca2+-free solution, the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were decreased in procaine (10 mM) and Ca2+-free solution treated preparations ( p < 0.05). BoP was found in Ca2+-free solution and procaine-treated preparations. Procaine still elicited BoP in preparations pretreated with Ca2+-free solution. The effects of intracellular injection of calcium ions on the potentials of the RP1 neuron were tested. The micropipettes containing calcium chloride (250 mM) were pressure injected using 200 mmHg for 2 s. Assuming that the volume of the RP1 neuron and the volume of calcium solution injected were 3.26 10 8 and 2.6 10 10 L, respectively, the injection increased the intracellular calcium concentration by 2 mM. The RMP was depolarized by 15.1 T1.3 mV (n = 7) immediately after intracellular injection, and BoP was also observed immediately after injection. The BoP elicited by intracellular injection of calcium ions were recovered to preinjection level 5 s after injection. The RP1 neuron was depolarized if injected for longer period of calcium ions. 3.6.3. High-magnesium solution Effects of high-magnesium solution (30 mM) on damphetamine-(270 AM) or procaine-(10 mM) elicited changes of the RMP, amplitude of action potential, and frequency of action potential on the RP1 neuron are shown in Tables 1– 3, respectively. High-magnesium solution (30 mM) did not alter the RMP, amplitude and frequency of the action potential of the RP1 neuron ( p > 0.05). Compared with the neurons treated with high-magnesium solution (30 mM), the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were increased in d-amphetamine (270 AM) and high-magnesium solution (30 mM) treated preparations ( p < 0.05). BoP was found in the high-magnesium solution and d-amphetamine-treated preparations. Highmagnesium solution did not alter the pattern of BoP elicited by d-amphetamine. Compared with the neurons treated with high-magnesium solution (30 mM), the RMP and amplitude of the spontaneously generated action potential of the RP1 neuron were decreased in procaine-(10 mM) and high-magnesium solution (30 mM) treated preparations ( p < 0.05). No BoP was found in the high-magnesium solution and the procainetreated preparations tested. For intracellular injection of magnesium ion, magnesium chloride was injected using 500 mM MgCl2 (200 mmHg for 2 s). The injection increased the intracellular magnesium concentration by 4 mM, assuming the volume of the RP1 neuron and the volume of magnesium ion injected were 3.26 10 8 and 2.6 10 10 L, respectively. The mean RMP of control neurons was 61.5 T 1.4 mV (n = 6). One and thirty minutes after intracellular injection of magnesium
ion, the RMP were 58.3 T 2.4 mV (n = 6) and 57.3 T 1.6 mV (n = 6), respectively. It appears that the RMP was not altered 1 and 30 min after intracellular magnesium injection. The frequency of the action potential of the RP1 neuron in 1 and 30 min after intracellular injection of magnesium ion were 68.7 T 3.4 pulses/min (n = 6) and 50.4 T 2.6 pulses/min (n = 6), respectively. It appears that intracellular injection of magnesium ion increased the frequency of spontaneously generated action potentials for short periods. The BoP elicited by procaine (10 mM) perfusion decreased immediately after intracellular magnesium ion injection. Then, BoP was resumed 30 min after injection. The BoP elicited by d-amphetamine perfusion decreased immediately after intracellular magnesium ion injection. Then, BoP was resumed 10 min after injection. This indicates that intra-neuronal injection of magnesium ion blocked the BoP elicited by either by procaine or by d-amphetamine. 3.7. Effects of adrenergic and cholinergic blocking agents on d-amphetamine-or procaine-elicited potential changes on the RP1 neuron To test whether adrenergic or cholinergic receptors were involved in the d-amphetamine or procaine-elicited burst activity, the effects of prazosin and propranolol, the adrenergic blocking agents or atropine and d-tubocurarine, the cholinergic blocking agents on the d-amphetamine-or procaine-treated RP1 neuron were tested. The RMP, amplitudes of action potential and the frequency of the spontaneously generated action potential of the RP1 neurons were not altered after 2h of incubation with prazosin (100 AM), propranolol (100 AM), atropine (1 mM), d-tubocurarine (100 AM). However, BoP elicited by procaine (10 mM) or d-amphetamine (270 AM), still existed in prazosin (100 AM), propranolol (100 AM), atropine (1 mM), or d-tubocurarine (100 AM) treated preparations. Similar results were found in 5 of the preparations tested. This indicates that procaine (10 mM) or d-amphetamine (270 AM) still elicited BoP on the RP1 neuron in the presence of prazosin (100 AM), propranolol (100 AM), atropine (1 mM), or d-tubocurarine (100 AM).
4. Discussion The present study presents, the modulation effects of damphetamine and procaine on the spontaneously generated action potentials in the central neuron of the giant African snail, A. fulica Ferussac. Both d-amphetamine (Tsai and Chen, 1995) and procaine reversibly elicited BoP on the same central neuron, whether d-amphetamine or procaine was applied by extra-neuronal perfusion or intra-neuronal injection. However, intracellular injection of procaine and damphetamine produced short term hyper-excitability which is not identical to bath application, which results in longlasting effects for tens of minutes. The delay of the effects
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after extra-cellular application of both drugs suggested that the sites of action of both drugs are inside of the neuron. Our previous study revealed that forskolin (adenyl – cyclase activator), rolipram (phosphodiesterase inhibitor) (Lin et al., 2004) and m-3M3FBS (PLC activator) (unpublished results) elicited BoP on central snail neuron. Those results suggested that cAMP and PLC activity in the neuron was closely associated with the BoP. The pattern of the BoP elicited by d-amphetamine and procaine were not altered after chelerythrine chloride, Ro 31-8220 treatments. Chelerythrine is commonly used as a protein kinase C (PKC) inhibitor (Gainer, 1972), and Ro 318220 is a synthetic PKC inhibitor (Hurd et al., 2002). These results suggested that the BoP elicited by d-amphetamine or procaine were not associated with the activation of protein kinase C. However, the pattern of the BoP elicited by damphetamine and procaine were decreased after replacing the sodium ion with lithium ion in physiological saline. These results may be due to the effect of the lithium ion on the intracellular calcium ion. Intracellular accumulation of lithium displaced intracellular sodium, which in turn, decreased intracellular calcium (Hussain et al., 2002). Intra-neuronal injection of magnesium ion also abolished the BoP elicited by d-amphetamine or procaine. Intracellular injection of EGTA abolished the BoP, and intracellular injection of calcium ion elicited BoP in the central snail neuron (Chen and Tsai, 1996; Jakab and Bowyer, 2002). Ca2+ and cAMP signaling pathways are tightly interconnected and they reciprocally modulate each other (Derlet et al., 1990). The reciprocal modulation of messengers in the central snail neuron remains an interesting subject for further study. The lithium ion has also been shown to inhibit turnover of arachidonic acid (AA) by phospholipase A2 (PLA2) (Kavalali et al., 1997). Pretreatment with KT-5720 or H89 blocked d-amphetamine-elicited BoP, whereas KT-5720 or H89 did not alter the pattern of BoP elicited by procaine. Pretreatment of H8 or anisomycin also blocked the BoP elicited by d-amphetamine (Chen and Tsai, 1997). Furthermore, KT-5720, H89 and H8 were commonly used as protein kinase A inhibitors (Kim et al., 2000). It appeared that the protein kinase A activity in the neuron was closely associated with the generation of BoP elicited by d-amphetamine (Chen and Tsai, 1996, 1997, 2000), whereas the PKA activity was not associated with the generation of BoP elicited by procaine. This view was further supported by the finding that (a) in the preparation treated with d-amphetamine and KT-5720, procaine still elicited BoP on the same neuron; and (b) in the preparation pretreated with procaine followed by U73122, neomycin or high-magnesium solution which blocked the BoP elicited by procaine and BoP was further observed if damphetamine was further added. Neomycin and U73122 are commonly used as phospholipase C inhibitors (Kodama et al., 2001). A high-magnesium solution (30 mM) decreased the transmitter-releasing process of the nerve terminal (Lee and Linstedt, 2000). Magnesium sulfate also inhibited the
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phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activity (McDonald et al., 1994). U73122, neomycin or high-magnesium solution decreased the BoP elicited by procaine, whereas these compounds did not alter the BoP elicited by d-amphetamine. These results suggest that the phospholipase activity in the neuron was closely associated with the generation of BoP elicited by procaine, and this effect was not directly associated with the protein kinase A activity. It is concluded that PKA and PLC in the same neuron were closely associated to the modulation of potential changes elicited by d-amphetamine or procaine. Acknowledgements We are grateful to Dr. R.L. Walsh (Research Technology Branch, National Institute of Drug Abuse, U.S.A.) for the generous supply of d-amphetamine and to KT Lee foundation for the development of science and technology, Taiwan for the generous suggestions of the manuscript. This work was supported by grant, NSC-93-2320-B-002-130 from National Science Council, Taipei, Taiwan. References Brown, A.M., McCrohan, C.R., 1991. Differential responses of two identified neurons of the pond snail Lymnaea stagnalis to the convulsant drug pentylenetetrazol. Brain Res. 565, 247 – 253. Chen, Y.H., Tsai, M.C., 1996. Bursting firing of action potentials in central snail neurons elicited by d-amphetamine: role of the intracellular calcium ions. Comp. Biochem. Physiol., A 115, 195 – 205. Chen, Y.H., Tsai, M.C., 1997. Bursting firing of action potentials in central snail neurons elicited by d-amphetamine: role of cytoplasmic second messengers. Neurosci. Res. 27, 295 – 304. Chen, Y.H., Tsai, M.C., 2000. Action potential bursts in central snail neurons elicited by d-amphetamine: role of ionic currents. Neuroscience 96, 237 – 248. Derlet, R.W., Albertson, T.E., Rice, P., 1990. The effect of SCH 23390 against toxic doses of cocaine, d-amphetamine and methamphetamine. Life Sci. 47, 821 – 827. Gainer, H., 1972. Electrophysiological behavior of an endogenously active neurosecretory cell. Brain Res. 39, 403 – 418. Hurd, W.W., Natarajan, V., Fischer, J.R., Singh, D.M., Gibbs, S.G., Fomin, V.P., 2002. Magnesium sulfate inhibits the oxytocin-induced production of inositol 1,4,5-trisphosphate in cultured human myometrial cells. Am. J. Obstet. Gynecol. 187, 419 – 424. Hussain, S., Assender, J.W., Bond, M., Wong, L.F., Murphy, D., Newby, A.C., 2002. Activation of protein kinase Czeta is essential for cytokine-induced metalloproteinase-1,-3, and-9 secretion from rabbit smooth muscle cells and inhibits proliferation. J. Biol. Chem. 277, 27345 – 27352. Jakab, R.L., Bowyer, J.F., 2002. Parvalbumin neuron circuits and microglia in three dopamine-poor cortical regions remain sensitive to amphetamine exposure in the absence of hyperthermia, seizure and stroke. Brain Res. 958, 52 – 69. Kavalali, E.T., Hwang, K.S., Plummer, M.R., 1997. cAMP-dependent enhancement of dihydropyridine-sensitive calcium channel availability in hippocampal neurons. J. Neurosci. 17, 5334 – 5348. Kim, K.H., Takeuchi, H., Kamatani, Y., Minakata, H., Nomoto, K., 1991. Slow inward current induced by achatin-I, an endogenous peptide with a D-Phe residue. Eur. J. Pharmacol. 194, 99 – 106.
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