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Effects of rolipram on induction of action potential bursts in central snail neurons
Experimental Neurology 194 (2005) 384 – 392 www.elsevier.com/locate/yexnr
Effects of rolipram on induction of action potential bursts in central snail neurons Chia-Hsien Lin, Pei-Jung Lin, Yi-Hung Chen, Pei-Lin Lin, I-Ming Chen, Kuan-Ling Lu, Yu-Chi Chang, Ming-Cheng TsaiT Department of Pharmacology, College of Medicine, National Taiwan University, No.1, Sec.1, Jen-Ai Road, Taipei, Taiwan Received 23 December 2004; revised 17 February 2005; accepted 25 February 2005 Available online 14 April 2005
Abstract Effects of rolipram, a selective inhibitor of phosphodiesterases (PDE) IV, on induction of action potential bursts were studied pharmacologically on the RP4 central neuron of the giant African snail (Achatina fulica Ferussac). Oscillations of membrane potential bursts were elicited by rolipram and forskolin. The bursts of potential elicited by rolipram were not inhibited after administration with (a) calciumfree solution, (b) high-magnesium solution (30 mM) or (c) U73122. However, the bursts of potential elicited by rolipram were inhibited by pretreatment with KT-5720 (10 AM). Voltage-clamp studies revealed that rolipram decreased the total inward current and steady-state outward currents of the RP4 neuron. The negative slope resistance (NSR) was not detectable in control or rolipram treated RP4 neurons. TEA elicited action potential bursts and an NSR at membrane potential between 50 mV and 30 mV. It is suggested that the bursts of potential elicited by rolipram were not due to (1) synaptic effects of neurotransmitters; (2) NSR of steady-state I–V curve; (3) phospholipase activity of the neuron. The rolipram-elicited bursts of potential were dependent on the phosphodiesterases inhibitory activity and the cAMP signaling pathway in the neuron. D 2005 Elsevier Inc. All rights reserved. Keywords: Second messenger; Phosphodiesterase inhibitors; Snail; Central neuron; Rolipram; cAMP; Bursts of potential; Protein kinase
Introduction Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) regulated many biological animal behavior and cellular functions (Maurice et al., 2003). Elevation of cAMP in neurons that express PDE4s attenuated the rewarding properties of cocaine and morphine (Thompson et al., 2004). Elevated cAMP levels in dunce mutants with reduced phosphodiesterase activity cause enhanced nerve terminal arborization at larval neuromuscular junctions of Drosophila (Zhong and Wu, 2004). Phosphodiesterases (PDE) are responsible for hydrolysis of the cyclic nucleotides cAMP and cGMP and the enzyme also associated with the Alzheimer’s disease brains (PerezTorres et al., 2003). Rolipram, a selective inhibitor of phosphodiesterases (PDE) IV, attenuate the endogenous T Corresponding author. Fax: +11 886 2 23915297. E-mail address: [email protected] (M.-C. Tsai). 0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.02.016
depression in the central nervous system (CNS) through elevation of intracellular cAMP and increasing synthesis and release of norepinephrine (Zhu et al., 2001). However, effect of rolipram on spontaneous action potential of central neuron was still unclear. Tetra-ethylammonium chloride (TEA), a blocker of the delayed outward K+ current, elicited action potential bursts and negative slope resistance (NSR) in the RP4 neuron. The TEA-elicited action potential bursts were closely related to its NSR activity (Chen and Tsai, 2000). Whether rolipram can elicit NSR in the central neuron remained unknown. The aims of the study are to characterize the effects of rolipram on spontaneous action potential of the central snail neuron. We found that rolipram-elicited action potential bursts in the RP4 neuron and the effects were associated with phosphodiesterase activity and elevation of intracellular cAMP in the neuron and the effects were not associated with synaptic effects of neurotransmitters or NSR of steady-state I–V curve or phospholipase activity in the neuron.
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Materials and methods Experiments were performed on the identified RP4 neuron from the subesophageal 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, 1996, 2000; Lin and Tsai, 2003). For intracellular recording, an Axoclamp 2A amplifier (Axon Instruments, Inc., USA) was used. Microelectrodes (5–6 MV) for recording membrane potentials were filled with 3 M KCl. The experimental chamber was perfused with control saline; composition (mM): NaCl, 85; KCl, 4; CaCl2, 8; MgCl2, 7; Tris–HCl, 10 (pH 7.6), at room temperature of 23–24, with perfusion speed of 6 ml/min. Calcium (Ca2+)free saline was substituted with CoCl2 for calcium ion in equimolar amounts (8 mM) (Kim et al., 1991). Highmagnesium solution (30 mM) was prepared by either decreasing extracellular sodium ion concentration (Aldenhoff et al., 1983) or adding hyper-osmotically to control saline (Murakami and Takahashi, 1983). For the quality of neurons tested, neurons were studied only if they had resting membrane potentials more negative than 50 mV with the time constant at about 5–8 ms and the rate of rise of the action potentials at about 5–8 DV/s. The same neurons were examined sequentially for control, drug treatments and in each of the tables (that is, tables present sequential experiments done on the same cells). The control result in physiological solution was recorded 60 min after electrophysiological recording on the RP4 neuron. The data for calcium-free solution were recorded 20 min after calcium-free solution treatment. The data recorded for rolipram in calcium-free solution was recorded in preparations pretreated with calcium-free solution for 20 min and further administration of rolipram (300 AM) for 20 min. To test the effect of synaptic transmission on the potential changes, preparation were perfused in calcium-free, highmagnesium solution (inhibition of transmitter releasing) for 20 min, and then, PDE inhibitors were further added in the perfusate on the RP4 neurons or as described in the text. To test the role of phospholipase on the potential changes of the neuron, the U73122 (phosphoinositide-specific phospholipase C inhibitor) (Ellershaw et al., 2002) was added after PDE inhibitors elicit bursts of potential. To test the effects of protein kinase A activity on the neuron, KT5720 (protein kinase A inhibitors) were added for 60 min before incubated with rolipram. The Control data in physiological solution were recorded 60 min after electrophysiological recording on the RP4 neuron. Data for rolipram (300 AM) and for KT-5720 (10 AM) were recorded 60 min after treatment. The data for KT-5720 (10 AM) control were recorded 60 min after KT-5720 (10 AM) treatment. The data for KT-5720 (10 AM) and rolipram (300 AM) were recorded in preparations pretreated with KT5720 (10 AM) for 60 min and further administration of
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rolipram (300 AM) for 60 min (Table 2). To test the effect of adenyl cyclase on the potential changes in RP4 neuron, different concentrations of forskolin (30, 100 AM), an adenylate cyclase activator were examined. The frequency of single spikes was presented as pulses per minute. The frequency of bursts was presented as bursts per minute. The bursts represent a change in the firing pattern from regularly spaced single spikes to one in which bursts of 2–20 action potentials were separated by large hyperpolarization of membrane potential (up to 6 mV) lasting 1– 20 s. The resting membrane potentials, amplitudes of action potentials and the frequency of single spikes of action potentials were recorded 20–60 min after drugs administration or as mentioned in the text. Amplitude of action potential was measured from resting membrane potential to the peak of action potential. Phosphodiesterase inhibitors and other drugs were applied by extracellular incubation. The ionic currents of the RP4 neurons were measured using two-electrode voltage-clamp method by means of a Gene clamp 500 amplifier (Axon Instrument Co.). All potentials and currents were recorded on tape via a digitalizing unit (Digidata 1200) and analyzed using a pCLAMP system (Axon Instruments, Inc., U.S.A.). For peak amplitude of total inward current, the currents were elicited by 70 ms depolarization of 50 to +30 mV from a holding potential of 60 mV. For steady-state outward currents, the currents were elicited by 5 s depolarization of 70 to +40 mV from a holding potential of 60 mV. The amplitude and frequency of the action potentials, resting membrane potentials and currents after various treatments were compared with the pre-drug control by means of Student’s two-tailed t test and paired t test. Student’s t test was used when the samples in the control and experimental conditions were from different groups of preparation. Student’s paired t test was used when the samples in the control and experimental condition were from the same groups of preparations. Differences were considered significant at P b 0.05. Rolipram (PDE4 selective), TEA and U73122 (1-[6[((17h)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]1H-pyrrole-2,5-dione) and forskolin were obtained from Sigma Chemical Company (St. Louis, MO, USA). KT-5720 was purchased from Calbiochem Ltd. (USA). TEA and forskolin were dissolved in distilled water. Rolipram, U73122 and KT-5720 were dissolved in dimethyl sulfoxide (DMSO). The concentration of DMSO we used (0.1%) did not affect the RMP, amplitude and frequency of spontaneous firing of action potentials in the RP4 neuron.
Results The RP4 neuron of Achatina fulica Ferussac The RP4 identified neuron had a resting membrane potential of 57.8 F 1.7 mV (n = 10) and the neuron
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Fig. 1. Effects of rolipram (30–300 AM) on the RP4 neuron of snail. Panels A, B, C at control showing the spontaneous action potentials of the RP4 neurons. Panels A, B, C at 30 min showed the potentials of the neurons after rolipram (30, 100, 300 AM) treatment for 30 min, respectively. Panels A, B, C at 60 min showed the potentials of the neurons after rolipram (30, 100, 300 AM) treatment for 60 min, respectively. Panels A, B, C at washing 120 min were potentials after 120 min of washing with normal saline from preparations incubated with 60 min rolipram of panels A, B, C respectively. The horizontal bars on the top left side of the figures represent the membrane potentials at 0 mM. Note that rolipram (300 AM) reversibly elicited bursts of potential on the RP4 neuron.
concentration rolipram decreased the amplitudes of the action potential (100 AM) of the RP4 neuron ( P b 0.05, n = 10) and elicited bursts firing of action potential (300 AM). The effect of rolipram (300 AM) on the neuronal activities was reversible. After 120 min of continuous washing with normal saline, the spontaneously generated spikes of the central neuron returned to control levels albeit with a slower frequency of spontaneous firing.
showed a spontaneous firing of action potential at a frequency of 34.8 F 2.8 pulses/min (n = 10). The action potentials occured as regularly spaced single spikes, no burst firing of action potentials was observed in the control RP4 neurons. The mean amplitude of the spontaneously generated action potential was 88.6 F 2.8 mV (n = 10). Effects of rolipram on the RP4 neuron
Effects of U73122 on rolipram-elicited potential changes in the RP4 neuron
Effects of rolipram (30–300 AM) on the resting membrane potential (RMP), amplitude and frequency of the spontaneously generated action potentials and frequency of bursts of potential on the RP4 neurons were shown in Fig. 1 and Table 1. Rolipram at low concentration (30 AM) did not affect the resting membrane potential, amplitude and spike frequency of the RP4 neuron ( P N 0.05). However, at higher
Effects of U73122 (10 AM) (the phospholipase C inhibitor) on rolipram elicited potentials changes were studied (Fig. 2A). Forty minutes after rolipram (300 AM) incubation, the spontaneous low frequency, regular firing became bursts of potential in the RP4 neuron. If U73122 (10 AM) was added,
Table 1 Effects of rolipram on the resting membrane potential, amplitude and frequency of the spontaneously generated action potential of the RP4 neurons Variable Rolipram (n = 10)
RMP (mV) Control 30 AM 100 AM 300 AM
57.8 57.0 54.2 64.5
F F F F
1.7 6.2 1.6 3.4a
Amplitudes (mV)
Frequency of single spikes (pulses/min)
Frequency of bursts (bursts/min)
88.6 86.1 83.6 97.5
34.8 F 3.6 38.0 F 8.3 38.4 F 6.9 –
– – – 8.5 F 0.9
F F F F
2.8 8.3 6.1a 7.5a
Values were expressed as the mean F SEM. n is the number of neurons tested. a Statistically significant compared with the data in physiological solution (control), P b 0.05.
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Fig. 2. Effects of U73122 (10 AM) (A), KT-5720 (10 AM) (B) and calcium-free solution (C) on rolipram (300 AM) elicited bursts of potential on the RP4 neuron. The same neuron was examined sequentially for drug treatment. Panels A1, B1 and C1 were control, showing the spontaneous action potential of the neurons. Panel A2 was the potentials after addition of rolipram (300 AM) for 40 min from A1. Panels A3 and A4 were the potentials after addition U73122 (10 AM) for 60 and 120 min respectively in the presence of rolipram (300 AM). Panel A5 was the potentials 120 min after washing with normal saline from panel A4. Panel B2 was the potentials after addition of KT-5720 (10 AM) for 60 min from panel B1. Panels B3 and B4 were the potentials after addition rolipram (300 AM) for 30 and 60 min respectively in the presence of KT-5720 (10 AM). Panel B5 was the potentials 120 min after washing off with normal saline from panel B4. Panel C2 was the potentials of the neuron 20 min after perfusion with calcium-free solution. Panel C3 was 20 min after further incubation with rolipram (300 AM) containing calcium-free solution in the neuron. Panel C4 was the potentials 30 min after washing off from panel C3 with normal saline. The horizontal bar on the top left side was the membrane potential at 0 mV. Note that U73122 (10 AM) and calcium-free solution did not, while KT-5720 (10 AM) did alter the bursts of potential elicited by rolipram (300 AM).
rolipram continued to elicit bursts of potential in the presence of U73122 even after 2 h of rolipram treatment (data not shown). Similar results were found in 5 of the preparations. U73122 did not alter the rolipram-elicited bursts of potential changes. The spontaneous action potential of the RP4 neuron had recovered to control level after 120 min of continuous washing with normal physiological solution. Effects of KT-5720 on rolipram-elicited potential changes in the RP4 neuron The effect of KT-5720 (10 AM), an inhibitor of protein kinase A on the RP4 neuron, and the effect of KT-5720 on rolipram (300 AM) elicited changes were studied. The resting membrane potential, amplitude and frequency of the spontaneously generated action potential of the RP4 neurons, as shown in Fig. 2B and Table 2. The control data in physiological solution were recorded 60 min after electrophysiological recording on the RP4 neuron. The data for rolipram (300 AM) were collected 60 min after rolipram (300 AM) treatment. The effect of KT-5720 (10 AM) control was recorded 60 min after KT-5720 (10 AM) treatment. The data
for KT-5720 (10 AM) and rolipram (300 AM) were recorded in preparations pretreated with KT-5720 (10 AM) for 60 min and further administration of rolipram (300 AM) for 60 min. The frequency of the spontaneously generated action potential of the RP4 neuron decreased in rolipram (300 AM) and KT-5720 (10 AM) treated preparations compared with the same neuron treated with KT-5720 (10 AM) ( P b 0.05). However, rolipram did not elicit burst of potential on the RP4 neuron in KT-5720 (10 AM) pretreated preparation (Fig. 2B). It appeared that KT-5720 abolishes the generation of bursts of potential by rolipram. Effects of calcium-free solution on rolipram-elicited potentials of the RP4 neuron Effects of calcium-free solution on rolipram (300 AM) elicited changes of the resting membrane potential, amplitude and frequency of the spontaneously generated action potential of the RP4 neurons are shown in Fig. 2C and Table 3. The resting membrane potential and amplitude of the spontaneously generated action potential of the RP4 neuron were decreased in rolipram (300 AM) and the
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Table 2 Effect of KT-5720 (10 AM) on rolipram (300 AM) elicited changes of the resting membrane potential, amplitude and frequency of the spontaneously generated action potential of the RP4 neurons Variable
Physiological solution (n = 6) Physiological solution
RMP (mV) Amplitudes (mV) Frequency of single spikes (pulses/min) Frequency of bursts (bursts/min)
57.8 F 4.2 83.0 F 1.7 42.0 F 3.5 –
Rolipram (300 AM)
KT-5720 (10 AM) control (n = 6)
61.5 F 3.3a 88.5 F 7.3a
KT-5720 (10 AM) + rolipram (300 AM) (n = 6) 54.0 F 6.7b,c 83.3 F 2.2b 32.0 F 2.0a,c
58.5 F 4.9 85.3 F 1.7 40.0 F 2.0
– 8.5 F 1.0
–
–
Note that KT-5720 blocked the rolipram-elicited bursts of potential changes. a Statistically significant compared with the data in physiological solution (control), P b 0.05. b Statistically significant compared with rolipram (300 AM) in physiological solution, P b 0.05. c Statistically significant compared with KT-5720 (10 AM) in physiological solution, P b 0.05.
calcium-free solution treated preparations compared with the same neuron treated with physiological solution ( P b 0.05). The amplitude and frequency of the spontaneously generated action potential of the RP4 neuron were both decreased in calcium-free solution. The effect of rolipram was then examined in the same neuron treated with calcium-free solution ( P b 0.05). We found that the pattern of the bursts of potential elicited by rolipram was not changed in the presence of the calcium-free solution in spite of a decrease in spike amplitude. Rolipram-elicited bursting potentials of the RP4 neuron in calcium-free solution were also reversible. After 30 min of continuous washing with normal saline, the amplitude of potentials had recovered to control levels. These results suggest that the action potential bursts inducted by rolipram do not depend on synaptic transmission or inward Ca2+ currents.
solution, rolipram still elicited bursts of potential even after 2 h of the high-magnesium solution treatment (Fig. 3A). It appears that the high-magnesium solution did not alter the rolipram-elicited bursts of potential changes. The spontaneous action potentials of the RP4 neuron had recovered to control level after 120 min of continuous washing with normal physiological solution. Effects of forskolin on the RP4 neuron In the physiological solution, the resting membrane potential, amplitude and frequency of the spontaneous action potential of the RP4 neuron were 56.7 F 1.6 mV, 81.3 F 2.3 mV and 40.0 F 5.3 pulses/min (n = 4), respectively. Forskolin at a lower concentration (30 AM) did not elicit bursts of potential in the neuron. However, 60 min after incubation with forskolin at 100 AM elicited bursts of potential in the RP4 neuron. The resting membrane potential, amplitude and frequency of bursts of potential were 56.3 F 1.2 mV, 70.3 F 4.9 mV and 8.1 F 5.3 bursts/min (n = 4), respectively. Therefore, forskolin (100 AM) did not affect the resting membrane potential ( P N 0.05), but elicited bursts firing of action potentials in the neuron (Fig. 3B).
Effects of high-magnesium solution on rolipram-elicited potential changes in the RP4 neuron Effects of a high-magnesium solution (30 mM) on rolipram-elicited potential changes were studied. High Mg2+ (30 mM) solution was prepared by either decreasing extracellular sodium ion concentration or using hyperosmotically to stock saline. Both solutions did not significantly alter the spontaneously generated action potential of the RP4 neuron. Forty minutes after rolipram (300 AM) incubation, bursts of potential were elicited in the RP4 neuron. Further addition of high magnesium to perfusion
Effect of rolipram on the fast peak of amplitudes of total inward current of the RP4 neuron Effect of rolipram (100–300 AM) on the peak amplitude of total inward current in the RP4 neurons was shown in
Table 3 Effect of calcium-free solution on rolipram (300 AM) elicited changes of the resting membrane potential, amplitude and frequency of the spontaneously generated action potential of the RP4 neurons Variable
Physiological solution RMP (mV) Amplitudes (mV) Frequency of single spikes (pulses/min) Frequency of bursts (bursts/min)
Ca2+-free solution (n = 6)
Physiological solution (n = 6)
60.2 F 3.5 92.3 F 7.2 38.0 F 2.0 –
Rolipram (300 AM)
Ca2+-free solution
a
52.3 F 1.5a 66.3 F 8.0a 52.0 F 5.3a
64.5 F 3.4 97.5 F 7.5a – 8.5 F 0.9
–
Rolipram (300 AM) 53.7 F 1.9a,b 55.7 F 5.6a,b,c – 2.0 F 0.1
Note that removing extracellular calcium ion did not interfere the pattern of the bursting firing of the action potentials elicited by rolipram. a Statistically significant compared with the data in physiological solution (control), P b 0.05. b Statistically significant compared with rolipram (300 AM) in physiological solution, P b 0.05. c Statistically significant compared with calcium-free solution, P b 0.05.
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Fig. 3. Effects of high Mg2+ (30 mM) solution and forskolin (30, 100 AM) on the RP4 neurons. Panels A1 and B1 at control showing the spontaneous action potentials of the RP4 neurons. Panel A2 was the potentials after addition of rolipram (300 AM) for 40 min from panel A1. Panels A3 and A4 were the potentials after addition of high Mg2+ (30 mM) for 30 and 60 min respectively in the presence of rolipram (300 AM). Panel A5 was the potentials 120 min after washing off with normal saline from panel A4. Panels B1 and B2 were control and saline, showing the spontaneous action potentials of the neuron. Panels B3 and B4 were the potentials of the RP4 neuron after addition of forskolin (30, 100 AM) for 60 min, respectively. Panel B5 was the potentials 120 min after washing off with normal saline from panel B4. The horizontal bar on the top left side was the membrane potential at 0 mV. Note that high Mg2+ (30 mM) did not alter the rolipram-elicited bursts of potential changes and forskolin (100 AM) did elicit bursts of potential on the RP4 neuron.
Figs. 4 and 5A. Membrane currents were elicited from a holding potentials of 60 mV to test potentials ( 50 to +30 mV) before and after rolipram application. The fast ionic currents of the RP4 neurons clamping at 70 ms durations were shown in Fig. 4. The membrane potentials were held at 60 mV and stepped to the testing potentials of 50 to +30 mV. Total inward currents were observed in various voltage clamping command steps lasting 70 ms and the peak amplitudes of inward currents are shown in Figs. 4 and 5A. Inward current was clearly observed if the potential stepped to a level more positive than 40 mV and the maximum peak inward current was found at 20 mV. The reversal potential of the total inward currents was around +45 mV. Effect of rolipram on steady-state outward currents of the RP4 neurons Effects of rolipram (100–300 AM) on steady-state outward currents are shown in Figs. 4 and 5B. The steady-state outward currents were elicited from a holding potential of 60 mV to stepping potential of 70 to +40 mV. A slow decaying outward current was found at potentials more positive than 30 mV. The steady-state outward currents in the voltage range of 30 to +40 mV were significantly decreased after 60 min of rolipram (100 or 300 AM) administration (Fig. 5B). Effects of TEA on the spontaneous action potentials in the RP4 neuron TEA exerted a similar effect on the spontaneous action potentials in the RP4 neuron as shown in Fig. 6. The resting
Fig. 4. Effects of rolipram on fast total inward and outward currents in the RP4 neuron. The membrane currents were elicited from a holding potential of 60 mV to (1) test potentials of 50, 40, 30, 20, 10, 0, 10, 20 and 30 mV for 70 ms or (2) test potentials of 70, 60, 50, 40, 30, 20, 10, 0, 10, 20, 30 and 40 mV for 5 s. (A) Control, fast total inward and outward currents recorded in normal physiological saline. (B) Fast inward and outward currents recorded 60 min after rolipram (100 AM) incubation from panel A. (C) Fast inward and outward currents recorded 60 min after rolipram (300 AM) incubation from panel B. (D) Voltage step commands.
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Fig. 6. Effects of TEA on the RP4 neurons. Panels A and B at control were potentials of the control RP4 neurons. Panels A and B at 30 min were potentials after 30 min of application of TEA (5, 7 mM), respectively. Panels A and B at 60 min were potentials after 60 min of application of TEA (5, 7 mM), respectively. Panels A and B at washing 120 min were potentials after 120 min of washing off with normal saline from preparations incubated with 60 min TEA of panels A and B respectively. The horizontal bar on the top left side is the membrane potential at 0 mV. Note that TEA (7 mM) elicited bursts of potential on the RP4 neuron.
membrane potentials of the RP4 neurons in control 60 min after TEA (5 and 7 mM) administration were 57.3 F 0.5 mV (n = 4), 55.5 F 0.5 mV (n = 4, P N 0.05) and 57.4 F 2.2 mV (n = 4, P N 0.05) respectively. The frequencies of the spontaneously generated action potential in control 60 min after TEA (5 and 7 mM) administration were 37.5 F 1.5 pulses/min (n = 4), 40.4 F 2.4 pulses/min (n = 4, P N 0.05) and 10.0 F 2.0 bursts/min (n = 4, P b 0.05), respectively. TEA elicited action potentials bursts in the RP4 neuron in a concentration dependent manner. TEA at 5 mM did not alter the resting membrane potential or the frequency of the spontaneously generated action potential of the RP4 neuron. However, at a higher concentration (7 mM), TEA elicited action potentials bursts in the RP4 neurons. The effects of TEA (7 mM) on the RP4 neurons were reversible. After washing off TEA at 120 min, the resting membrane potentials and the patterns of the action potentials of the RP4 neuron returned to control level. Effects of TEA on steady-state I–V curve are shown in Fig. 5C. The currents were elicited by a 5 s duration of commanding step from a holding potential of 60 mV to stepping potentials, ranging from 70 mV to +40 mV at intervals of 10 mV. Compared with pre-drug controls, the steady-state I–V curve did not significantly change 40 min after TEA (5 mM) treatment (data not shown) but were clearly affected 60 min after TEA (7 mM) treatment. The steady-state I–V curve revealed an N-shaped appearance
(the negative slope resistance, NSR) in the steps ranging from 50 to 30 mV, after TEA (7 mM) treatment (Fig. 5C). The effects of TEA on the NSR response are reversible. After washing off TEA for 120 min, the steady-state membrane currents resumed and the NSR of the steadystate I–V curve elicited by TEA was reversed. It appears that TEA (7 mM) has a differentiation that elicits a negative slope region (NSR) in the steady-state current–voltage curve, which was not seen in rolipram-treated neruon.
Discussion In the present study, we characterized rolipram-elicited bursts of potential on the RP4 neuron of snail. The same neuQ rons were examined sequentially for drug treatment in each of the tables (that is, tables present sequential experiments done on the same neurons). Therefore, some of the control values varied according to different batches of samples. Rolipram is a selective inhibitor of phosphodiesterases (PDE) IV and the phosphodiesterase is responsible for hydrolysis of the cyclic nucleotides cAMP and cGMP in nerve cells. Rolipram-induced elevation of intracellular cAMP may have elicited the potential bursts. Other PDE inhibitors, e.g., vinpocetine (PDE1 selective), erythro-9-(2hydroxy-3-nonyl) adenine (EHNA, PDE2 selective), milri-
Fig. 5. Effect of rolipram and TEA on I–V curves. Panel A shows the current–voltage relationships of the peak fast total inward currents before (.), after rolipram (100 AM) (n) and rolipram (300 AM) (q) application for 60 min. Panel B shows the current–voltage relationships of the steady-state outward currents before (.), after rolipram (100 AM) (n) and rolipram (300 AM) (q) application for 60 min. Panel C shows the current–voltage relationships of the steady-state outward currents before (.) and after TEA (7 mM) (5) application for 60 min. Note that rolipram decreased the peak fast total inward currents and steady-state outward currents by a concentration dependent manner. No NSR was observed in control or rolipram-treated preparation while TEA (7 mM) elicited a negative slope region (NSR) at membrane potential between 50 mV and 30 mV. *P b 0.05 versus control.
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none (PDE3 selective) have also been shown to elicit bursts of potential on the RP4 neurons (Lin et al., 2004). Besides, forskolin, the adenyl-cyclase activator, elicited bursts of potential on the same neuron. It is interesting to note that cyclic AMP, the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor rolipram have been reported to enhance afterdischarges (ADs) induced in the CA1 region of rat hippocampal slices (Higashima et al., 2002). Voltage-clamp studies revealed that rolipram decreased the fast total inward current and steady-state outward currents of the RP4 neuron. However, no negative slope resistance (NSR) in the steady-state I–V curve was found in control or rolipram treated neurons. TEA, a blocker of the delayed outward K+ current, elicited action potential bursts and NSR in the RP4 neuron. It appears that the action potential bursts elicited by rolipram are not directly related to the NSR of the steady-state current–voltage relationship. The phosphodiesterase type 7 and 8 isozymes are also associated with the Alzheimer’s disease brains (Perez-Torres et al., 2003). The role of phosphodiesterase type 7 and 8 in the potential changes of central neurons remained an interesting subject for further study. The rolipram-elicited bursts of potential were not altered after addition of either a calcium-free solution, highmagnesium solution (which inhibited synaptic transmission) (Gainer, 1972) or in the presence of U73122 (phosphoinositide-specific phospholipase C inhibitor). The experimental results suggested that the bursts of potential elicited by rolipram were not in association with the synaptic transmission process of the ganglia or the phospholipase C activity in the RP4 neuron. The finding that the action potential bursts elicited by rolipram were blocked if KT-5720 (protein kinase A inhibitors) were pre-administrated. The results suggested that protein kinase A activity in the neuron play important roles on the bursts of potential elicited by rolipram. In calcium-free solution containing 8 mM Co2+ or highmagnesium solution which blocks the synaptic neurotransmission (Gainer, 1972), RP4 neurons still generate pace making action potentials that could be converted into bursting activity by rolipram. In addition, Co2+ also inhibited calcium current and the calcium-free solution severely reduced the calcium inward currents and calcium dependent outward potassium currents. The result suggests that sodium inward currents and voltage dependent potassium currents are the major targets for cAMP mediated neuro-modulation. It is suggested that the bursts of potential elicited by rolipram were not due to (1) synaptic effects of neurotransmitters, (2) NSR of steady-state I–V curve and (3) phospholipase activity of the neuron. The rolipramelicited bursts of potential were dependent on the
phosphodiesterases inhibitory activity and cAMP in the neuron.
Acknowledgment This work was supported by grant NSC-93-2320-B-002130 from National Science Council, Taipei, Taiwan.
References Aldenhoff, J.B., Hofmeier, G., Lux, H.D., Swandulla, D., 1983. Stimulation of a sodium influx by cAMP in Helix neurons. Brain Res. 276, 289 – 296. Chen, Y.H., Tsai, M.C., 1996. Bursting firing of action potential in central snail neuron 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. Ellershaw, D.C., Greenwood, I.A., Large, W.A., 2002. Modulation of volume-sensitive chloride current by noradrenaline in rabbit portal vein myocytes. J. Physiol. 542, 537 – 547. Gainer, H., 1972. Electrophysiological behavior of an endogenously active neurosecretory cell. Brain Res. 39, 403 – 418. Higashima, M., Ohno, K., Koshino, Y., 2002. Cyclic AMP-mediated modulation of epileptiform afterdischarge generation in rat hippocampal slices. Brain Res. 949, 157 – 161. 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. Lin, C.H., Tsai, M.C., 2003. d-amphetamine-elicited action potential bursts in central snail neurons: role of second messenger systems. J. Formos. Med. Assoc. 102, 394 – 403. 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. 9, 162 – 173. Maurice, D.H., Palmer, D., Tilley, D.G., Dunkerley, H.A., Netherton, S.J., Raymond, D.R., Elbatarny, H.S., Jimmo, S.L., 2003. Cyclic nucleotide phosphodiesterase activity, expression, and targeting in cells of the cardiovascular system. Mol. Pharmacol. 64, 533 – 546. Murakami, N., Takahashi, K., 1983. Circadian rhythm of adenosine-3V,5Vmonophosphate content in suprachiasmatic nucleus (SCN) and ventromedial hypothalamus (VMH) in the rat. Brain Res. 276, 297 – 304. Perez-Torres, S., Cortes, R., Tolnay, M., Probst, A., Palacios, J.M., Mengod, G., 2003. Alterations on phosphodiesterase type 7 and 8 isozyme mRNA expression in Alzheimer’s disease brains examined by in situ hybridization. Exp. Neurol. 182, 322 – 334. Thompson, B.E., Sachs, B.D., Kantak, K.M., Cherry, J.A., 2004. The Type IV phosphodiesterase inhibitor rolipram interferes with drug-induced conditioned place preference but not immediate early gene induction in mice. Eur. J. Neurosci. 19, 2561 – 2568. Zhong, Y., Wu, C.F., 2004. Neuronal activity and adenylyl cyclase in environment-dependent plasticity of axonal outgrowth in Drosophila. J. Neurosci. 24, 1439 – 1445. Zhu, J., Mix, E., Winblad, B., 2001. The antidepressant and antiinflammatory effects of rolipram in the central nervous system. CNS Drug Rev. 7, 387 – 398.
Effects of rolipram on induction of action potential bursts in central snail neurons Chia-Hsien Lin, Pei-Jung Lin, Yi-Hung Chen, Pei-Lin Lin, I-Ming Chen, Kuan-Ling Lu, Yu-Chi Chang, Ming-Cheng TsaiT Department of Pharmacology, College of Medicine, National Taiwan University, No.1, Sec.1, Jen-Ai Road, Taipei, Taiwan Received 23 December 2004; revised 17 February 2005; accepted 25 February 2005 Available online 14 April 2005
Abstract Effects of rolipram, a selective inhibitor of phosphodiesterases (PDE) IV, on induction of action potential bursts were studied pharmacologically on the RP4 central neuron of the giant African snail (Achatina fulica Ferussac). Oscillations of membrane potential bursts were elicited by rolipram and forskolin. The bursts of potential elicited by rolipram were not inhibited after administration with (a) calciumfree solution, (b) high-magnesium solution (30 mM) or (c) U73122. However, the bursts of potential elicited by rolipram were inhibited by pretreatment with KT-5720 (10 AM). Voltage-clamp studies revealed that rolipram decreased the total inward current and steady-state outward currents of the RP4 neuron. The negative slope resistance (NSR) was not detectable in control or rolipram treated RP4 neurons. TEA elicited action potential bursts and an NSR at membrane potential between 50 mV and 30 mV. It is suggested that the bursts of potential elicited by rolipram were not due to (1) synaptic effects of neurotransmitters; (2) NSR of steady-state I–V curve; (3) phospholipase activity of the neuron. The rolipram-elicited bursts of potential were dependent on the phosphodiesterases inhibitory activity and the cAMP signaling pathway in the neuron. D 2005 Elsevier Inc. All rights reserved. Keywords: Second messenger; Phosphodiesterase inhibitors; Snail; Central neuron; Rolipram; cAMP; Bursts of potential; Protein kinase
Introduction Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) regulated many biological animal behavior and cellular functions (Maurice et al., 2003). Elevation of cAMP in neurons that express PDE4s attenuated the rewarding properties of cocaine and morphine (Thompson et al., 2004). Elevated cAMP levels in dunce mutants with reduced phosphodiesterase activity cause enhanced nerve terminal arborization at larval neuromuscular junctions of Drosophila (Zhong and Wu, 2004). Phosphodiesterases (PDE) are responsible for hydrolysis of the cyclic nucleotides cAMP and cGMP and the enzyme also associated with the Alzheimer’s disease brains (PerezTorres et al., 2003). Rolipram, a selective inhibitor of phosphodiesterases (PDE) IV, attenuate the endogenous T Corresponding author. Fax: +11 886 2 23915297. E-mail address: [email protected] (M.-C. Tsai). 0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.02.016
depression in the central nervous system (CNS) through elevation of intracellular cAMP and increasing synthesis and release of norepinephrine (Zhu et al., 2001). However, effect of rolipram on spontaneous action potential of central neuron was still unclear. Tetra-ethylammonium chloride (TEA), a blocker of the delayed outward K+ current, elicited action potential bursts and negative slope resistance (NSR) in the RP4 neuron. The TEA-elicited action potential bursts were closely related to its NSR activity (Chen and Tsai, 2000). Whether rolipram can elicit NSR in the central neuron remained unknown. The aims of the study are to characterize the effects of rolipram on spontaneous action potential of the central snail neuron. We found that rolipram-elicited action potential bursts in the RP4 neuron and the effects were associated with phosphodiesterase activity and elevation of intracellular cAMP in the neuron and the effects were not associated with synaptic effects of neurotransmitters or NSR of steady-state I–V curve or phospholipase activity in the neuron.
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Materials and methods Experiments were performed on the identified RP4 neuron from the subesophageal 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, 1996, 2000; Lin and Tsai, 2003). For intracellular recording, an Axoclamp 2A amplifier (Axon Instruments, Inc., USA) was used. Microelectrodes (5–6 MV) for recording membrane potentials were filled with 3 M KCl. The experimental chamber was perfused with control saline; composition (mM): NaCl, 85; KCl, 4; CaCl2, 8; MgCl2, 7; Tris–HCl, 10 (pH 7.6), at room temperature of 23–24, with perfusion speed of 6 ml/min. Calcium (Ca2+)free saline was substituted with CoCl2 for calcium ion in equimolar amounts (8 mM) (Kim et al., 1991). Highmagnesium solution (30 mM) was prepared by either decreasing extracellular sodium ion concentration (Aldenhoff et al., 1983) or adding hyper-osmotically to control saline (Murakami and Takahashi, 1983). For the quality of neurons tested, neurons were studied only if they had resting membrane potentials more negative than 50 mV with the time constant at about 5–8 ms and the rate of rise of the action potentials at about 5–8 DV/s. The same neurons were examined sequentially for control, drug treatments and in each of the tables (that is, tables present sequential experiments done on the same cells). The control result in physiological solution was recorded 60 min after electrophysiological recording on the RP4 neuron. The data for calcium-free solution were recorded 20 min after calcium-free solution treatment. The data recorded for rolipram in calcium-free solution was recorded in preparations pretreated with calcium-free solution for 20 min and further administration of rolipram (300 AM) for 20 min. To test the effect of synaptic transmission on the potential changes, preparation were perfused in calcium-free, highmagnesium solution (inhibition of transmitter releasing) for 20 min, and then, PDE inhibitors were further added in the perfusate on the RP4 neurons or as described in the text. To test the role of phospholipase on the potential changes of the neuron, the U73122 (phosphoinositide-specific phospholipase C inhibitor) (Ellershaw et al., 2002) was added after PDE inhibitors elicit bursts of potential. To test the effects of protein kinase A activity on the neuron, KT5720 (protein kinase A inhibitors) were added for 60 min before incubated with rolipram. The Control data in physiological solution were recorded 60 min after electrophysiological recording on the RP4 neuron. Data for rolipram (300 AM) and for KT-5720 (10 AM) were recorded 60 min after treatment. The data for KT-5720 (10 AM) control were recorded 60 min after KT-5720 (10 AM) treatment. The data for KT-5720 (10 AM) and rolipram (300 AM) were recorded in preparations pretreated with KT5720 (10 AM) for 60 min and further administration of
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rolipram (300 AM) for 60 min (Table 2). To test the effect of adenyl cyclase on the potential changes in RP4 neuron, different concentrations of forskolin (30, 100 AM), an adenylate cyclase activator were examined. The frequency of single spikes was presented as pulses per minute. The frequency of bursts was presented as bursts per minute. The bursts represent a change in the firing pattern from regularly spaced single spikes to one in which bursts of 2–20 action potentials were separated by large hyperpolarization of membrane potential (up to 6 mV) lasting 1– 20 s. The resting membrane potentials, amplitudes of action potentials and the frequency of single spikes of action potentials were recorded 20–60 min after drugs administration or as mentioned in the text. Amplitude of action potential was measured from resting membrane potential to the peak of action potential. Phosphodiesterase inhibitors and other drugs were applied by extracellular incubation. The ionic currents of the RP4 neurons were measured using two-electrode voltage-clamp method by means of a Gene clamp 500 amplifier (Axon Instrument Co.). All potentials and currents were recorded on tape via a digitalizing unit (Digidata 1200) and analyzed using a pCLAMP system (Axon Instruments, Inc., U.S.A.). For peak amplitude of total inward current, the currents were elicited by 70 ms depolarization of 50 to +30 mV from a holding potential of 60 mV. For steady-state outward currents, the currents were elicited by 5 s depolarization of 70 to +40 mV from a holding potential of 60 mV. The amplitude and frequency of the action potentials, resting membrane potentials and currents after various treatments were compared with the pre-drug control by means of Student’s two-tailed t test and paired t test. Student’s t test was used when the samples in the control and experimental conditions were from different groups of preparation. Student’s paired t test was used when the samples in the control and experimental condition were from the same groups of preparations. Differences were considered significant at P b 0.05. Rolipram (PDE4 selective), TEA and U73122 (1-[6[((17h)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]1H-pyrrole-2,5-dione) and forskolin were obtained from Sigma Chemical Company (St. Louis, MO, USA). KT-5720 was purchased from Calbiochem Ltd. (USA). TEA and forskolin were dissolved in distilled water. Rolipram, U73122 and KT-5720 were dissolved in dimethyl sulfoxide (DMSO). The concentration of DMSO we used (0.1%) did not affect the RMP, amplitude and frequency of spontaneous firing of action potentials in the RP4 neuron.
Results The RP4 neuron of Achatina fulica Ferussac The RP4 identified neuron had a resting membrane potential of 57.8 F 1.7 mV (n = 10) and the neuron
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Fig. 1. Effects of rolipram (30–300 AM) on the RP4 neuron of snail. Panels A, B, C at control showing the spontaneous action potentials of the RP4 neurons. Panels A, B, C at 30 min showed the potentials of the neurons after rolipram (30, 100, 300 AM) treatment for 30 min, respectively. Panels A, B, C at 60 min showed the potentials of the neurons after rolipram (30, 100, 300 AM) treatment for 60 min, respectively. Panels A, B, C at washing 120 min were potentials after 120 min of washing with normal saline from preparations incubated with 60 min rolipram of panels A, B, C respectively. The horizontal bars on the top left side of the figures represent the membrane potentials at 0 mM. Note that rolipram (300 AM) reversibly elicited bursts of potential on the RP4 neuron.
concentration rolipram decreased the amplitudes of the action potential (100 AM) of the RP4 neuron ( P b 0.05, n = 10) and elicited bursts firing of action potential (300 AM). The effect of rolipram (300 AM) on the neuronal activities was reversible. After 120 min of continuous washing with normal saline, the spontaneously generated spikes of the central neuron returned to control levels albeit with a slower frequency of spontaneous firing.
showed a spontaneous firing of action potential at a frequency of 34.8 F 2.8 pulses/min (n = 10). The action potentials occured as regularly spaced single spikes, no burst firing of action potentials was observed in the control RP4 neurons. The mean amplitude of the spontaneously generated action potential was 88.6 F 2.8 mV (n = 10). Effects of rolipram on the RP4 neuron
Effects of U73122 on rolipram-elicited potential changes in the RP4 neuron
Effects of rolipram (30–300 AM) on the resting membrane potential (RMP), amplitude and frequency of the spontaneously generated action potentials and frequency of bursts of potential on the RP4 neurons were shown in Fig. 1 and Table 1. Rolipram at low concentration (30 AM) did not affect the resting membrane potential, amplitude and spike frequency of the RP4 neuron ( P N 0.05). However, at higher
Effects of U73122 (10 AM) (the phospholipase C inhibitor) on rolipram elicited potentials changes were studied (Fig. 2A). Forty minutes after rolipram (300 AM) incubation, the spontaneous low frequency, regular firing became bursts of potential in the RP4 neuron. If U73122 (10 AM) was added,
Table 1 Effects of rolipram on the resting membrane potential, amplitude and frequency of the spontaneously generated action potential of the RP4 neurons Variable Rolipram (n = 10)
RMP (mV) Control 30 AM 100 AM 300 AM
57.8 57.0 54.2 64.5
F F F F
1.7 6.2 1.6 3.4a
Amplitudes (mV)
Frequency of single spikes (pulses/min)
Frequency of bursts (bursts/min)
88.6 86.1 83.6 97.5
34.8 F 3.6 38.0 F 8.3 38.4 F 6.9 –
– – – 8.5 F 0.9
F F F F
2.8 8.3 6.1a 7.5a
Values were expressed as the mean F SEM. n is the number of neurons tested. a Statistically significant compared with the data in physiological solution (control), P b 0.05.
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Fig. 2. Effects of U73122 (10 AM) (A), KT-5720 (10 AM) (B) and calcium-free solution (C) on rolipram (300 AM) elicited bursts of potential on the RP4 neuron. The same neuron was examined sequentially for drug treatment. Panels A1, B1 and C1 were control, showing the spontaneous action potential of the neurons. Panel A2 was the potentials after addition of rolipram (300 AM) for 40 min from A1. Panels A3 and A4 were the potentials after addition U73122 (10 AM) for 60 and 120 min respectively in the presence of rolipram (300 AM). Panel A5 was the potentials 120 min after washing with normal saline from panel A4. Panel B2 was the potentials after addition of KT-5720 (10 AM) for 60 min from panel B1. Panels B3 and B4 were the potentials after addition rolipram (300 AM) for 30 and 60 min respectively in the presence of KT-5720 (10 AM). Panel B5 was the potentials 120 min after washing off with normal saline from panel B4. Panel C2 was the potentials of the neuron 20 min after perfusion with calcium-free solution. Panel C3 was 20 min after further incubation with rolipram (300 AM) containing calcium-free solution in the neuron. Panel C4 was the potentials 30 min after washing off from panel C3 with normal saline. The horizontal bar on the top left side was the membrane potential at 0 mV. Note that U73122 (10 AM) and calcium-free solution did not, while KT-5720 (10 AM) did alter the bursts of potential elicited by rolipram (300 AM).
rolipram continued to elicit bursts of potential in the presence of U73122 even after 2 h of rolipram treatment (data not shown). Similar results were found in 5 of the preparations. U73122 did not alter the rolipram-elicited bursts of potential changes. The spontaneous action potential of the RP4 neuron had recovered to control level after 120 min of continuous washing with normal physiological solution. Effects of KT-5720 on rolipram-elicited potential changes in the RP4 neuron The effect of KT-5720 (10 AM), an inhibitor of protein kinase A on the RP4 neuron, and the effect of KT-5720 on rolipram (300 AM) elicited changes were studied. The resting membrane potential, amplitude and frequency of the spontaneously generated action potential of the RP4 neurons, as shown in Fig. 2B and Table 2. The control data in physiological solution were recorded 60 min after electrophysiological recording on the RP4 neuron. The data for rolipram (300 AM) were collected 60 min after rolipram (300 AM) treatment. The effect of KT-5720 (10 AM) control was recorded 60 min after KT-5720 (10 AM) treatment. The data
for KT-5720 (10 AM) and rolipram (300 AM) were recorded in preparations pretreated with KT-5720 (10 AM) for 60 min and further administration of rolipram (300 AM) for 60 min. The frequency of the spontaneously generated action potential of the RP4 neuron decreased in rolipram (300 AM) and KT-5720 (10 AM) treated preparations compared with the same neuron treated with KT-5720 (10 AM) ( P b 0.05). However, rolipram did not elicit burst of potential on the RP4 neuron in KT-5720 (10 AM) pretreated preparation (Fig. 2B). It appeared that KT-5720 abolishes the generation of bursts of potential by rolipram. Effects of calcium-free solution on rolipram-elicited potentials of the RP4 neuron Effects of calcium-free solution on rolipram (300 AM) elicited changes of the resting membrane potential, amplitude and frequency of the spontaneously generated action potential of the RP4 neurons are shown in Fig. 2C and Table 3. The resting membrane potential and amplitude of the spontaneously generated action potential of the RP4 neuron were decreased in rolipram (300 AM) and the
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Table 2 Effect of KT-5720 (10 AM) on rolipram (300 AM) elicited changes of the resting membrane potential, amplitude and frequency of the spontaneously generated action potential of the RP4 neurons Variable
Physiological solution (n = 6) Physiological solution
RMP (mV) Amplitudes (mV) Frequency of single spikes (pulses/min) Frequency of bursts (bursts/min)
57.8 F 4.2 83.0 F 1.7 42.0 F 3.5 –
Rolipram (300 AM)
KT-5720 (10 AM) control (n = 6)
61.5 F 3.3a 88.5 F 7.3a
KT-5720 (10 AM) + rolipram (300 AM) (n = 6) 54.0 F 6.7b,c 83.3 F 2.2b 32.0 F 2.0a,c
58.5 F 4.9 85.3 F 1.7 40.0 F 2.0
– 8.5 F 1.0
–
–
Note that KT-5720 blocked the rolipram-elicited bursts of potential changes. a Statistically significant compared with the data in physiological solution (control), P b 0.05. b Statistically significant compared with rolipram (300 AM) in physiological solution, P b 0.05. c Statistically significant compared with KT-5720 (10 AM) in physiological solution, P b 0.05.
calcium-free solution treated preparations compared with the same neuron treated with physiological solution ( P b 0.05). The amplitude and frequency of the spontaneously generated action potential of the RP4 neuron were both decreased in calcium-free solution. The effect of rolipram was then examined in the same neuron treated with calcium-free solution ( P b 0.05). We found that the pattern of the bursts of potential elicited by rolipram was not changed in the presence of the calcium-free solution in spite of a decrease in spike amplitude. Rolipram-elicited bursting potentials of the RP4 neuron in calcium-free solution were also reversible. After 30 min of continuous washing with normal saline, the amplitude of potentials had recovered to control levels. These results suggest that the action potential bursts inducted by rolipram do not depend on synaptic transmission or inward Ca2+ currents.
solution, rolipram still elicited bursts of potential even after 2 h of the high-magnesium solution treatment (Fig. 3A). It appears that the high-magnesium solution did not alter the rolipram-elicited bursts of potential changes. The spontaneous action potentials of the RP4 neuron had recovered to control level after 120 min of continuous washing with normal physiological solution. Effects of forskolin on the RP4 neuron In the physiological solution, the resting membrane potential, amplitude and frequency of the spontaneous action potential of the RP4 neuron were 56.7 F 1.6 mV, 81.3 F 2.3 mV and 40.0 F 5.3 pulses/min (n = 4), respectively. Forskolin at a lower concentration (30 AM) did not elicit bursts of potential in the neuron. However, 60 min after incubation with forskolin at 100 AM elicited bursts of potential in the RP4 neuron. The resting membrane potential, amplitude and frequency of bursts of potential were 56.3 F 1.2 mV, 70.3 F 4.9 mV and 8.1 F 5.3 bursts/min (n = 4), respectively. Therefore, forskolin (100 AM) did not affect the resting membrane potential ( P N 0.05), but elicited bursts firing of action potentials in the neuron (Fig. 3B).
Effects of high-magnesium solution on rolipram-elicited potential changes in the RP4 neuron Effects of a high-magnesium solution (30 mM) on rolipram-elicited potential changes were studied. High Mg2+ (30 mM) solution was prepared by either decreasing extracellular sodium ion concentration or using hyperosmotically to stock saline. Both solutions did not significantly alter the spontaneously generated action potential of the RP4 neuron. Forty minutes after rolipram (300 AM) incubation, bursts of potential were elicited in the RP4 neuron. Further addition of high magnesium to perfusion
Effect of rolipram on the fast peak of amplitudes of total inward current of the RP4 neuron Effect of rolipram (100–300 AM) on the peak amplitude of total inward current in the RP4 neurons was shown in
Table 3 Effect of calcium-free solution on rolipram (300 AM) elicited changes of the resting membrane potential, amplitude and frequency of the spontaneously generated action potential of the RP4 neurons Variable
Physiological solution RMP (mV) Amplitudes (mV) Frequency of single spikes (pulses/min) Frequency of bursts (bursts/min)
Ca2+-free solution (n = 6)
Physiological solution (n = 6)
60.2 F 3.5 92.3 F 7.2 38.0 F 2.0 –
Rolipram (300 AM)
Ca2+-free solution
a
52.3 F 1.5a 66.3 F 8.0a 52.0 F 5.3a
64.5 F 3.4 97.5 F 7.5a – 8.5 F 0.9
–
Rolipram (300 AM) 53.7 F 1.9a,b 55.7 F 5.6a,b,c – 2.0 F 0.1
Note that removing extracellular calcium ion did not interfere the pattern of the bursting firing of the action potentials elicited by rolipram. a Statistically significant compared with the data in physiological solution (control), P b 0.05. b Statistically significant compared with rolipram (300 AM) in physiological solution, P b 0.05. c Statistically significant compared with calcium-free solution, P b 0.05.
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Fig. 3. Effects of high Mg2+ (30 mM) solution and forskolin (30, 100 AM) on the RP4 neurons. Panels A1 and B1 at control showing the spontaneous action potentials of the RP4 neurons. Panel A2 was the potentials after addition of rolipram (300 AM) for 40 min from panel A1. Panels A3 and A4 were the potentials after addition of high Mg2+ (30 mM) for 30 and 60 min respectively in the presence of rolipram (300 AM). Panel A5 was the potentials 120 min after washing off with normal saline from panel A4. Panels B1 and B2 were control and saline, showing the spontaneous action potentials of the neuron. Panels B3 and B4 were the potentials of the RP4 neuron after addition of forskolin (30, 100 AM) for 60 min, respectively. Panel B5 was the potentials 120 min after washing off with normal saline from panel B4. The horizontal bar on the top left side was the membrane potential at 0 mV. Note that high Mg2+ (30 mM) did not alter the rolipram-elicited bursts of potential changes and forskolin (100 AM) did elicit bursts of potential on the RP4 neuron.
Figs. 4 and 5A. Membrane currents were elicited from a holding potentials of 60 mV to test potentials ( 50 to +30 mV) before and after rolipram application. The fast ionic currents of the RP4 neurons clamping at 70 ms durations were shown in Fig. 4. The membrane potentials were held at 60 mV and stepped to the testing potentials of 50 to +30 mV. Total inward currents were observed in various voltage clamping command steps lasting 70 ms and the peak amplitudes of inward currents are shown in Figs. 4 and 5A. Inward current was clearly observed if the potential stepped to a level more positive than 40 mV and the maximum peak inward current was found at 20 mV. The reversal potential of the total inward currents was around +45 mV. Effect of rolipram on steady-state outward currents of the RP4 neurons Effects of rolipram (100–300 AM) on steady-state outward currents are shown in Figs. 4 and 5B. The steady-state outward currents were elicited from a holding potential of 60 mV to stepping potential of 70 to +40 mV. A slow decaying outward current was found at potentials more positive than 30 mV. The steady-state outward currents in the voltage range of 30 to +40 mV were significantly decreased after 60 min of rolipram (100 or 300 AM) administration (Fig. 5B). Effects of TEA on the spontaneous action potentials in the RP4 neuron TEA exerted a similar effect on the spontaneous action potentials in the RP4 neuron as shown in Fig. 6. The resting
Fig. 4. Effects of rolipram on fast total inward and outward currents in the RP4 neuron. The membrane currents were elicited from a holding potential of 60 mV to (1) test potentials of 50, 40, 30, 20, 10, 0, 10, 20 and 30 mV for 70 ms or (2) test potentials of 70, 60, 50, 40, 30, 20, 10, 0, 10, 20, 30 and 40 mV for 5 s. (A) Control, fast total inward and outward currents recorded in normal physiological saline. (B) Fast inward and outward currents recorded 60 min after rolipram (100 AM) incubation from panel A. (C) Fast inward and outward currents recorded 60 min after rolipram (300 AM) incubation from panel B. (D) Voltage step commands.
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Fig. 6. Effects of TEA on the RP4 neurons. Panels A and B at control were potentials of the control RP4 neurons. Panels A and B at 30 min were potentials after 30 min of application of TEA (5, 7 mM), respectively. Panels A and B at 60 min were potentials after 60 min of application of TEA (5, 7 mM), respectively. Panels A and B at washing 120 min were potentials after 120 min of washing off with normal saline from preparations incubated with 60 min TEA of panels A and B respectively. The horizontal bar on the top left side is the membrane potential at 0 mV. Note that TEA (7 mM) elicited bursts of potential on the RP4 neuron.
membrane potentials of the RP4 neurons in control 60 min after TEA (5 and 7 mM) administration were 57.3 F 0.5 mV (n = 4), 55.5 F 0.5 mV (n = 4, P N 0.05) and 57.4 F 2.2 mV (n = 4, P N 0.05) respectively. The frequencies of the spontaneously generated action potential in control 60 min after TEA (5 and 7 mM) administration were 37.5 F 1.5 pulses/min (n = 4), 40.4 F 2.4 pulses/min (n = 4, P N 0.05) and 10.0 F 2.0 bursts/min (n = 4, P b 0.05), respectively. TEA elicited action potentials bursts in the RP4 neuron in a concentration dependent manner. TEA at 5 mM did not alter the resting membrane potential or the frequency of the spontaneously generated action potential of the RP4 neuron. However, at a higher concentration (7 mM), TEA elicited action potentials bursts in the RP4 neurons. The effects of TEA (7 mM) on the RP4 neurons were reversible. After washing off TEA at 120 min, the resting membrane potentials and the patterns of the action potentials of the RP4 neuron returned to control level. Effects of TEA on steady-state I–V curve are shown in Fig. 5C. The currents were elicited by a 5 s duration of commanding step from a holding potential of 60 mV to stepping potentials, ranging from 70 mV to +40 mV at intervals of 10 mV. Compared with pre-drug controls, the steady-state I–V curve did not significantly change 40 min after TEA (5 mM) treatment (data not shown) but were clearly affected 60 min after TEA (7 mM) treatment. The steady-state I–V curve revealed an N-shaped appearance
(the negative slope resistance, NSR) in the steps ranging from 50 to 30 mV, after TEA (7 mM) treatment (Fig. 5C). The effects of TEA on the NSR response are reversible. After washing off TEA for 120 min, the steady-state membrane currents resumed and the NSR of the steadystate I–V curve elicited by TEA was reversed. It appears that TEA (7 mM) has a differentiation that elicits a negative slope region (NSR) in the steady-state current–voltage curve, which was not seen in rolipram-treated neruon.
Discussion In the present study, we characterized rolipram-elicited bursts of potential on the RP4 neuron of snail. The same neuQ rons were examined sequentially for drug treatment in each of the tables (that is, tables present sequential experiments done on the same neurons). Therefore, some of the control values varied according to different batches of samples. Rolipram is a selective inhibitor of phosphodiesterases (PDE) IV and the phosphodiesterase is responsible for hydrolysis of the cyclic nucleotides cAMP and cGMP in nerve cells. Rolipram-induced elevation of intracellular cAMP may have elicited the potential bursts. Other PDE inhibitors, e.g., vinpocetine (PDE1 selective), erythro-9-(2hydroxy-3-nonyl) adenine (EHNA, PDE2 selective), milri-
Fig. 5. Effect of rolipram and TEA on I–V curves. Panel A shows the current–voltage relationships of the peak fast total inward currents before (.), after rolipram (100 AM) (n) and rolipram (300 AM) (q) application for 60 min. Panel B shows the current–voltage relationships of the steady-state outward currents before (.), after rolipram (100 AM) (n) and rolipram (300 AM) (q) application for 60 min. Panel C shows the current–voltage relationships of the steady-state outward currents before (.) and after TEA (7 mM) (5) application for 60 min. Note that rolipram decreased the peak fast total inward currents and steady-state outward currents by a concentration dependent manner. No NSR was observed in control or rolipram-treated preparation while TEA (7 mM) elicited a negative slope region (NSR) at membrane potential between 50 mV and 30 mV. *P b 0.05 versus control.
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none (PDE3 selective) have also been shown to elicit bursts of potential on the RP4 neurons (Lin et al., 2004). Besides, forskolin, the adenyl-cyclase activator, elicited bursts of potential on the same neuron. It is interesting to note that cyclic AMP, the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor rolipram have been reported to enhance afterdischarges (ADs) induced in the CA1 region of rat hippocampal slices (Higashima et al., 2002). Voltage-clamp studies revealed that rolipram decreased the fast total inward current and steady-state outward currents of the RP4 neuron. However, no negative slope resistance (NSR) in the steady-state I–V curve was found in control or rolipram treated neurons. TEA, a blocker of the delayed outward K+ current, elicited action potential bursts and NSR in the RP4 neuron. It appears that the action potential bursts elicited by rolipram are not directly related to the NSR of the steady-state current–voltage relationship. The phosphodiesterase type 7 and 8 isozymes are also associated with the Alzheimer’s disease brains (Perez-Torres et al., 2003). The role of phosphodiesterase type 7 and 8 in the potential changes of central neurons remained an interesting subject for further study. The rolipram-elicited bursts of potential were not altered after addition of either a calcium-free solution, highmagnesium solution (which inhibited synaptic transmission) (Gainer, 1972) or in the presence of U73122 (phosphoinositide-specific phospholipase C inhibitor). The experimental results suggested that the bursts of potential elicited by rolipram were not in association with the synaptic transmission process of the ganglia or the phospholipase C activity in the RP4 neuron. The finding that the action potential bursts elicited by rolipram were blocked if KT-5720 (protein kinase A inhibitors) were pre-administrated. The results suggested that protein kinase A activity in the neuron play important roles on the bursts of potential elicited by rolipram. In calcium-free solution containing 8 mM Co2+ or highmagnesium solution which blocks the synaptic neurotransmission (Gainer, 1972), RP4 neurons still generate pace making action potentials that could be converted into bursting activity by rolipram. In addition, Co2+ also inhibited calcium current and the calcium-free solution severely reduced the calcium inward currents and calcium dependent outward potassium currents. The result suggests that sodium inward currents and voltage dependent potassium currents are the major targets for cAMP mediated neuro-modulation. It is suggested that the bursts of potential elicited by rolipram were not due to (1) synaptic effects of neurotransmitters, (2) NSR of steady-state I–V curve and (3) phospholipase activity of the neuron. The rolipramelicited bursts of potential were dependent on the
phosphodiesterases inhibitory activity and cAMP in the neuron.
Acknowledgment This work was supported by grant NSC-93-2320-B-002130 from National Science Council, Taipei, Taiwan.
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