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Nitric oxide increases excitability by depressing a calcium activated potassium current in snail neurons
Neuroscience Letters 295 (2000) 85±88
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Nitric oxide increases excitability by depressing a calcium activated potassium current in snail neurons Andrea Zsombok a,b, Siegfried Schrofner a, Anton Hermann a, Hubert H. Kerschbaum a,* a
Department of Molecular Neurobiology and Cellular Physiology, Institute of Zoology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria b Janus Pannonius University, Department of General Zoology and Neurobiology, University of PeÂcs, PeÂcs, Hungary Received 21 July 2000; received in revised form 9 October 2000; accepted 9 October 2000
Abstract In gastropods, the interneuronal messenger, nitric oxide (NO), modulates spike frequency and synaptic transmission. We have characterized the effect of NO on ion currents underlying neuronal excitability, using current-clamp and twoelectrode voltage-clamp techniques. Identi®ed neurons of the pulmonate snail, Helix pomatia, respond to the NO donor sodium nitroprusside (SNP) by increasing the ®ring frequency and decreasing the latency. Voltage-clamp experiments revealed that SNP or S-nitro-N-acetylpenicillamine (SNAP) depressed the macroscopic outward current, while the control compound N-acetylpenicillamine (NAP) had no effect. Current voltage curves generated from voltage steps to different membrane potentials ranging from 240 to 1180 mV showed an N-shaped outward current. Superfusion of ganglia with Ca 21 free Helix solution abolished the N-shape, indicating the contribution of a Ca 21 activated K 1 current (IK,Ca). Exposure of neurons to SNP or SNAP diminished the N-shape, indicating that NO affects IK,Ca. The depressing effect of SNP on the outward current was slow and reached steady state in about 5 min. In conclusion, our ®ndings indicate that NO enhances excitability in Helix nervous system by decreasing IK,Ca. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nitric oxide; Gastropods; Invertebrate; Neuron; Excitability; Ion currents
Nitric oxide (NO), a gaseous, membrane permeant interand intracellular messenger, is generated enzymatically by nitric oxide synthase (NOS), which oxidizes l-arginine to l-citrulline and NO using O2 and nicotinamide adenine dinucleotide phosphate (NADPH) [2]. In the nervous system, NO affects morphogenesis and electrical properties [6,8]. By altering the NO-cyclic guanosine monophosphate (cGMP)-PKG pathway, NO may modulate the cellular orchestration of behavior and regulate neuronal plasticity [4,16,20]. Although a neuronal NOS has been characterized in snails [12,15], only few studies addressed NO-dependent modulation of excitability and synaptic plasticity in gastropods [4,6,16]. Recently, it has been shown that NO and the NO-cGMP pathway mediates chemosensory activation of feeding in Lymnaea, olfactory processing in Limax, and * Corresponding author. Tel.: 143-662-8044-5667; fax: 143662-8044-5698. E-mail address: [email protected] (H.H. Kerschbaum).
hypersensibility in Aplysia [4,7,16]. Whereas these studies demonstrate the capacity of NO to alter neuronal excitability, less is known about the speci®c ion-currents involved. Here we report that NO tonically depresses IK,Ca in Helix neurons. In contrast to the depression of IK,Ca, the voltage gated ICa was not affected by NO. These ®ndings demonstrate that generation of NO increases excitability in the snail nervous system by depressing IK,Ca. Specimen of the pulmonate snail, Helix pomatia, were purchased from commercial suppliers (Exoterra, Deringen, Germany and O. Navratil, Austria) and kept under laboratory conditions. Subesophageal ganglia were dissected, pinned to the ¯oor of a recording chamber coated with Sylgard (Dow±Corning, Midland, MI), and perfused with Helix saline (in mM: 80 NaCl, 4 KCl, 10 CaCl2, 5 MgCl2, 5 Tris±HCl, 10 sucrose; pH 7.5). The connective tissue was mechanically removed. The epineurium was softened by 5 min treatment with protease (type XIV, Sigma) and dissected with micro-scissors. Following protease treatment, ganglia were thoroughly washed in Helix saline.
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 60 6- 2
86
A. Zsombok et al. / Neuroscience Letters 295 (2000) 85±88
All recordings were made from somata of visually identi®ed neurons from Helix subesophageal ganglia. For current-clamp and voltage-clamp experiments, individual somata were penetrated with a single or with two glass micropipettes, respectively. The glass microelectrodes were ®lled with 3 M KCl (resistance 3±7 MV). Hyperpolarizing current steps were used to study the passive electronic properties, while depolarizing current steps were used to measure spike threshold, frequency, duration, amplitude, and after hyperpolarization. The ®rst hyperpolarizing current step was 0.5 nA, then current steps were incremented in 0.5 nA steps. Depolarizing current steps started with 0.5 nA, then incremented with 0.5 nA steps. Input resistance was determined by injecting a 2.7 s, 0.5 nA current step. For two-electrode voltage-clamp experiments, the membrane potential was clamped at 240 mV and current responses were elicited by depolarizing voltage pulses of 100 ms duration to membrane potentials between 230 and 1180 mV. Pulses were delivered every 30 s. Voltage gated Ca 21 currents were isolated by superfusion of ganglia with (in mM: 40 CaCl2, 5 MgCl2, 4 KCl, 45 tetraethylammoniumchloride (TEA), 5 4-aminopyridine (4-AP), 5 Tris± HCl, 5 sucrose; pH 7.5). Recordings were made using an Axoclamp-2A ampli®er (Axon Instruments, Foster City, CA). We used the identi®ed subesophageal neurons rPa1, rPa5, lPa1±4 [14] to study the effect of NO on excitability. The most obvious effects of the NO donor sodium nitroprusside (SNP) (1 mM) on neuronal excitability were a decrease in action potential latency and an increase in the ®ring frequency (Fig. 1A,B) (n 10). SNP had neither no effect or induced only a small increase of the input resistance (,10%) (n 10). These experiments show that NO increases excitability in Helix neurones, similar to results in Lymnaea and Aplysia [4,16,17]. The increase in excitability could in principle arise by decreasing the activity of K 1 channels or by increasing
Fig. 1. Exogenous application of SNP increases excitability in neuron lPa2. Action potentials were monitored during depolarizing pulses in Helix saline (A) and in the presence of 1 mM SNP (B).
the activity of Na 1 or Ca 21 channels. To discriminate between these possibilities, we used a two-electrode voltage-clamp technique to analyze ion currents. As shown in Fig. 2, peak current amplitudes derived from depolarizing pulses from 240 to 80 mV delivered every 30 s were plotted against time. Application of 1 mM SNP tonically depressed the outward current by about 25% (n 5). About 5 min were required to reach the maximum effect of SNP (n 5). Because of the slow time course of NO-dependent depression, we studied the effect of NO on a family of ion currents 10 min after application of the NO donor. Fig. 3A shows a family of inward current and outward current. Plotting the peak current against the membrane potential reveals an N-shaped outward current, which is a hallmark for the contribution of a IK,Ca current to the outward current. Superfusion of the neuron with Ca 21 free Helix saline abolished the N-shape, further indicating a contribution IK,Ca to the total outward current (not shown). Exposure of neurons to 1 mM SNP (n 8) or 1 mM S-nitro-N-acetylpenicillamine (SNAP) (n 4) signi®cantly depressed the N-shape, indicating that NO affects mainly IK,Ca (Fig. 3B,C). N-acetylpenicillamine (NAP) (1 mM), a compound similar in structure to SNAP but without NO activity, did not signi®cantly affect the current (Fig. 3D) (n 4). To study the effect of NO on voltage gated K 1 current, neurons were incubated in Ca 21 free Helix saline. Under these conditions, SNP had little or no effect on the outward current. In additional experiments, we isolated a voltage gated Ca 21 current by blocking K 1 channels with 4-AP and TEA and substituting choline for Na 1. As shown in Fig. 4, SNP did not affect the voltage-gated Ca 21 current (n 4). Taken together, these experiments demonstrate that NO increases excitability in Helix neurons by decreasing a IK,Ca.
Fig. 2. SNP tonically decreases the macroscopic outward current in neuron lPa3. The plot shows peak currents (open circles) during depolarizing voltage steps from 240 to 180 mV delivered every 30 s. After the current stabilized in the presence of Helix saline, the neuron was exposed 1 mM SNP (bar). The NO donor, SNP, produces a tonic decrease in the outward current.
A. Zsombok et al. / Neuroscience Letters 295 (2000) 85±88
87
Fig. 3. SNP and SNAP but not NAP decrease the macroscopic outward current in neuron lPa2 (A,B) and lPa3 (C,D). (A) Family of ion currents. Ion currents were elicited by steps from 230 to 1180 mV in 10 mV increments from a holding potential of 240 mV in the absence (upper panel) and in the presence of 1 mM SNP (lower panel). Current traces were not corrected for leak current. (B) Currentvoltage relationship of the peak current amplitudes from the cell shown in (A) in the absence (squares) and presence (circles) of SNP (1 mM). (C) Current-voltage relationship of the peak current amplitudes before (squares) and 10 min after application of 1 mM SNAP (circles). (D) Peak current amplitude plotted against membrane potential before and 10 min after application of NAP (1 mM).
We have found that exposure of Helix ganglia to the NO donors SNP or SNAP increases neuronal, electrical excitability by blocking the outward current. These ®ndings complement previous electrophysiological studies showing a NO-dependent increase in excitability in gastropod neurons [4,16,17]. In the present study, we found that NO depresses IK,Ca in
Fig. 4. SNP does not affect the voltage gated Ca 21 current in neuron rPa3. Peak current amplitudes in the absence (squares) and presence (circles) of 1 mM SNP are plotted against the membrane potential.
Helix neurons. IK,Ca affects repolarization, after hyperpolarization, frequency of action potentials and adaptation [19]. In snail neurons, IK,Ca has been biophysically and pharmacologically characterized [9,11]. Depending on the neuron investigated and, possibly, on the speci®c type of Ca 21 activated potassium channel, NO activates or inhibits IK,Ca [1,5,18]. NO donors enhance electrical discharge activity in snail neurons involved in the generation of the buccal motor pattern and chemosensory activation of a ®ctive feeding behavior in Lymnaea stagnalis, and induce hyperexcitability in sensory neurons of Aplysia californica [4,16,17]. Furthermore, NO modulates synaptic transmission in A. californica [6]. In a previous study, we showed that NO increases the intracellular cGMP level in the Helix nervous system [13]. Preliminary experiments showed that exposure of neurons to the guanylylcyclase inhibitor, methylene blue, did not completely block the effect of SNP on the outward current (unpublished results). Therefore, it appears possible that NO has direct (via protein nitrosylation) and indirect (via cGMP signaling cascade) effects on IK,Ca. In neurons of H. pomatia, activation of IK,Ca terminates ®ring [3]. Therefore, blocking of IK,Ca, with NO should
88
A. Zsombok et al. / Neuroscience Letters 295 (2000) 85±88
increase the ®ring frequency of neurons. Thus, NO may shift the nervous system of gastropods toward an increased excitability. Since different types of identi®ed neurons exhibit quantitative differences in their K 1 outward currents [10], it is feasible that NO has a different effect on excitability. Neurons with a predominant IK,Ca will respond more strongly to NO compared to cells with a predominant voltage-dependent K 1 current. We thank Professor Dr W. Klimesch for comments on the manuscript. This work was supported by the FWF grant 13395 to HHK and by the Austrian Academic Exchange Service to A.Z. [1] Ahern, G.P., Hsu, S.F. and Jackson, M.B., Direct actions of nitric oxide on rat neurohypophysical K 1 channels, J. Physiol. (Lond.), 520 (1999) 165±176. [2] Bredt, D.S. and Snyder, S.H., Nitric oxide: a physiologic messenger molecule, Annu. Rev. Biochem., 63 (1994) 175±195. [3] Crest, M. and Gola, M., Large conductance Ca 21-activated K 1 channels are involved in both spike shaping and ®ring regulation in Helix neurones, J. Physiol. (Lond.), 465 (1993) 265±287. [4] Elphick, M.R., Kemenes, G., Staras, K. and O'Shea, M., Behavioral role for nitric oxide in chemosensory activation of feeding in a mollusc, J. Neurosci., 15 (1995) 7653±7664. [5] Erdemli, G. and Krnjevic, K., Nitric oxide tonically depresses a voltage- and Ca-dependent outward current in hippocampal slices, Neurosci. Lett., 201 (1995) 57±60. [6] Fossier, P., Tauc, L. and Baux, G., Calcium transients and neurotransmitter release at an identi®ed synapse, Trends Neurosci., 22 (1999) 161±166. [7] Gelperin, A., Nitric oxide mediates network oscillations of olfactory interneurons in a terrestrial mollusc, Nature, 369 (1994) 61±63. [8] Gibbs, S.M. and Truman, J.W., Nitric oxide and cyclic GMP regulate retinal patterning in the optic lobe of Drosophila, Neuron, 20 (1998) 83±93.
[9] Gola, M., Ducreux, C. and Chagneux, H., Ca 21-activated K 1 current involvement in neuronal function revealed by in situ single-channel analysis in Helix neurones, J. Physiol. (Lond.), 420 (1990) 73±109. [10] Gorman, A.L. and Hermann, A., Quantitative differences in the currents of bursting and beating molluscan pace-maker neurones, J. Physiol. (Lond.), 333 (1982) 681±699. [11] Hermann, A. and Erxleben, C., Charybdotoxin selectively blocks small Ca-activated K channels in Aplysia neurons, J. Gen. Physiol., 90 (1987) 27±47. [12] Huang, S., Kerschbaum, H.H., Engel, E. and Hermann, A., Biochemical characterization and histochemical localization of nitric oxide synthase in the nervous system of the snail, Helix pomatia, J. Neurochem., 69 (1997) 2516±2528. [13] Huang, S., Kerschbaum, H.H. and Hermann, A., Nitric oxidemediated cGMP synthesis in Helix neural ganglia, Brain Res., 780 (1998) 329±336. [14] Johansen, J., Jensen, L.H. and Holm, C., Morphological and electrophysiological mapping of giant neurons in the suboesophageal ganglia of Helix pomatia, Comp. Biochem. Physiol. A, 71 (1982) 283±291. [15] Korneev, S.A., Piper, M.R., Picot, J., Phillips, R., Korneeva, E.I. and O'Shea, M., Molecular characterization of NOS in a mollusc: expression in a giant modulatory neuron, J. Neurobiol., 35 (1998) 65±76. [16] Lewin, M.R. and Walters, E.T., Cyclic GMP pathway is critical for inducing long-term sensitization of nociceptive sensory neurons, Nat. Neurosci., 2 (1999) 18±23. [17] Moroz, L.L., Park, J.H. and Winlow, W., Nitric oxide activates buccal motor patterns in Lymnaea stagnalis, NeuroReport, 4 (1993) 643±646. [18] Murray, J.A., Shibata, E.F., Buresh, T.L., Picken, H., O'Meara, B.W. and Conklin, J.L., Nitric oxide modulates a calcium-activated potassium current in muscle cells from opossum esophagus, Am. J. Physiol., 269 (1995) 606± 612. [19] Sah, P., Ca 21-activated K 1 currents in neurones: types, physiological roles and modulation, Trends Neurosci., 19 (1996) 150±154. [20] Zorumski, C.F. and Izumi, Y., Modulation of LTP induction by NMDA receptor activation and nitric oxide release, Prog. Brain Res., 118 (1998) 173±182.
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Nitric oxide increases excitability by depressing a calcium activated potassium current in snail neurons Andrea Zsombok a,b, Siegfried Schrofner a, Anton Hermann a, Hubert H. Kerschbaum a,* a
Department of Molecular Neurobiology and Cellular Physiology, Institute of Zoology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria b Janus Pannonius University, Department of General Zoology and Neurobiology, University of PeÂcs, PeÂcs, Hungary Received 21 July 2000; received in revised form 9 October 2000; accepted 9 October 2000
Abstract In gastropods, the interneuronal messenger, nitric oxide (NO), modulates spike frequency and synaptic transmission. We have characterized the effect of NO on ion currents underlying neuronal excitability, using current-clamp and twoelectrode voltage-clamp techniques. Identi®ed neurons of the pulmonate snail, Helix pomatia, respond to the NO donor sodium nitroprusside (SNP) by increasing the ®ring frequency and decreasing the latency. Voltage-clamp experiments revealed that SNP or S-nitro-N-acetylpenicillamine (SNAP) depressed the macroscopic outward current, while the control compound N-acetylpenicillamine (NAP) had no effect. Current voltage curves generated from voltage steps to different membrane potentials ranging from 240 to 1180 mV showed an N-shaped outward current. Superfusion of ganglia with Ca 21 free Helix solution abolished the N-shape, indicating the contribution of a Ca 21 activated K 1 current (IK,Ca). Exposure of neurons to SNP or SNAP diminished the N-shape, indicating that NO affects IK,Ca. The depressing effect of SNP on the outward current was slow and reached steady state in about 5 min. In conclusion, our ®ndings indicate that NO enhances excitability in Helix nervous system by decreasing IK,Ca. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nitric oxide; Gastropods; Invertebrate; Neuron; Excitability; Ion currents
Nitric oxide (NO), a gaseous, membrane permeant interand intracellular messenger, is generated enzymatically by nitric oxide synthase (NOS), which oxidizes l-arginine to l-citrulline and NO using O2 and nicotinamide adenine dinucleotide phosphate (NADPH) [2]. In the nervous system, NO affects morphogenesis and electrical properties [6,8]. By altering the NO-cyclic guanosine monophosphate (cGMP)-PKG pathway, NO may modulate the cellular orchestration of behavior and regulate neuronal plasticity [4,16,20]. Although a neuronal NOS has been characterized in snails [12,15], only few studies addressed NO-dependent modulation of excitability and synaptic plasticity in gastropods [4,6,16]. Recently, it has been shown that NO and the NO-cGMP pathway mediates chemosensory activation of feeding in Lymnaea, olfactory processing in Limax, and * Corresponding author. Tel.: 143-662-8044-5667; fax: 143662-8044-5698. E-mail address: [email protected] (H.H. Kerschbaum).
hypersensibility in Aplysia [4,7,16]. Whereas these studies demonstrate the capacity of NO to alter neuronal excitability, less is known about the speci®c ion-currents involved. Here we report that NO tonically depresses IK,Ca in Helix neurons. In contrast to the depression of IK,Ca, the voltage gated ICa was not affected by NO. These ®ndings demonstrate that generation of NO increases excitability in the snail nervous system by depressing IK,Ca. Specimen of the pulmonate snail, Helix pomatia, were purchased from commercial suppliers (Exoterra, Deringen, Germany and O. Navratil, Austria) and kept under laboratory conditions. Subesophageal ganglia were dissected, pinned to the ¯oor of a recording chamber coated with Sylgard (Dow±Corning, Midland, MI), and perfused with Helix saline (in mM: 80 NaCl, 4 KCl, 10 CaCl2, 5 MgCl2, 5 Tris±HCl, 10 sucrose; pH 7.5). The connective tissue was mechanically removed. The epineurium was softened by 5 min treatment with protease (type XIV, Sigma) and dissected with micro-scissors. Following protease treatment, ganglia were thoroughly washed in Helix saline.
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 60 6- 2
86
A. Zsombok et al. / Neuroscience Letters 295 (2000) 85±88
All recordings were made from somata of visually identi®ed neurons from Helix subesophageal ganglia. For current-clamp and voltage-clamp experiments, individual somata were penetrated with a single or with two glass micropipettes, respectively. The glass microelectrodes were ®lled with 3 M KCl (resistance 3±7 MV). Hyperpolarizing current steps were used to study the passive electronic properties, while depolarizing current steps were used to measure spike threshold, frequency, duration, amplitude, and after hyperpolarization. The ®rst hyperpolarizing current step was 0.5 nA, then current steps were incremented in 0.5 nA steps. Depolarizing current steps started with 0.5 nA, then incremented with 0.5 nA steps. Input resistance was determined by injecting a 2.7 s, 0.5 nA current step. For two-electrode voltage-clamp experiments, the membrane potential was clamped at 240 mV and current responses were elicited by depolarizing voltage pulses of 100 ms duration to membrane potentials between 230 and 1180 mV. Pulses were delivered every 30 s. Voltage gated Ca 21 currents were isolated by superfusion of ganglia with (in mM: 40 CaCl2, 5 MgCl2, 4 KCl, 45 tetraethylammoniumchloride (TEA), 5 4-aminopyridine (4-AP), 5 Tris± HCl, 5 sucrose; pH 7.5). Recordings were made using an Axoclamp-2A ampli®er (Axon Instruments, Foster City, CA). We used the identi®ed subesophageal neurons rPa1, rPa5, lPa1±4 [14] to study the effect of NO on excitability. The most obvious effects of the NO donor sodium nitroprusside (SNP) (1 mM) on neuronal excitability were a decrease in action potential latency and an increase in the ®ring frequency (Fig. 1A,B) (n 10). SNP had neither no effect or induced only a small increase of the input resistance (,10%) (n 10). These experiments show that NO increases excitability in Helix neurones, similar to results in Lymnaea and Aplysia [4,16,17]. The increase in excitability could in principle arise by decreasing the activity of K 1 channels or by increasing
Fig. 1. Exogenous application of SNP increases excitability in neuron lPa2. Action potentials were monitored during depolarizing pulses in Helix saline (A) and in the presence of 1 mM SNP (B).
the activity of Na 1 or Ca 21 channels. To discriminate between these possibilities, we used a two-electrode voltage-clamp technique to analyze ion currents. As shown in Fig. 2, peak current amplitudes derived from depolarizing pulses from 240 to 80 mV delivered every 30 s were plotted against time. Application of 1 mM SNP tonically depressed the outward current by about 25% (n 5). About 5 min were required to reach the maximum effect of SNP (n 5). Because of the slow time course of NO-dependent depression, we studied the effect of NO on a family of ion currents 10 min after application of the NO donor. Fig. 3A shows a family of inward current and outward current. Plotting the peak current against the membrane potential reveals an N-shaped outward current, which is a hallmark for the contribution of a IK,Ca current to the outward current. Superfusion of the neuron with Ca 21 free Helix saline abolished the N-shape, further indicating a contribution IK,Ca to the total outward current (not shown). Exposure of neurons to 1 mM SNP (n 8) or 1 mM S-nitro-N-acetylpenicillamine (SNAP) (n 4) signi®cantly depressed the N-shape, indicating that NO affects mainly IK,Ca (Fig. 3B,C). N-acetylpenicillamine (NAP) (1 mM), a compound similar in structure to SNAP but without NO activity, did not signi®cantly affect the current (Fig. 3D) (n 4). To study the effect of NO on voltage gated K 1 current, neurons were incubated in Ca 21 free Helix saline. Under these conditions, SNP had little or no effect on the outward current. In additional experiments, we isolated a voltage gated Ca 21 current by blocking K 1 channels with 4-AP and TEA and substituting choline for Na 1. As shown in Fig. 4, SNP did not affect the voltage-gated Ca 21 current (n 4). Taken together, these experiments demonstrate that NO increases excitability in Helix neurons by decreasing a IK,Ca.
Fig. 2. SNP tonically decreases the macroscopic outward current in neuron lPa3. The plot shows peak currents (open circles) during depolarizing voltage steps from 240 to 180 mV delivered every 30 s. After the current stabilized in the presence of Helix saline, the neuron was exposed 1 mM SNP (bar). The NO donor, SNP, produces a tonic decrease in the outward current.
A. Zsombok et al. / Neuroscience Letters 295 (2000) 85±88
87
Fig. 3. SNP and SNAP but not NAP decrease the macroscopic outward current in neuron lPa2 (A,B) and lPa3 (C,D). (A) Family of ion currents. Ion currents were elicited by steps from 230 to 1180 mV in 10 mV increments from a holding potential of 240 mV in the absence (upper panel) and in the presence of 1 mM SNP (lower panel). Current traces were not corrected for leak current. (B) Currentvoltage relationship of the peak current amplitudes from the cell shown in (A) in the absence (squares) and presence (circles) of SNP (1 mM). (C) Current-voltage relationship of the peak current amplitudes before (squares) and 10 min after application of 1 mM SNAP (circles). (D) Peak current amplitude plotted against membrane potential before and 10 min after application of NAP (1 mM).
We have found that exposure of Helix ganglia to the NO donors SNP or SNAP increases neuronal, electrical excitability by blocking the outward current. These ®ndings complement previous electrophysiological studies showing a NO-dependent increase in excitability in gastropod neurons [4,16,17]. In the present study, we found that NO depresses IK,Ca in
Fig. 4. SNP does not affect the voltage gated Ca 21 current in neuron rPa3. Peak current amplitudes in the absence (squares) and presence (circles) of 1 mM SNP are plotted against the membrane potential.
Helix neurons. IK,Ca affects repolarization, after hyperpolarization, frequency of action potentials and adaptation [19]. In snail neurons, IK,Ca has been biophysically and pharmacologically characterized [9,11]. Depending on the neuron investigated and, possibly, on the speci®c type of Ca 21 activated potassium channel, NO activates or inhibits IK,Ca [1,5,18]. NO donors enhance electrical discharge activity in snail neurons involved in the generation of the buccal motor pattern and chemosensory activation of a ®ctive feeding behavior in Lymnaea stagnalis, and induce hyperexcitability in sensory neurons of Aplysia californica [4,16,17]. Furthermore, NO modulates synaptic transmission in A. californica [6]. In a previous study, we showed that NO increases the intracellular cGMP level in the Helix nervous system [13]. Preliminary experiments showed that exposure of neurons to the guanylylcyclase inhibitor, methylene blue, did not completely block the effect of SNP on the outward current (unpublished results). Therefore, it appears possible that NO has direct (via protein nitrosylation) and indirect (via cGMP signaling cascade) effects on IK,Ca. In neurons of H. pomatia, activation of IK,Ca terminates ®ring [3]. Therefore, blocking of IK,Ca, with NO should
88
A. Zsombok et al. / Neuroscience Letters 295 (2000) 85±88
increase the ®ring frequency of neurons. Thus, NO may shift the nervous system of gastropods toward an increased excitability. Since different types of identi®ed neurons exhibit quantitative differences in their K 1 outward currents [10], it is feasible that NO has a different effect on excitability. Neurons with a predominant IK,Ca will respond more strongly to NO compared to cells with a predominant voltage-dependent K 1 current. We thank Professor Dr W. Klimesch for comments on the manuscript. This work was supported by the FWF grant 13395 to HHK and by the Austrian Academic Exchange Service to A.Z. [1] Ahern, G.P., Hsu, S.F. and Jackson, M.B., Direct actions of nitric oxide on rat neurohypophysical K 1 channels, J. Physiol. (Lond.), 520 (1999) 165±176. [2] Bredt, D.S. and Snyder, S.H., Nitric oxide: a physiologic messenger molecule, Annu. Rev. Biochem., 63 (1994) 175±195. [3] Crest, M. and Gola, M., Large conductance Ca 21-activated K 1 channels are involved in both spike shaping and ®ring regulation in Helix neurones, J. Physiol. (Lond.), 465 (1993) 265±287. [4] Elphick, M.R., Kemenes, G., Staras, K. and O'Shea, M., Behavioral role for nitric oxide in chemosensory activation of feeding in a mollusc, J. Neurosci., 15 (1995) 7653±7664. [5] Erdemli, G. and Krnjevic, K., Nitric oxide tonically depresses a voltage- and Ca-dependent outward current in hippocampal slices, Neurosci. Lett., 201 (1995) 57±60. [6] Fossier, P., Tauc, L. and Baux, G., Calcium transients and neurotransmitter release at an identi®ed synapse, Trends Neurosci., 22 (1999) 161±166. [7] Gelperin, A., Nitric oxide mediates network oscillations of olfactory interneurons in a terrestrial mollusc, Nature, 369 (1994) 61±63. [8] Gibbs, S.M. and Truman, J.W., Nitric oxide and cyclic GMP regulate retinal patterning in the optic lobe of Drosophila, Neuron, 20 (1998) 83±93.
[9] Gola, M., Ducreux, C. and Chagneux, H., Ca 21-activated K 1 current involvement in neuronal function revealed by in situ single-channel analysis in Helix neurones, J. Physiol. (Lond.), 420 (1990) 73±109. [10] Gorman, A.L. and Hermann, A., Quantitative differences in the currents of bursting and beating molluscan pace-maker neurones, J. Physiol. (Lond.), 333 (1982) 681±699. [11] Hermann, A. and Erxleben, C., Charybdotoxin selectively blocks small Ca-activated K channels in Aplysia neurons, J. Gen. Physiol., 90 (1987) 27±47. [12] Huang, S., Kerschbaum, H.H., Engel, E. and Hermann, A., Biochemical characterization and histochemical localization of nitric oxide synthase in the nervous system of the snail, Helix pomatia, J. Neurochem., 69 (1997) 2516±2528. [13] Huang, S., Kerschbaum, H.H. and Hermann, A., Nitric oxidemediated cGMP synthesis in Helix neural ganglia, Brain Res., 780 (1998) 329±336. [14] Johansen, J., Jensen, L.H. and Holm, C., Morphological and electrophysiological mapping of giant neurons in the suboesophageal ganglia of Helix pomatia, Comp. Biochem. Physiol. A, 71 (1982) 283±291. [15] Korneev, S.A., Piper, M.R., Picot, J., Phillips, R., Korneeva, E.I. and O'Shea, M., Molecular characterization of NOS in a mollusc: expression in a giant modulatory neuron, J. Neurobiol., 35 (1998) 65±76. [16] Lewin, M.R. and Walters, E.T., Cyclic GMP pathway is critical for inducing long-term sensitization of nociceptive sensory neurons, Nat. Neurosci., 2 (1999) 18±23. [17] Moroz, L.L., Park, J.H. and Winlow, W., Nitric oxide activates buccal motor patterns in Lymnaea stagnalis, NeuroReport, 4 (1993) 643±646. [18] Murray, J.A., Shibata, E.F., Buresh, T.L., Picken, H., O'Meara, B.W. and Conklin, J.L., Nitric oxide modulates a calcium-activated potassium current in muscle cells from opossum esophagus, Am. J. Physiol., 269 (1995) 606± 612. [19] Sah, P., Ca 21-activated K 1 currents in neurones: types, physiological roles and modulation, Trends Neurosci., 19 (1996) 150±154. [20] Zorumski, C.F. and Izumi, Y., Modulation of LTP induction by NMDA receptor activation and nitric oxide release, Prog. Brain Res., 118 (1998) 173±182.