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Neurohypophysial peptides selectively depressed high voltage-activated Ca-current in snail neurons
Neuroscience Letters, 120 (1990) 9-12
9
ElsevierScientificPublishers Ireland Ltd. NSL 07307
Neurohypophysial peptides selectively depressed high voltage-activated Ca-current in snail neurons Oleg N. O s i p e n k o 1 a n d T. Kiss 2 1Department of General Physiology of the Nervous System, A.A. Bogomoletz Institute, Academy of Sciences of the Ukrainian S.S.R., Kiev (U.S.S.R.) and 2Balaton Limnological Research Institute of the Hungarian Academy of Sciences, Tihany (Hungary)
(Received8 June 1990;Revisedversionreceived30 July 1990;Accepted 1 August 1990) Key words." Ca-current;HVA channel; Neurohypophysialpeptide; Voltage-clamp;Helix pomatia neuron
It was found that neurohypophysialpeptides oxytocin and Lys-vasopressinselectivelydecreased the high voltage-activatedcomponent of the inward Ca-currentin voltage-clampedHelix pomatia identifiedneurons in a dose- and time-dependentmanner. This effectwas unaffectedor further enhanced applying phosphodiesterases inhibitors. It is suggested that suppression of high voltage-activatedcurrent was due to the activation of the adenylatecyclasesystem,likelyprotein kinase A.
It is well known that oxytocin (OXT) and vasopressin (LVP) are the most important nonapeptides secreted from the posterior pituitary. In several molluscan species extracts of the cerebral and pleuro-pedal ganglia have been recently shown to contain immunoreactivity which resembles that o f vasotocin or LVP [14], which suggests that some peptides may have been well conserved through evolution [1]. In invertebrates the effects of OXT and LVP may not correspond to those in vertebrates. For example, OXT and LVP regulate the osmotic pressure of hemolymph in the earthworm [3], regulate the diuresis in insects [15] or excite 'and inhibit some giant nerve cells in molluscs [1, 2, 6, 11, 16]. Previously it was described that the effect of OXT, LVP and related peptides is to initiate or enhance the bursting activity, causing an N-shaped current-voltage curve [1, 2]. Furthermore it was found that OXT and LVP initiate steady-state currents in voltage-clamped snail neurons [11], and that the amplitudes of the cAMP-evoked current were also enhanced upon application of OXT [12]. It was concluded, therefore, that this modulatory response of the peptide was due to the direct effect on the cAMP-dependent protein kinase activity [12]. Ca-channels in molluscan neurons are not easily grouped compared with those of the vertebrate Ca-channels [10]. Nevertheless the existence of low voltage-activated
Correspondence: O.N. Osipenko, Department of General Physiology of the Nervous System, A.A. BogomoletzInstitute, Academy of Sciences of the Ukrainian S.R.R., Kiev-24,GSP, 252601, U.S.S.R.
0304-3940/90/$03.50 O 1990ElsevierScientificPublishers Ireland Ltd.
(LVA) and high voltage-activated (HVA) channels was demonstrated in H e l i x neurons [8, 9]. Since the metabolic dependence of the HVA is already established [9, 10], the aim of the present experiments was to examine the OXT and LVP actions on HVA currents. Experiments were performed on identified neurons of the suboesophageal ganglia of the snail H e l i x pomatia L [13]. The preparation procedure and two-microelectrode voltage-clamp recording technique were described in detail earlier [4]. Oxytocin (Gedeon Richter A.O. or Organon VB) and lysin-vasopressin (Sandoz) were dissolved in control saline immediately before each experiment. The experimental chamber was perfused with a gravityfed perfusion system both with control and peptide-containing salines at a rate of 1 ml/min. Data acquisition was made using an IBM XT PC and the results were plotted on an X - Y plotter (Hewlett-Packard 74 75A). Fig. 1A presents a family of Ca-currents recorded at different step depolarizations from a holding potential of - 9 0 mV. It is supposed that currents recorded at voltages more positive than - 1 0 mV are largely carried through HVA Ca-channels. Application of OXT reduces the current between potentials of 0 and + 50 mV by about 20-35%. In Fig. 1B it can be seen that the effect of OXT is concentration-dependent. In Fig. 1C the current-voltage characteristics ( I - V curves) of the difference currents (i.e. the currents which were blocked by OXT) are also plotted. The activation and inactivation ranges and also the peak value of the I - V curves are very similar to those of the HVA current [8-10]. However, the rate of inactivation was decreased, which is clearly
lO
B
A -10
-2(
20
4,o
6,0 U(mV)
---voxt
oxt / • 10.6 M • 10-7M + conlrol
p O nA Ito H
oxt
1
10
- 100. ~ . . _ ~ _ (oxt
I
oxt - 150.
30
50
-200
_-6o
-4.0
-20
_
I(nA)
2,0
40
6o
,
U( m Vl • 10-6 M o 10-7M
OXt - 50
l(nA)
Fig. 1. A: effect of OXT on Ca-currents in an RPal neuron. Current traces were recorded at the potentials indicated in the right upper comer of each trace. Records were taken 20 min after the perfusion had started with peptide-containing saline. B: I-V curves of peak currents recorded before (+) and after peptide application: 10-7 M ((3) and 10-6 M (•). C: the potential dependence of the differencecurrents, which was blocked by peptide. Leakagecurrents have been substracted.
demonstrated in Fig. 1A at 4-10 mV c o m m a n d potential. Application of LVP caused similar changes in the individual current records (Fig. 2A) and in the I - V curve (Fig. 2B) too. In this series of experiments the difference currents were also plotted (Fig. 2C) which showed the same properties as after OXT blocking. Both effects were reversible after 30 min washing with control saline. Similar results were obtained in salines in which Ca-ions were replaced for Ba 2+. Ba-ions do not substitute Ca-ions in the activation of Ca-dependent potassium current. Therefore the decrease of HVA currents was not a result of the activtion of Ca-activated conductances. It is suggested that both peptides exerted their effect through the same mechanism. The dose-response relationships show that both peptides are effective between 10 -8 and 10 -5 M (Fig. 3A). Also the time course of the peptide action was measured. In Fig. 3B the time dependence of the actions can be referred. 50% O X T blockade was observed between 25 and 35 min after peptide application. In the
case of LVP, the time required to reach 50% depression was between 15 and 20 min. The neuropeptides O X T and LVP depressed part of the H V A Ca-currents in snail neurons, leaving the LVA channels unaltered. The onset of the peptide actions was slow and the effect of the peptides was long-lasting indicating that the neuron neither has a means of rapidly inactivating O X T and LVP nor becomes desensitized to them. Both peptides decreased the H V A Ca-current amplitude in a dose- and time-dependent manner, without affecting the activation. The long-time effect suggests likely involvement of the second messenger system in the conductance changes upon O X T and LVP application. Therefore the effects of both peptides were tested on neurons which were pretreated with phosphodiesterase inhibitors (theophylline, caffeine or 1-methylxanthine (1 mM), which, as was found earlier, increase the intracellular c A M P level [7] and decrease Ca-current [5] in molluscan neurons. We have also observed suppressing effect of these inhibitors which was unaffected or further enhanced applying pep-
11
A
B -10
-20
eo -e,O -4p
-3,
70
20
40
, U(mVl
I
LVP
~P
~
[
nA
• •
,°
1 0 - 6 M kVP 10"7M
÷ control
0 LVP
10[
/ LVP
final
-500
5O
3O
-po-sp -op -4p -2p -
Lvp 10-7.
• •
29
--,=~
4,o
sO , U{mVI
10 -6 M LVP 10-7M
I1 nA}, _ 3 0 0 Fig. 2. A: effect of LVP on Ca-currents in an RPal neuron. Current traces were recorded at the potentials indicated in the fight upper comer of each trace. Records were taken 20 min after the perfusion was started with peptide-containing saline. B: I-Vcurves of peak currents recorded before ( + ) and after peptide application: 10 -7 (O) and 10 -6 M ( e ) . C: the potential dependence of the difference currents, which was blocked by peptide. Leakage currents have been substracted.
tides. According to these results and those obtained earlier [12] we propose that peptides decrease HVA current due to their action on protein kinase A.
Our findings may indicate therefore that OXT and LVP suppressed selectively the HVA current in the neurons investigated.
A i/imax
oxt
I/I max
B
LVP I/Imax
1.0!
oxt
1.0( o
•
1.0
[3
o
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o
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LVP
o
g 0.5~
qmax
•
0.5.
o
o
o
•
@
o
0
M
10-9 ";0-8 10-7 10-6
M 10-9 10-8 10-7 10-6
0
lb
2'0
.rain
30
40
0
1'0
2'0
ab
,min
4o
Fig. 3. Concentration (A) and time (B) dependence of OXT and LVP actions on calcium current. Points show the values of modified current peak amplitude normalized to control value. I and/max are maximal values of Ca-current measured in the presence of a peptide at different concentrations or at different times after starting the peptide application and in control, respectively. Different symbols denote different neurons. Test potential was + 30 mV.
12 1 Barker, J.L. and Gainer, H., Peptide regulation of bursting pacemaker activity in a molluscan neurosecretory cell, Science, 184 (1974) 1371-1372. 2 Barker, J.L., Ishin, M.S. and Gainer, H., Studies on bursting pacemaker potential activity in molluscan neurons III. Effects of hormones. Brain Res., 84 (1975) 501-513. 3 Kinoshita, K. and Kawashima, S., Research for the function of AVP- and OXT-immunoreactive cells in the ganglia of the earthworm, Zool. Sci., 2 (1985) 971. 4 Kiss, T., Catch-relaxing peptide (CARP) decreases the Ca-permeability of snail neuronal membrane, Experientia, 44 (1988) 998I000. 5 Kononenko, N.I., Kostyuk, P.G. and Shcherbatko, A.D., The effect of intracellular cAMP injections on stationary membrane conductance and voltage- and time-dependent ionic currents in identified snail neurons, Brain Res., 268 (1983) 321-338. 6 Ku, B.S., Takeuchi, H., Yanaihara, N., Munekata, E. and Ariyoshi, Y., Effects of synthetic peptides on giant neurone identified in the ganglia of an African giant snail (Achatinafulica Ferussac), Comp. Biochem. Physiol., 84C (1986) 331 396. 7 Levitan, I.B., Bergstroem, E. and Simonet, M., Adenylate cyclase in Helix and Aplysia ganglia: characteristics of its stimulation by a peptide-containing nervous system, J. Neurochem., 31 (1978) 1353-1359. 8 Lozinskaya, I.M. and Shuba, Y.M., Low- and high-threshold calcium current in the membrane of cultured neurons of Helix pomatia, Neurophysiologia, 21 (1989) 127 129.
9 Mironov, S.L., Tepikin, A.V. and Grishchenko, A.V., Two calcium currents in the somatic membrane of the mollusc neurons, Neurophysiologia, 17 (1985) 627-633. 10 Nowycky, M.C., Fox, A.P. and Tsien, R.W., Three types of neuronal calcium channel with different calcium agonist selectivity, Nature, 316 (1985) 440-443. l I Osipenko, O.N., Depolarization and hyperpolarization of the neuronal membrane in the snail Helix pomatia identified neurons induced by oxytocin application to the soma of these neurons, Comp. Biochem. Physiol., 94C (1989) 655-661. 12 Osipenko, O.N., Modulatory action of oxytocin on electrical responses of the neurons in the snail Helix pomatia, Comp. Biochem. Physiol., 95C (1990) 9-14. 13 Sakharov, D.A. and Salanki, J., Physiological and pharmacological identification of neurons in the central nervous system of Helix pomatia. L., Acta Physiol. Acad. Sci. Hung., 35 (1969) 19-30. 14 Sawyer, W.H., Deyrup-Olsen, I. and Martin, A.W., Immunological and biological characteristics of the vasotocin-like activity in the head ganglia of gastropoda molluscs, Gen. Comp. Endocrinol., 54 (1984) 97-108. 15 Strambi, C., Rougon-Rapuzzi, G., Cupo, A., Martin, N. and Strambi, A., Mise en 6vidence immunocytologique d'un compose apparente ~. la vasopressine dans le syst~me nerveux du grillon Acheta domesticus, C.R. Acad. Sci. Paris, 288D (1979) 131-133. 16 Takeda, N., Takayanagi, H., Hasegawa, K., Miczuno, J. and Fujii, K., Neuropeptides and their actions on neurons in the central nervous system of the snail, Aehatinafulica. In, Abstracts of the Symposium on Molluscan Neurobiology, Amsterdam, The Netherlands, 1986, pp. 45.
9
ElsevierScientificPublishers Ireland Ltd. NSL 07307
Neurohypophysial peptides selectively depressed high voltage-activated Ca-current in snail neurons Oleg N. O s i p e n k o 1 a n d T. Kiss 2 1Department of General Physiology of the Nervous System, A.A. Bogomoletz Institute, Academy of Sciences of the Ukrainian S.S.R., Kiev (U.S.S.R.) and 2Balaton Limnological Research Institute of the Hungarian Academy of Sciences, Tihany (Hungary)
(Received8 June 1990;Revisedversionreceived30 July 1990;Accepted 1 August 1990) Key words." Ca-current;HVA channel; Neurohypophysialpeptide; Voltage-clamp;Helix pomatia neuron
It was found that neurohypophysialpeptides oxytocin and Lys-vasopressinselectivelydecreased the high voltage-activatedcomponent of the inward Ca-currentin voltage-clampedHelix pomatia identifiedneurons in a dose- and time-dependentmanner. This effectwas unaffectedor further enhanced applying phosphodiesterases inhibitors. It is suggested that suppression of high voltage-activatedcurrent was due to the activation of the adenylatecyclasesystem,likelyprotein kinase A.
It is well known that oxytocin (OXT) and vasopressin (LVP) are the most important nonapeptides secreted from the posterior pituitary. In several molluscan species extracts of the cerebral and pleuro-pedal ganglia have been recently shown to contain immunoreactivity which resembles that o f vasotocin or LVP [14], which suggests that some peptides may have been well conserved through evolution [1]. In invertebrates the effects of OXT and LVP may not correspond to those in vertebrates. For example, OXT and LVP regulate the osmotic pressure of hemolymph in the earthworm [3], regulate the diuresis in insects [15] or excite 'and inhibit some giant nerve cells in molluscs [1, 2, 6, 11, 16]. Previously it was described that the effect of OXT, LVP and related peptides is to initiate or enhance the bursting activity, causing an N-shaped current-voltage curve [1, 2]. Furthermore it was found that OXT and LVP initiate steady-state currents in voltage-clamped snail neurons [11], and that the amplitudes of the cAMP-evoked current were also enhanced upon application of OXT [12]. It was concluded, therefore, that this modulatory response of the peptide was due to the direct effect on the cAMP-dependent protein kinase activity [12]. Ca-channels in molluscan neurons are not easily grouped compared with those of the vertebrate Ca-channels [10]. Nevertheless the existence of low voltage-activated
Correspondence: O.N. Osipenko, Department of General Physiology of the Nervous System, A.A. BogomoletzInstitute, Academy of Sciences of the Ukrainian S.R.R., Kiev-24,GSP, 252601, U.S.S.R.
0304-3940/90/$03.50 O 1990ElsevierScientificPublishers Ireland Ltd.
(LVA) and high voltage-activated (HVA) channels was demonstrated in H e l i x neurons [8, 9]. Since the metabolic dependence of the HVA is already established [9, 10], the aim of the present experiments was to examine the OXT and LVP actions on HVA currents. Experiments were performed on identified neurons of the suboesophageal ganglia of the snail H e l i x pomatia L [13]. The preparation procedure and two-microelectrode voltage-clamp recording technique were described in detail earlier [4]. Oxytocin (Gedeon Richter A.O. or Organon VB) and lysin-vasopressin (Sandoz) were dissolved in control saline immediately before each experiment. The experimental chamber was perfused with a gravityfed perfusion system both with control and peptide-containing salines at a rate of 1 ml/min. Data acquisition was made using an IBM XT PC and the results were plotted on an X - Y plotter (Hewlett-Packard 74 75A). Fig. 1A presents a family of Ca-currents recorded at different step depolarizations from a holding potential of - 9 0 mV. It is supposed that currents recorded at voltages more positive than - 1 0 mV are largely carried through HVA Ca-channels. Application of OXT reduces the current between potentials of 0 and + 50 mV by about 20-35%. In Fig. 1B it can be seen that the effect of OXT is concentration-dependent. In Fig. 1C the current-voltage characteristics ( I - V curves) of the difference currents (i.e. the currents which were blocked by OXT) are also plotted. The activation and inactivation ranges and also the peak value of the I - V curves are very similar to those of the HVA current [8-10]. However, the rate of inactivation was decreased, which is clearly
lO
B
A -10
-2(
20
4,o
6,0 U(mV)
---voxt
oxt / • 10.6 M • 10-7M + conlrol
p O nA Ito H
oxt
1
10
- 100. ~ . . _ ~ _ (oxt
I
oxt - 150.
30
50
-200
_-6o
-4.0
-20
_
I(nA)
2,0
40
6o
,
U( m Vl • 10-6 M o 10-7M
OXt - 50
l(nA)
Fig. 1. A: effect of OXT on Ca-currents in an RPal neuron. Current traces were recorded at the potentials indicated in the right upper comer of each trace. Records were taken 20 min after the perfusion had started with peptide-containing saline. B: I-V curves of peak currents recorded before (+) and after peptide application: 10-7 M ((3) and 10-6 M (•). C: the potential dependence of the differencecurrents, which was blocked by peptide. Leakagecurrents have been substracted.
demonstrated in Fig. 1A at 4-10 mV c o m m a n d potential. Application of LVP caused similar changes in the individual current records (Fig. 2A) and in the I - V curve (Fig. 2B) too. In this series of experiments the difference currents were also plotted (Fig. 2C) which showed the same properties as after OXT blocking. Both effects were reversible after 30 min washing with control saline. Similar results were obtained in salines in which Ca-ions were replaced for Ba 2+. Ba-ions do not substitute Ca-ions in the activation of Ca-dependent potassium current. Therefore the decrease of HVA currents was not a result of the activtion of Ca-activated conductances. It is suggested that both peptides exerted their effect through the same mechanism. The dose-response relationships show that both peptides are effective between 10 -8 and 10 -5 M (Fig. 3A). Also the time course of the peptide action was measured. In Fig. 3B the time dependence of the actions can be referred. 50% O X T blockade was observed between 25 and 35 min after peptide application. In the
case of LVP, the time required to reach 50% depression was between 15 and 20 min. The neuropeptides O X T and LVP depressed part of the H V A Ca-currents in snail neurons, leaving the LVA channels unaltered. The onset of the peptide actions was slow and the effect of the peptides was long-lasting indicating that the neuron neither has a means of rapidly inactivating O X T and LVP nor becomes desensitized to them. Both peptides decreased the H V A Ca-current amplitude in a dose- and time-dependent manner, without affecting the activation. The long-time effect suggests likely involvement of the second messenger system in the conductance changes upon O X T and LVP application. Therefore the effects of both peptides were tested on neurons which were pretreated with phosphodiesterase inhibitors (theophylline, caffeine or 1-methylxanthine (1 mM), which, as was found earlier, increase the intracellular c A M P level [7] and decrease Ca-current [5] in molluscan neurons. We have also observed suppressing effect of these inhibitors which was unaffected or further enhanced applying pep-
11
A
B -10
-20
eo -e,O -4p
-3,
70
20
40
, U(mVl
I
LVP
~P
~
[
nA
• •
,°
1 0 - 6 M kVP 10"7M
÷ control
0 LVP
10[
/ LVP
final
-500
5O
3O
-po-sp -op -4p -2p -
Lvp 10-7.
• •
29
--,=~
4,o
sO , U{mVI
10 -6 M LVP 10-7M
I1 nA}, _ 3 0 0 Fig. 2. A: effect of LVP on Ca-currents in an RPal neuron. Current traces were recorded at the potentials indicated in the fight upper comer of each trace. Records were taken 20 min after the perfusion was started with peptide-containing saline. B: I-Vcurves of peak currents recorded before ( + ) and after peptide application: 10 -7 (O) and 10 -6 M ( e ) . C: the potential dependence of the difference currents, which was blocked by peptide. Leakage currents have been substracted.
tides. According to these results and those obtained earlier [12] we propose that peptides decrease HVA current due to their action on protein kinase A.
Our findings may indicate therefore that OXT and LVP suppressed selectively the HVA current in the neurons investigated.
A i/imax
oxt
I/I max
B
LVP I/Imax
1.0!
oxt
1.0( o
•
1.0
[3
o
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o
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o
g 0.5~
qmax
•
0.5.
o
o
o
•
@
o
0
M
10-9 ";0-8 10-7 10-6
M 10-9 10-8 10-7 10-6
0
lb
2'0
.rain
30
40
0
1'0
2'0
ab
,min
4o
Fig. 3. Concentration (A) and time (B) dependence of OXT and LVP actions on calcium current. Points show the values of modified current peak amplitude normalized to control value. I and/max are maximal values of Ca-current measured in the presence of a peptide at different concentrations or at different times after starting the peptide application and in control, respectively. Different symbols denote different neurons. Test potential was + 30 mV.
12 1 Barker, J.L. and Gainer, H., Peptide regulation of bursting pacemaker activity in a molluscan neurosecretory cell, Science, 184 (1974) 1371-1372. 2 Barker, J.L., Ishin, M.S. and Gainer, H., Studies on bursting pacemaker potential activity in molluscan neurons III. Effects of hormones. Brain Res., 84 (1975) 501-513. 3 Kinoshita, K. and Kawashima, S., Research for the function of AVP- and OXT-immunoreactive cells in the ganglia of the earthworm, Zool. Sci., 2 (1985) 971. 4 Kiss, T., Catch-relaxing peptide (CARP) decreases the Ca-permeability of snail neuronal membrane, Experientia, 44 (1988) 998I000. 5 Kononenko, N.I., Kostyuk, P.G. and Shcherbatko, A.D., The effect of intracellular cAMP injections on stationary membrane conductance and voltage- and time-dependent ionic currents in identified snail neurons, Brain Res., 268 (1983) 321-338. 6 Ku, B.S., Takeuchi, H., Yanaihara, N., Munekata, E. and Ariyoshi, Y., Effects of synthetic peptides on giant neurone identified in the ganglia of an African giant snail (Achatinafulica Ferussac), Comp. Biochem. Physiol., 84C (1986) 331 396. 7 Levitan, I.B., Bergstroem, E. and Simonet, M., Adenylate cyclase in Helix and Aplysia ganglia: characteristics of its stimulation by a peptide-containing nervous system, J. Neurochem., 31 (1978) 1353-1359. 8 Lozinskaya, I.M. and Shuba, Y.M., Low- and high-threshold calcium current in the membrane of cultured neurons of Helix pomatia, Neurophysiologia, 21 (1989) 127 129.
9 Mironov, S.L., Tepikin, A.V. and Grishchenko, A.V., Two calcium currents in the somatic membrane of the mollusc neurons, Neurophysiologia, 17 (1985) 627-633. 10 Nowycky, M.C., Fox, A.P. and Tsien, R.W., Three types of neuronal calcium channel with different calcium agonist selectivity, Nature, 316 (1985) 440-443. l I Osipenko, O.N., Depolarization and hyperpolarization of the neuronal membrane in the snail Helix pomatia identified neurons induced by oxytocin application to the soma of these neurons, Comp. Biochem. Physiol., 94C (1989) 655-661. 12 Osipenko, O.N., Modulatory action of oxytocin on electrical responses of the neurons in the snail Helix pomatia, Comp. Biochem. Physiol., 95C (1990) 9-14. 13 Sakharov, D.A. and Salanki, J., Physiological and pharmacological identification of neurons in the central nervous system of Helix pomatia. L., Acta Physiol. Acad. Sci. Hung., 35 (1969) 19-30. 14 Sawyer, W.H., Deyrup-Olsen, I. and Martin, A.W., Immunological and biological characteristics of the vasotocin-like activity in the head ganglia of gastropoda molluscs, Gen. Comp. Endocrinol., 54 (1984) 97-108. 15 Strambi, C., Rougon-Rapuzzi, G., Cupo, A., Martin, N. and Strambi, A., Mise en 6vidence immunocytologique d'un compose apparente ~. la vasopressine dans le syst~me nerveux du grillon Acheta domesticus, C.R. Acad. Sci. Paris, 288D (1979) 131-133. 16 Takeda, N., Takayanagi, H., Hasegawa, K., Miczuno, J. and Fujii, K., Neuropeptides and their actions on neurons in the central nervous system of the snail, Aehatinafulica. In, Abstracts of the Symposium on Molluscan Neurobiology, Amsterdam, The Netherlands, 1986, pp. 45.