- Email: [email protected]
Mechanism of long lasting synaptic inhibition of a silent neuron of Helix pomatia
Camp. Biochem. Phvsiol. Vol. Printed in Great Biitain
79A, No.
3, pp. 425-430,
1984 0
0300-9629184 $3.00 + 0.00 1984 Pergamon Press Ltd
MECHANISM OF LONG LASTING SYNAPTIC INHIBITION OF A SILENT NEURON OF HELIX POA4AZl4 CARSTEN JUEL
Zoophysiolo~cal Laboratorium B, August Krogh Institutet, 13, Universitetsparken, DK-2100 Copenhagen 0, Denmark. Telephone: 01-37-7000 (Received 20 February 1984) Abstract-l. Long lasting synaptic inhibitions (LLI) were recorded in the silent cells LPLI and RPLl of Helix pomatia. 2. During LLI the excitability of the tell was strongly reduced. 3. The membrane conductance changes during LLI were characterized using the voltage-clamp technique. 4. LLI in the silent cell LPLI is mediated bv, a svnaptically induced inhibition of the voltage-dependent , inward Ca*+ -current.
INTRODUCTION
METHODS
Long lasting hyperpolarizations, LLH (or long lasting inhibitions, LLI) are synaptically mediated responses recorded in certain molluscan neurons. They are characterized by their long duration and the requirement of several stimulations of the presynaptic cell. In most cells LLH is preceded by a short K-dependent h~e~ola~~tion (ILD). Several cells showing LLH have been described: cell R15 of Aplysiu (Parnas et a/., 1974), L,-L, in Aplysia (Wilson and Wachtel, 1978), F, in Helix aspersa (Lambert, 1975) and the homologous cell RPAI in Helix pomatia (Christoffersen and Simonsen, 1983). These cells are all bursting neurons showing periodic trains of action potentials. Several mechanisms underlying LLH have been proposed: a selective increase in K +-conductance (Parnas and Strumwasser, 1975; Lambert, 1975; Christoffersen and Simonsen, 1983), activation of the electrogenic Na-K pump (Pinsker and Kandel, 1969), reduction of the regenerative current underlying the slow oscillations of membrane potentials in bursting cells (Wilson and Wachtel, 1978) or a decrease in voltage-dependent inward current (Adams et al., 1980). LLH has been shown to be dependent on the level of intracellular CAMP (Drake and Treistman, 1981). and LLH can be mimicked by a guanylylimidodiphosphate (GMP-PNP) induced increase in intracellular CAMP (Treistman and Levitan, 1976). It has, however, in some of these experiments been difficult to separate CAMP-dependent effects on the burst firing mechanism and the mechanism underlying LLH. Recently an increase in intracellular Ca*+ activity during LLH has been reported (Christoffersen and Simonsen, 1983). The present work shows that LLI can also be evoked in the silent cells LPLI and RPLI in Helix pomutiu. It will be shown that LLI in these cells is due to a decrease in the inward current carried by Ca* + , causing a strong reduction in the excitability of the cell.
The suboesophageal ganglia of Helix pomatia were isolated and pinned in a chamber containing Ringer’s solution. The right pallial nerve was stimulated (duration 0.1 msec) and synaptic potentials were recorded in the silent cells LPLl and RPLl (named by Sakharov and Salanki, 1969). Voltage-clamp experiments were carried out using double-barrelled microelectrodes (theta alass) filled with ’ 2.5 M K-acetate, resistance S-10 Mohm. Current-voltage (I-V) curves were obtained by sweeping the voltage from - 100 mV to - 20 mV (IO mV/sec). This procedure ensures a good time resolution because sweeping time is short relative to the duration of LLI. Intracellular injection of test substances (CAMP. GMPPNP or Ca’+) was done by pressure injection’using a second double-barrelled microelectrode. A syringe was connected to the barrel containing the test substance by means of a plastic tubing sealed with epoxy glue. In the other barrel, filled with K-acetate, was placed an Ag/AgCl electrode for recording of membrane potentials. The Ringer’s solution had the following composition (mM): NaCl, 60; KCl, 3; CaCl,, 7; MgCl,, 13; t&-Cl buffer, 5. pH 7.8.
RESULTS
The synaptic potentials
Single stimulation of the right pallial nerve leads to a two-phased synaptic potential in cell LPLl. The first phase (E-phase) is excitatory having a reversal potential of -4OmV, while the second phase is inhibitory having a reversal potential of -80 to -70 mV (Fig. 1A). The first phase is Cl--dependent, the second phase K+-dependent (Juel, 1982). The pharmacology of these dopaminergic responses has previously been described (Juel, 1983). The inhibitory phase, lasting 5-10 set, is usually called ILD (inhibition of long duration). Repetitive stimulation as a rule leads to an additional hyperpolarizing phase lasting several minutes (Fig. IC). Similar long lasting inhibitions can be evoked in the symmetrically placed cell, RPLI. This last phase corresponds to the long lasting hyperpoiarizations (LLH) which can be recorded in bursting Aplysia and Helix neurons (Parnas et al., 1974;
425
426
A.
CARSTEN JUEL
-35
mVV_
-60mV--G_1°mv~ 10 set
-100
Voltage dependency of ILD and LLI
mV -IL
Cell LPLl was voltage-clamped to different membrane potential levels. ILD was evoked by a single stimulation while LLI was evoked by ten stimulations (1 Hz). ILD was decreased by hyperpolarization of the cell, and clearly reversed sign in strongly hyperpolarized neurons (Fig. 3A). The reverse potential was always found in the interval -65 to -80 mV, and the amplitude versus potential curve was rectilinear (Fig. 3C). The amplitude of LLI was measured 60 set after the stimulation. LLI decreased by hyperpolarization and was never observed for potentials more negative than - 80 mV. LLI never reversed sign (Fig. 3B, C).
B.e C.
potential changed gradually from -58 to -65 mV and returned within 20min. The excitability reduction during LLI far exceeded what would be expected from the increase in total membrane conductance. The time constant for inactivation of LLI in LPLl was 4-5 min, which seems to be characteristic for Helix neurons showing LLI (Christoffersen and Simonsen, 1983).
10 mV
L 3 min
Fig. 1. Synaptic potentials recorded in cell LPLl. (A) A single stimulation of the pallial nerve leads to a two-phased synaptic potential (the E- and ILD-phases). The figure shows the synaptic potentials at three different levels of the membrane potential which was changed by current injection (B) The effect of twenty stimulations (1 Hz). The Eand ILD-phases are summed. The figure demonstrates the delayed onset of the long lasting inhibition, LLI. Note different time scales. (C) LLI evoked by ten stimulations. Resting membrane potential - 54 mV.
Lambert, 1975). LLH is also named LLI, long-lasting inhibition (Adams et al., 1980). LLI in cell LPLl was preceeded by a potential resulting from a summation of the E- and ILD-phases (Fig. 1B). Occasionally LLI can be evoked by a single stimulation, but ten stimulations are always needed to obtain the maximal amplitude and duration.
Current-voltage characteristics of LLZ The changes in membrane current during LLI were analysed using the following procedure: The dynamic current-voltage (I-V) characteristic for the resting LPLl neuron was obtained by sweeping the voltage from - 100 to -20 mV (10 mV/sec). The pallial nerve was then stimulated ten times (1 Hz), and a second I-V curve recorded. A third I-V curve was recorded 60 set after the stimulation (60 set being the time needed for LLI to reach maximal amplitude). One example of a set of three I-V curves is shown in Fig. 4A. Data from eight sets of I-V curves (from four different LPLl cells) were pooled, and the slope conductance (G,) curves constructed (Fig. 4B).
mV
5L
I 2L
; I
-LO.
Change in excitability during LLZ Cell excitability can be characterized by two parameters: The excitation threshold (in mV) and the current (in nA) needed to reach the excitation threshold. The excitability of cell LPLl was investigated using double-barrelled electrodes, one barrel being used for passing 1 set square current-pulses and the other for measuring the resulting membrane potential. At rest the current needed to excite cell LPLl was 0.5-1.0 nA (depending on resting membrane potential (RMP), and total membrane resistance). The excitation threshold was - 50 to -46 mV and seems to be independent of RMP (- 55 to - 70 mV). Figure 2 shows the changes in excitability during LLI evoked by ten stimulations (1 Hz). In the experiment illustrated the excitation threshold rose from -48 to -29 mV within the first minute following stimulation. The excitation current increased from 0.5 to 5.4nA in the same period. The membrane
-30#
Oh
1L
: iL
06
-50
-60-
(
v--
t 0
5
10
15
Fig. 2. The effects of repetitive stimulation (which evokes LLI) on excitability and resting membrane potential. Before stimulation the excitation threshold was -48 mV and RMP - 58 mV. The arrow shows onset of LLI evoked by ten stimulations. The upper curve shows the excitation threshold, the lower curve shows membrane potential. On the upper curve is given the current (in nA) needed to reach the excitation threshold.
Long
A.
lasting
synaptic
-61 -T-91 mV
-7
-71 mV rnvL
1 nA
inhibition
L
in Helix neuron
427
can be seen in Figs 1B and 3B) or an increase in K+ permeability (reversal potential - 75 mV) participating in the very first phase of LLI.
20 set
=v-----
V
-E+ILD
Analysis
of inward current during voltage-clamp
The preceding experiments showed a remarkable decrease in slope conductance during LLI. This may reflect a decrease in potential-dependent inward current carried by Na + or Ca* + . The following experiments were designed to characterize the ionic nature of the voltage-dependent currents during voltageclamp of the resting neurone. The effect on slope conductance of Na substitution with Tris (eliminating inward Na-current) or inhibition of Ca2 +-current with Co and Cd (Meech and Standen, 1975; Kostyuk and Krishtal, 1977) is shown in Fig. 5. The experiments show that Na+ is responsible for less than 20% of the voltage-dependent inward current, whereas Ca2+ is charge carrier for the main part of the voltage-dependent inward current. This Ca2+-current is strongly inhibited during LLI. Effect of CAMP Phosphodiesterase inhibitors and derivatives of CAMP have been reported to augment LLI in bursting cells (Treistman and Levitan, 1976; Levitan and
Fig. 3. Voltage dependency of ILD and LLI. The cell is voltage-clamped to different levels of the membrane potential. Upward deflections means hyperpolarizing membrane current. (A) Voltage dependency of ILD evoked by a single stimulation. (B) Voltage dependency of LLI evoked by ten stimulation (I Hz). Note the summation of the E- and ILD-phases, and the slow onset of LLI. (C) The voltage dependency of ILD (single stimulation) and LLI (amplitude 60 set after ten stimulations).
A.
I
0.5 nA
The I-V and G,-V curves for the unstimulated cell are characterized by a reduced positive slopeconductance in the interval - 60 to - 50 mV, and a gradual increase in G, for potentials more positive than - 50 mV (voltage-dependent inward current). Immediately following stimulation (second I-V curve) the G,-V curve showed a high degree of rectilinearity. When compared to the first curve three changes can be seen. In the voltage range more negative than - 70 mV G, is increased by 30%, the characteristic reduction in positive slope-G, between - 60 and - 50 mV has disappeared, and G, is decreased for potentials more positive than -40 mV. The I-V curves showed a reversal potential at - 75 mV. One minute after stimulation (maximal LLI) the I-V and G,,-V curves are still nearly rectilinear, but no G, change can be seen in the hyperpolarized interval, whereas the region showing reduced positive slope-G, is still lacking. G, is still decreased in the depolarized region. No reversal potential is seen between - 100 and -20 mV. In can be concluded that LLI is due to a reduced G,,, for potentials more positive than -40 mV, and a G, increase (lack of reduction in positive slope-G,) in the potential range near RMP. Immediately following stimulation a conductance increase in the hyperpolarized region is also seen. This may reflect either a summation of E-ILD (which
-90
-60
-30
Fig. 4. Current-voltage (I-V) relationship and membrane slope conductance during LLI. (A) I-V curves obtained before stimulation (l), immediately after ten stimulations (2) and 60 set after stimulation (3). The voltage was increased from - 100 to -20 mV (10 mV/sec). (B) Membrane slope conductance (G,) calculated from 8 sets of I-V curves obtained in 4 different LPLl cells. Same procedure as in A.
428
CARSTEN
IG,
0’
(MQ-‘1
P
-20
-60
-90
mV
Fig. 5. Analysis of membrane slope conductance. Cell LPLl. (1) Control values of G,-V (pooled data from 10 runs). (2) The effect of substituting Na by Tris. Lower curve (3): the effect of 1OmM Co added to the Ringer. Similar effects were obtained with 1 mM Cd.
1980). Isobutylmethylxanthine, IBMX Norman, (10 -4 M), or dibuturyl-CAMP, dbcAMP (10 4 M), increased slope conductance of cell LPLl in the hyperpolarized voltage interval (increased Kconductance), but failed to mimic the LLI effect in the depolarized interval. Guanylylimidodiphosphate (GMP-PNP), which activates adenylate cyclase, has been shown to mimic LLI when injected into cell R15 of Aplysiu (Treistman and Levitan, 1976). Injection of GMP-PNP into LPLl was carried out during voltage clamp. Figure 6 shows changes in slope conductance induced by GMP-PNP. Before the experiment RMP was - 59 mV and the current leading excitation 0.6 nA. Fifteen minutes after injection the membrane potential had fallen to -76 mV and the excitation current was 8.0 nA. The effect of GMP-PNP on slope conductance (Fig. 6) showed great similarities to the LLI-effect (Fig. 4B, curve 2 and 3). Injection
JUEL
resolution. Cell LPLl was impaled with one doublebarrelled electrode for voltage-clamp and with a second double-barrelled electrode for pressure injection of CAMP (0.5 M in distilled water) and recording of membrane potential. Short pulses (l-2 set) of CAMP evoked an inward current at RMP. When the neuron was depolarized to -40 mV and pulses of 10 set duration were employed, a second hyperpolarizing phase could be distinguished (Fig. 7A). The first phase reversed at -20 mV and the second phase at - 60 to - 70 mV. Similar results have been reported by Kononenko and Mironov (1981) also using cell LPLl . The current-voltage curve obtained 60sec after the CAMP injection (during the last phase) showed a small conductance increase (possibly K-mediated) at hyperpolarized potentials, but no significant effect at depolarized levels. Ca2 + injection The following experiments were designed to determine whether the conductance changes underlying LLI could be triggered by a Ca2 +-injection. One barrel of the pressure injection electrode was filled with 0.1 M CaCl, and a second double-barrelled electrode was used for voltage-clamp. The following experimental schedule was employed. The cell soma was impaled with the double-barrelled electrode for voltage-clamp. When stability was obtained the first I-V sweep was carried out. The cell was then penetrated with the CaCl,-electrode. When the clamp current was constant (with the cell clamped to initial membrane potential), the second I-V sweep was carried out. A comparison of the first and second I-V curves allowed a characterization of effects induced by a leak of Ca2+ from the electrode. A 20 set pressure injection of Ca2+ was done, and the third I-V curve obtained (Fig. 8A). Ca2+ injection caused a fast inward current followed by a slow outward current. Figure 8B shows
A.
-LOmV
H
of CAMP -9OmV
Since phosphodiesterase inhibitors are unspecific (increasing both CAMP and cGMP) and the effect of
H
--Try-
GMP-PNP is slow, injections of CAMP were carried out to obtain more specific effects and a better time
0.25 nA
L
20 set
G, ( M R-’ )
B.
0.5
1
/
Control
&GMp-pNp -9.0
-60
-9OmV
30 mV
Fig. 6. The effect of guanylyl-imidodiphosphate (GMPPNP) injection on membrane slope conductance. The GMPPNP curve was obtained 10 min after the injection. The G, value was calculated for each 10mV interval.
-
Fig. 7. CAMP injection into LPLI cell soma. The pressure injection of CAMP was carried out using double-barrelled electrodes. One barrel was filled with 0.5 M CAMP. A second double-barrelled electrode was used for voltageclamp. Upward deflection means hyperpolarizing membrane current. (A) The effect of 5 set injections at different levels of the membrane potential. (B) 20 set injections. The bar shows time of pressure injection.
Long lasting synaptic inhibition in Helix neuron
A.
0.5 nA
ii
i
i
L
20 set
--f--f/-
B.
The voltage dependent inward currents seen, when cell LPLl is depolarized during voltage-clamp, were shown to be carried mainly by Ca*+ (Fig. 5). The conductance decrease seen during LLI therefore reflects a reduction of voltage-dependent inward Ca2+-current. This conclusion is illustrated by the great similarity between the effects of LLI and “Ca* + -blockers” (Co and Cd) on the current-voltage relation of cell LPLl. This model explains why LLI can be reduced in size but not inverted, when the neuron is hyperpolarized by current injection. No effect of stimulation will be seen if the neuron is hyperpolarized to the voltage where no potential-dependent inward current is present. The mechanism behind LLI reported here is an example of a synaptic inhibition of ionic channels involved in excitability processes, whereas most other synaptic effects involve separate synaptic channels. The involvement
0
J -90,
-60
-30 mV
Fig. 8. Effect of CaC1, injection. (A) Clamp current. Upward deflection means hyperpolarizing membrane current. The control I-V value (1) was obtained with only the voltage-clamp electrode in the cell. At (*) the Ca* + electrode was inserted. (2) shows the time of the second I-V run. The bar shows the &st CaCl, injection. The third I-V run (3) was carried out 60 set after the Ca*+ injection. (B) Membrane slope conductance. (1) Control. (2) Effect of Ca*+ leak from the electrode. (3) Effect of Ca2+ injection.
The the Ca* + mediated effects on slope conductance. Ca*+ effect, obtained 60 set after the injection, was clearly a slope conductance decrease at depolarized potentials, whereas no conductance changes were seen for potentials more negative than - 80 mV. It is concluded that a Ca* + -injection mimics the conductance decrease seen during maximal development of LLI in cell LPLl. The transmitter for LLI Dopamine (10m4 M), but not serotonin when added before stimulation, attenuated ILD and LLI with more than SO’%.This shows that dopamine receptors are involved in LLI. It has previously been reported that ILD recorded in LPLl is dopaminergic (Juel, 1983).
DISCUSSION
The conductance
changes underlying LLZ
The current-voltage experiments demonstrate that LLI evoked in cell LPLl is mediated by a characteristic change of total cell conductance. In the very first phase of LLI an increase in outward current (Kcurrent) is seen. This current seems to reflect a summation of the ILD responses. During the main part of LLI the only effect is a decrease of inward current.
429
of Ca2+ and CAMP
The synaptically mediated decrease in the voltageunderlying LLI Ca*+ -current dependent inward seems to involve the whole soma-membrane implying an intracellular second messenger spreading from the synaptic region to the entire cell soma. It has been proposed that CAMP functions as a messenger during LLI. This was supported by the finding that phosphodiesterase inhibitors and CAMP derivatives modulates LLI and bursting activity (Levitan, 1978; Treistaman and Levitan, 1976; Drummond et al., 1980; Levitan and Norman, 1980; Drake and Treistman, 1980, 1981). Many of these effects seem, however, to be manipulations of the burst firing mechanism (increase in duration and depth of the interburst intervals), rather than activation of a distinct long lasting hyperpolarization. In bursting cells, of course, LLI may be identical to an enlarged interburst period (Wilson and Wachtel, 1978). Injection of GMP-PNP, which selectively increase the adenylate cyclase activity up to 700x, is reported to mimic LLI in the bursting neuron Rl5 in Aplysia (Triestman and Levitan, 1976). Injection of CAMP into molluscan neurons has shown varying responses dependent on the cell used. In none of these experiments a clear effect comparable to LLI was obtained. In the silent cell LPLl injection of CAMP caused a fast inward current followed by a weak outward current (Kononenko and Mironov, 1981, and Fig. 7A, B in the present report). The second phase seems to be K-dependent and is therefore distinct from LLI. Injection of GMP-PNP, on the other hand, was capable of mediating a conductance decrease similar to LLI. It has recently been shown that the intracellular concentration of free Ca2+ increases during LLI. Typically a doubling of the intracellular Ca* + activity was seen during LLI (Christoffersen and Simonsen, 1983). It is generally believed that internal CAMP increases intracellular free Ca*+ (Berridge, 1975) although injection of CAMP into molluscan neurons failed to show a direct effect of CAMP on Ca* + level (Hockberger and Conner, 1983).
430
CARSTEN JUEL
The results presented here show that a small increase in CAMP content (induced by IBMX and dbcAMP) mimicked the first phase of LLI, and that a strong increase in CAMP level (induced by GMPPNP) could give rise to the main phase as well. Injection of Ca* + , on the other hand, mimicked the last phase characterized by a decrease in voltagedependent inward Ca* + -current, whereas an increase in K-current was absent. It is concluded, therefore, that the main phase of LLI is dependent on an increase in intracellular level of Ca2 + . The conductance decrease could conceivably be obtained by a Ca* + -mediated inhibition of the voltage-dependent Ca*+ -influx; such a mechanism has been described for molluscan neurons (Kostyuk and Kristal, 1977; Standen, 1981). In bursting cells Ca2+ -injection activates a K-current (Hofmeier and Lux, 1981). This effect may be regarded as an activation of the membrane oscillation mechanism underlying burst firing (Eckert and Lux, 1976). A Ca2 + -activated K-current seems to be weak or absent in the silent cell LPLl at RMP. It is characteristic for LLI that repeated stimulations are needed to obtain the full amplitude. In parallel, a large increase in CAMP content is needed to trigger LLI. This can be explained by the existence of a barrier between ILD and LLI. Exceeding this barrier (accumulation of CAMP) could trigger a regenerative process leading to intracellular Ca2 + liberation, which would inhibit voltage-dependent inward Ca* + -current. The excitability
reduction
The LLI mechanism involving an inhibition of inward voltage-dependent Ca2 + -current gives an explanation to the dramatically decreased excitability during LLI (Fig. 2). Acknowledgement-This work was supported Natural Science Research Council.
by the Danish
REFERENCES
Adams W. B., Parnas I. and Levitan I. B. (1980) Mechanism of long lasting synaptic inhibition in Ap[ysia neuron R15. J. Neurophysiol. 44, 1148-l 160. Berridge M. J. (1975) The interaction of cyclic nucleotides and calcium in the control of cellular activity. In Advances in Cyclic Nucleotide Research (Edited by Greengaard R. and Robinson G. A.), Vol. 6, pp. 1-98. Raven Press, New York. Christoffersen G. R. J. and Simonsen L. (1983) Intracellular Ca activity during slow synaptic hyperpolarizations in Helix porn&a. Comp. Biochem Physiol. 76C, 351-364. Drake P. F. and Treistman S. N. (1980) Alteration of neuronal activity in response to cyclic nucleotide agents in Aplysia neurons. J. Neurobiol. 11, 476482. Drake P. F. and Treistman S. N. (1981) Mechanisms of action of cyclic nucleotides on a bursting pacemaker and a silent neuron in Aplysia. Brain Res. 218, 243-254.
Drummond A. H., Benzon J. A. and Levitan I. B. (1980) Serotonin-induced hyperpolarization of an identified Aplysia neuron is mediated by cyclic AMP. Proc. natn Acad. Sci. USA 77, 5013-5017. Eckert R. and Lux H. D. (1976) A voltage-sensitive persistent calcium conductance in neuronal somata of Helix. J. Physiol. 254, 129- 15 1. Hockberger P. and Conner J. A. (1983) Intracellular calcium measurements with arsenazo III during cyclic AMP iniections into molluscan neurons. Science 2i9, 869-871._ Hofmeier G. and Lux H. D. (1981) The time courses of intracellular free calcium and related electrical effects after injection of CaCl, into neurons of the snail, Helix pomatia. Pfliigers Arch. ges. Physiol. 391, 242-251. Juel C. (1982) Evidence for the existence of dopaminergic autoreceptors in Helix pomatia. Camp. Biochem Physiol. 73C, 157-160. Juel C. (1983) Pre- and postsynaptic effects of dopamine antagonists on dopaminergic synaptic transmission in Helix pomatia. Camp. Biochem. Physiol. 76C, 203-208. Kononenko N. I. and Mironov S. L. (1981) Effect of intracellular injection of cyclic AMP on electrical characteristics of identified neurons in Helix pomatia. Neurophysiology 12, 332-339. Kostyuk P. G. and Krishtal 0. A. (1977) Separation of sodium and calcium currents in the somatic membrane of molluscan neurones. J. Physiol. 270, 545-568. Lambert J. D. C. (1975) A long lasting hyperpolarization evoked in an identified neurone of Helix porn&a. Brain Res. 87, 118-122. Levitan I. B. (1978) Modulation of neuronal activity by peptides and neurotransmitters: possible role of cyclic nucleotides. J. Physiol. (Paris) 74, 521-525. Levitan I. B. and Norman J. (1980) Different effects of CAMP and cGMP derivatives on the activity of an identified neuron: biochemical and electrophysiological analysis. Brain Res. 187, 415429. Meech R. W. and Standen N. B. (1975) Potassium activation in Helix aspersa neurones under voltage clamp: a component mediated by calcium influx. J. Physiol. 249, 21 l-239. Parnas I., Armstrong D. and Strumwasser F. (1974) Prolonged excitatory and inhibitory synaptic modulation of a bursting pacemaker neuron. J. Neurophysiol. 37, 594608. Parnas I. and Strumwasser F. (1974) Mechanisms of longlasting inhibition of a bursting pacemaker neuron. J. Neurophysiol. 37, 609-620. Pinsker H. and Kandel E. R. (1969) Synaptic activation of an electrogenic sodium pump. Science 163, 931-933. Sakharov D. A. and Salanki J. (1969) Physiological and pharmacololgical identification of neurones in the central nervous system of Helix pornaria. Acta Physiol. Hung. 35, 19-30. Standen N. B. (1981) Ca channel inactivation by intracellular Ca injection into Helix neurones. Nature, Lond. 293, 158-159. Treistman S. N. and Levitan I. B. (1976) Intraneuronal guanylyl-imidodiphosphate injection mimics long term synaptic hyperpolarization in Aplysia. Proc. natn. Acad. Sci. USA 73, 46894692. Wilson W. A. and Wachtel H. (1978) Prolonged inhibition in burst firing neurons: synaptic inactivation of the slow regenerative inward current. Science 202, 772-775.
79A, No.
3, pp. 425-430,
1984 0
0300-9629184 $3.00 + 0.00 1984 Pergamon Press Ltd
MECHANISM OF LONG LASTING SYNAPTIC INHIBITION OF A SILENT NEURON OF HELIX POA4AZl4 CARSTEN JUEL
Zoophysiolo~cal Laboratorium B, August Krogh Institutet, 13, Universitetsparken, DK-2100 Copenhagen 0, Denmark. Telephone: 01-37-7000 (Received 20 February 1984) Abstract-l. Long lasting synaptic inhibitions (LLI) were recorded in the silent cells LPLI and RPLl of Helix pomatia. 2. During LLI the excitability of the tell was strongly reduced. 3. The membrane conductance changes during LLI were characterized using the voltage-clamp technique. 4. LLI in the silent cell LPLI is mediated bv, a svnaptically induced inhibition of the voltage-dependent , inward Ca*+ -current.
INTRODUCTION
METHODS
Long lasting hyperpolarizations, LLH (or long lasting inhibitions, LLI) are synaptically mediated responses recorded in certain molluscan neurons. They are characterized by their long duration and the requirement of several stimulations of the presynaptic cell. In most cells LLH is preceded by a short K-dependent h~e~ola~~tion (ILD). Several cells showing LLH have been described: cell R15 of Aplysiu (Parnas et a/., 1974), L,-L, in Aplysia (Wilson and Wachtel, 1978), F, in Helix aspersa (Lambert, 1975) and the homologous cell RPAI in Helix pomatia (Christoffersen and Simonsen, 1983). These cells are all bursting neurons showing periodic trains of action potentials. Several mechanisms underlying LLH have been proposed: a selective increase in K +-conductance (Parnas and Strumwasser, 1975; Lambert, 1975; Christoffersen and Simonsen, 1983), activation of the electrogenic Na-K pump (Pinsker and Kandel, 1969), reduction of the regenerative current underlying the slow oscillations of membrane potentials in bursting cells (Wilson and Wachtel, 1978) or a decrease in voltage-dependent inward current (Adams et al., 1980). LLH has been shown to be dependent on the level of intracellular CAMP (Drake and Treistman, 1981). and LLH can be mimicked by a guanylylimidodiphosphate (GMP-PNP) induced increase in intracellular CAMP (Treistman and Levitan, 1976). It has, however, in some of these experiments been difficult to separate CAMP-dependent effects on the burst firing mechanism and the mechanism underlying LLH. Recently an increase in intracellular Ca*+ activity during LLH has been reported (Christoffersen and Simonsen, 1983). The present work shows that LLI can also be evoked in the silent cells LPLI and RPLI in Helix pomutiu. It will be shown that LLI in these cells is due to a decrease in the inward current carried by Ca* + , causing a strong reduction in the excitability of the cell.
The suboesophageal ganglia of Helix pomatia were isolated and pinned in a chamber containing Ringer’s solution. The right pallial nerve was stimulated (duration 0.1 msec) and synaptic potentials were recorded in the silent cells LPLl and RPLl (named by Sakharov and Salanki, 1969). Voltage-clamp experiments were carried out using double-barrelled microelectrodes (theta alass) filled with ’ 2.5 M K-acetate, resistance S-10 Mohm. Current-voltage (I-V) curves were obtained by sweeping the voltage from - 100 mV to - 20 mV (IO mV/sec). This procedure ensures a good time resolution because sweeping time is short relative to the duration of LLI. Intracellular injection of test substances (CAMP. GMPPNP or Ca’+) was done by pressure injection’using a second double-barrelled microelectrode. A syringe was connected to the barrel containing the test substance by means of a plastic tubing sealed with epoxy glue. In the other barrel, filled with K-acetate, was placed an Ag/AgCl electrode for recording of membrane potentials. The Ringer’s solution had the following composition (mM): NaCl, 60; KCl, 3; CaCl,, 7; MgCl,, 13; t&-Cl buffer, 5. pH 7.8.
RESULTS
The synaptic potentials
Single stimulation of the right pallial nerve leads to a two-phased synaptic potential in cell LPLl. The first phase (E-phase) is excitatory having a reversal potential of -4OmV, while the second phase is inhibitory having a reversal potential of -80 to -70 mV (Fig. 1A). The first phase is Cl--dependent, the second phase K+-dependent (Juel, 1982). The pharmacology of these dopaminergic responses has previously been described (Juel, 1983). The inhibitory phase, lasting 5-10 set, is usually called ILD (inhibition of long duration). Repetitive stimulation as a rule leads to an additional hyperpolarizing phase lasting several minutes (Fig. IC). Similar long lasting inhibitions can be evoked in the symmetrically placed cell, RPLI. This last phase corresponds to the long lasting hyperpoiarizations (LLH) which can be recorded in bursting Aplysia and Helix neurons (Parnas et al., 1974;
425
426
A.
CARSTEN JUEL
-35
mVV_
-60mV--G_1°mv~ 10 set
-100
Voltage dependency of ILD and LLI
mV -IL
Cell LPLl was voltage-clamped to different membrane potential levels. ILD was evoked by a single stimulation while LLI was evoked by ten stimulations (1 Hz). ILD was decreased by hyperpolarization of the cell, and clearly reversed sign in strongly hyperpolarized neurons (Fig. 3A). The reverse potential was always found in the interval -65 to -80 mV, and the amplitude versus potential curve was rectilinear (Fig. 3C). The amplitude of LLI was measured 60 set after the stimulation. LLI decreased by hyperpolarization and was never observed for potentials more negative than - 80 mV. LLI never reversed sign (Fig. 3B, C).
B.e C.
potential changed gradually from -58 to -65 mV and returned within 20min. The excitability reduction during LLI far exceeded what would be expected from the increase in total membrane conductance. The time constant for inactivation of LLI in LPLl was 4-5 min, which seems to be characteristic for Helix neurons showing LLI (Christoffersen and Simonsen, 1983).
10 mV
L 3 min
Fig. 1. Synaptic potentials recorded in cell LPLl. (A) A single stimulation of the pallial nerve leads to a two-phased synaptic potential (the E- and ILD-phases). The figure shows the synaptic potentials at three different levels of the membrane potential which was changed by current injection (B) The effect of twenty stimulations (1 Hz). The Eand ILD-phases are summed. The figure demonstrates the delayed onset of the long lasting inhibition, LLI. Note different time scales. (C) LLI evoked by ten stimulations. Resting membrane potential - 54 mV.
Lambert, 1975). LLH is also named LLI, long-lasting inhibition (Adams et al., 1980). LLI in cell LPLl was preceeded by a potential resulting from a summation of the E- and ILD-phases (Fig. 1B). Occasionally LLI can be evoked by a single stimulation, but ten stimulations are always needed to obtain the maximal amplitude and duration.
Current-voltage characteristics of LLZ The changes in membrane current during LLI were analysed using the following procedure: The dynamic current-voltage (I-V) characteristic for the resting LPLl neuron was obtained by sweeping the voltage from - 100 to -20 mV (10 mV/sec). The pallial nerve was then stimulated ten times (1 Hz), and a second I-V curve recorded. A third I-V curve was recorded 60 set after the stimulation (60 set being the time needed for LLI to reach maximal amplitude). One example of a set of three I-V curves is shown in Fig. 4A. Data from eight sets of I-V curves (from four different LPLl cells) were pooled, and the slope conductance (G,) curves constructed (Fig. 4B).
mV
5L
I 2L
; I
-LO.
Change in excitability during LLZ Cell excitability can be characterized by two parameters: The excitation threshold (in mV) and the current (in nA) needed to reach the excitation threshold. The excitability of cell LPLl was investigated using double-barrelled electrodes, one barrel being used for passing 1 set square current-pulses and the other for measuring the resulting membrane potential. At rest the current needed to excite cell LPLl was 0.5-1.0 nA (depending on resting membrane potential (RMP), and total membrane resistance). The excitation threshold was - 50 to -46 mV and seems to be independent of RMP (- 55 to - 70 mV). Figure 2 shows the changes in excitability during LLI evoked by ten stimulations (1 Hz). In the experiment illustrated the excitation threshold rose from -48 to -29 mV within the first minute following stimulation. The excitation current increased from 0.5 to 5.4nA in the same period. The membrane
-30#
Oh
1L
: iL
06
-50
-60-
(
v--
t 0
5
10
15
Fig. 2. The effects of repetitive stimulation (which evokes LLI) on excitability and resting membrane potential. Before stimulation the excitation threshold was -48 mV and RMP - 58 mV. The arrow shows onset of LLI evoked by ten stimulations. The upper curve shows the excitation threshold, the lower curve shows membrane potential. On the upper curve is given the current (in nA) needed to reach the excitation threshold.
Long
A.
lasting
synaptic
-61 -T-91 mV
-7
-71 mV rnvL
1 nA
inhibition
L
in Helix neuron
427
can be seen in Figs 1B and 3B) or an increase in K+ permeability (reversal potential - 75 mV) participating in the very first phase of LLI.
20 set
=v-----
V
-E+ILD
Analysis
of inward current during voltage-clamp
The preceding experiments showed a remarkable decrease in slope conductance during LLI. This may reflect a decrease in potential-dependent inward current carried by Na + or Ca* + . The following experiments were designed to characterize the ionic nature of the voltage-dependent currents during voltageclamp of the resting neurone. The effect on slope conductance of Na substitution with Tris (eliminating inward Na-current) or inhibition of Ca2 +-current with Co and Cd (Meech and Standen, 1975; Kostyuk and Krishtal, 1977) is shown in Fig. 5. The experiments show that Na+ is responsible for less than 20% of the voltage-dependent inward current, whereas Ca2+ is charge carrier for the main part of the voltage-dependent inward current. This Ca2+-current is strongly inhibited during LLI. Effect of CAMP Phosphodiesterase inhibitors and derivatives of CAMP have been reported to augment LLI in bursting cells (Treistman and Levitan, 1976; Levitan and
Fig. 3. Voltage dependency of ILD and LLI. The cell is voltage-clamped to different levels of the membrane potential. Upward deflections means hyperpolarizing membrane current. (A) Voltage dependency of ILD evoked by a single stimulation. (B) Voltage dependency of LLI evoked by ten stimulation (I Hz). Note the summation of the E- and ILD-phases, and the slow onset of LLI. (C) The voltage dependency of ILD (single stimulation) and LLI (amplitude 60 set after ten stimulations).
A.
I
0.5 nA
The I-V and G,-V curves for the unstimulated cell are characterized by a reduced positive slopeconductance in the interval - 60 to - 50 mV, and a gradual increase in G, for potentials more positive than - 50 mV (voltage-dependent inward current). Immediately following stimulation (second I-V curve) the G,-V curve showed a high degree of rectilinearity. When compared to the first curve three changes can be seen. In the voltage range more negative than - 70 mV G, is increased by 30%, the characteristic reduction in positive slope-G, between - 60 and - 50 mV has disappeared, and G, is decreased for potentials more positive than -40 mV. The I-V curves showed a reversal potential at - 75 mV. One minute after stimulation (maximal LLI) the I-V and G,,-V curves are still nearly rectilinear, but no G, change can be seen in the hyperpolarized interval, whereas the region showing reduced positive slope-G, is still lacking. G, is still decreased in the depolarized region. No reversal potential is seen between - 100 and -20 mV. In can be concluded that LLI is due to a reduced G,,, for potentials more positive than -40 mV, and a G, increase (lack of reduction in positive slope-G,) in the potential range near RMP. Immediately following stimulation a conductance increase in the hyperpolarized region is also seen. This may reflect either a summation of E-ILD (which
-90
-60
-30
Fig. 4. Current-voltage (I-V) relationship and membrane slope conductance during LLI. (A) I-V curves obtained before stimulation (l), immediately after ten stimulations (2) and 60 set after stimulation (3). The voltage was increased from - 100 to -20 mV (10 mV/sec). (B) Membrane slope conductance (G,) calculated from 8 sets of I-V curves obtained in 4 different LPLl cells. Same procedure as in A.
428
CARSTEN
IG,
0’
(MQ-‘1
P
-20
-60
-90
mV
Fig. 5. Analysis of membrane slope conductance. Cell LPLl. (1) Control values of G,-V (pooled data from 10 runs). (2) The effect of substituting Na by Tris. Lower curve (3): the effect of 1OmM Co added to the Ringer. Similar effects were obtained with 1 mM Cd.
1980). Isobutylmethylxanthine, IBMX Norman, (10 -4 M), or dibuturyl-CAMP, dbcAMP (10 4 M), increased slope conductance of cell LPLl in the hyperpolarized voltage interval (increased Kconductance), but failed to mimic the LLI effect in the depolarized interval. Guanylylimidodiphosphate (GMP-PNP), which activates adenylate cyclase, has been shown to mimic LLI when injected into cell R15 of Aplysiu (Treistman and Levitan, 1976). Injection of GMP-PNP into LPLl was carried out during voltage clamp. Figure 6 shows changes in slope conductance induced by GMP-PNP. Before the experiment RMP was - 59 mV and the current leading excitation 0.6 nA. Fifteen minutes after injection the membrane potential had fallen to -76 mV and the excitation current was 8.0 nA. The effect of GMP-PNP on slope conductance (Fig. 6) showed great similarities to the LLI-effect (Fig. 4B, curve 2 and 3). Injection
JUEL
resolution. Cell LPLl was impaled with one doublebarrelled electrode for voltage-clamp and with a second double-barrelled electrode for pressure injection of CAMP (0.5 M in distilled water) and recording of membrane potential. Short pulses (l-2 set) of CAMP evoked an inward current at RMP. When the neuron was depolarized to -40 mV and pulses of 10 set duration were employed, a second hyperpolarizing phase could be distinguished (Fig. 7A). The first phase reversed at -20 mV and the second phase at - 60 to - 70 mV. Similar results have been reported by Kononenko and Mironov (1981) also using cell LPLl . The current-voltage curve obtained 60sec after the CAMP injection (during the last phase) showed a small conductance increase (possibly K-mediated) at hyperpolarized potentials, but no significant effect at depolarized levels. Ca2 + injection The following experiments were designed to determine whether the conductance changes underlying LLI could be triggered by a Ca2 +-injection. One barrel of the pressure injection electrode was filled with 0.1 M CaCl, and a second double-barrelled electrode was used for voltage-clamp. The following experimental schedule was employed. The cell soma was impaled with the double-barrelled electrode for voltage-clamp. When stability was obtained the first I-V sweep was carried out. The cell was then penetrated with the CaCl,-electrode. When the clamp current was constant (with the cell clamped to initial membrane potential), the second I-V sweep was carried out. A comparison of the first and second I-V curves allowed a characterization of effects induced by a leak of Ca2+ from the electrode. A 20 set pressure injection of Ca2+ was done, and the third I-V curve obtained (Fig. 8A). Ca2+ injection caused a fast inward current followed by a slow outward current. Figure 8B shows
A.
-LOmV
H
of CAMP -9OmV
Since phosphodiesterase inhibitors are unspecific (increasing both CAMP and cGMP) and the effect of
H
--Try-
GMP-PNP is slow, injections of CAMP were carried out to obtain more specific effects and a better time
0.25 nA
L
20 set
G, ( M R-’ )
B.
0.5
1
/
Control
&GMp-pNp -9.0
-60
-9OmV
30 mV
Fig. 6. The effect of guanylyl-imidodiphosphate (GMPPNP) injection on membrane slope conductance. The GMPPNP curve was obtained 10 min after the injection. The G, value was calculated for each 10mV interval.
-
Fig. 7. CAMP injection into LPLI cell soma. The pressure injection of CAMP was carried out using double-barrelled electrodes. One barrel was filled with 0.5 M CAMP. A second double-barrelled electrode was used for voltageclamp. Upward deflection means hyperpolarizing membrane current. (A) The effect of 5 set injections at different levels of the membrane potential. (B) 20 set injections. The bar shows time of pressure injection.
Long lasting synaptic inhibition in Helix neuron
A.
0.5 nA
ii
i
i
L
20 set
--f--f/-
B.
The voltage dependent inward currents seen, when cell LPLl is depolarized during voltage-clamp, were shown to be carried mainly by Ca*+ (Fig. 5). The conductance decrease seen during LLI therefore reflects a reduction of voltage-dependent inward Ca2+-current. This conclusion is illustrated by the great similarity between the effects of LLI and “Ca* + -blockers” (Co and Cd) on the current-voltage relation of cell LPLl. This model explains why LLI can be reduced in size but not inverted, when the neuron is hyperpolarized by current injection. No effect of stimulation will be seen if the neuron is hyperpolarized to the voltage where no potential-dependent inward current is present. The mechanism behind LLI reported here is an example of a synaptic inhibition of ionic channels involved in excitability processes, whereas most other synaptic effects involve separate synaptic channels. The involvement
0
J -90,
-60
-30 mV
Fig. 8. Effect of CaC1, injection. (A) Clamp current. Upward deflection means hyperpolarizing membrane current. The control I-V value (1) was obtained with only the voltage-clamp electrode in the cell. At (*) the Ca* + electrode was inserted. (2) shows the time of the second I-V run. The bar shows the &st CaCl, injection. The third I-V run (3) was carried out 60 set after the Ca*+ injection. (B) Membrane slope conductance. (1) Control. (2) Effect of Ca*+ leak from the electrode. (3) Effect of Ca2+ injection.
The the Ca* + mediated effects on slope conductance. Ca*+ effect, obtained 60 set after the injection, was clearly a slope conductance decrease at depolarized potentials, whereas no conductance changes were seen for potentials more negative than - 80 mV. It is concluded that a Ca* + -injection mimics the conductance decrease seen during maximal development of LLI in cell LPLl. The transmitter for LLI Dopamine (10m4 M), but not serotonin when added before stimulation, attenuated ILD and LLI with more than SO’%.This shows that dopamine receptors are involved in LLI. It has previously been reported that ILD recorded in LPLl is dopaminergic (Juel, 1983).
DISCUSSION
The conductance
changes underlying LLZ
The current-voltage experiments demonstrate that LLI evoked in cell LPLl is mediated by a characteristic change of total cell conductance. In the very first phase of LLI an increase in outward current (Kcurrent) is seen. This current seems to reflect a summation of the ILD responses. During the main part of LLI the only effect is a decrease of inward current.
429
of Ca2+ and CAMP
The synaptically mediated decrease in the voltageunderlying LLI Ca*+ -current dependent inward seems to involve the whole soma-membrane implying an intracellular second messenger spreading from the synaptic region to the entire cell soma. It has been proposed that CAMP functions as a messenger during LLI. This was supported by the finding that phosphodiesterase inhibitors and CAMP derivatives modulates LLI and bursting activity (Levitan, 1978; Treistaman and Levitan, 1976; Drummond et al., 1980; Levitan and Norman, 1980; Drake and Treistman, 1980, 1981). Many of these effects seem, however, to be manipulations of the burst firing mechanism (increase in duration and depth of the interburst intervals), rather than activation of a distinct long lasting hyperpolarization. In bursting cells, of course, LLI may be identical to an enlarged interburst period (Wilson and Wachtel, 1978). Injection of GMP-PNP, which selectively increase the adenylate cyclase activity up to 700x, is reported to mimic LLI in the bursting neuron Rl5 in Aplysia (Triestman and Levitan, 1976). Injection of CAMP into molluscan neurons has shown varying responses dependent on the cell used. In none of these experiments a clear effect comparable to LLI was obtained. In the silent cell LPLl injection of CAMP caused a fast inward current followed by a weak outward current (Kononenko and Mironov, 1981, and Fig. 7A, B in the present report). The second phase seems to be K-dependent and is therefore distinct from LLI. Injection of GMP-PNP, on the other hand, was capable of mediating a conductance decrease similar to LLI. It has recently been shown that the intracellular concentration of free Ca2+ increases during LLI. Typically a doubling of the intracellular Ca* + activity was seen during LLI (Christoffersen and Simonsen, 1983). It is generally believed that internal CAMP increases intracellular free Ca*+ (Berridge, 1975) although injection of CAMP into molluscan neurons failed to show a direct effect of CAMP on Ca* + level (Hockberger and Conner, 1983).
430
CARSTEN JUEL
The results presented here show that a small increase in CAMP content (induced by IBMX and dbcAMP) mimicked the first phase of LLI, and that a strong increase in CAMP level (induced by GMPPNP) could give rise to the main phase as well. Injection of Ca* + , on the other hand, mimicked the last phase characterized by a decrease in voltagedependent inward Ca* + -current, whereas an increase in K-current was absent. It is concluded, therefore, that the main phase of LLI is dependent on an increase in intracellular level of Ca2 + . The conductance decrease could conceivably be obtained by a Ca* + -mediated inhibition of the voltage-dependent Ca*+ -influx; such a mechanism has been described for molluscan neurons (Kostyuk and Kristal, 1977; Standen, 1981). In bursting cells Ca2+ -injection activates a K-current (Hofmeier and Lux, 1981). This effect may be regarded as an activation of the membrane oscillation mechanism underlying burst firing (Eckert and Lux, 1976). A Ca2 + -activated K-current seems to be weak or absent in the silent cell LPLl at RMP. It is characteristic for LLI that repeated stimulations are needed to obtain the full amplitude. In parallel, a large increase in CAMP content is needed to trigger LLI. This can be explained by the existence of a barrier between ILD and LLI. Exceeding this barrier (accumulation of CAMP) could trigger a regenerative process leading to intracellular Ca2 + liberation, which would inhibit voltage-dependent inward Ca* + -current. The excitability
reduction
The LLI mechanism involving an inhibition of inward voltage-dependent Ca2 + -current gives an explanation to the dramatically decreased excitability during LLI (Fig. 2). Acknowledgement-This work was supported Natural Science Research Council.
by the Danish
REFERENCES
Adams W. B., Parnas I. and Levitan I. B. (1980) Mechanism of long lasting synaptic inhibition in Ap[ysia neuron R15. J. Neurophysiol. 44, 1148-l 160. Berridge M. J. (1975) The interaction of cyclic nucleotides and calcium in the control of cellular activity. In Advances in Cyclic Nucleotide Research (Edited by Greengaard R. and Robinson G. A.), Vol. 6, pp. 1-98. Raven Press, New York. Christoffersen G. R. J. and Simonsen L. (1983) Intracellular Ca activity during slow synaptic hyperpolarizations in Helix porn&a. Comp. Biochem Physiol. 76C, 351-364. Drake P. F. and Treistman S. N. (1980) Alteration of neuronal activity in response to cyclic nucleotide agents in Aplysia neurons. J. Neurobiol. 11, 476482. Drake P. F. and Treistman S. N. (1981) Mechanisms of action of cyclic nucleotides on a bursting pacemaker and a silent neuron in Aplysia. Brain Res. 218, 243-254.
Drummond A. H., Benzon J. A. and Levitan I. B. (1980) Serotonin-induced hyperpolarization of an identified Aplysia neuron is mediated by cyclic AMP. Proc. natn Acad. Sci. USA 77, 5013-5017. Eckert R. and Lux H. D. (1976) A voltage-sensitive persistent calcium conductance in neuronal somata of Helix. J. Physiol. 254, 129- 15 1. Hockberger P. and Conner J. A. (1983) Intracellular calcium measurements with arsenazo III during cyclic AMP iniections into molluscan neurons. Science 2i9, 869-871._ Hofmeier G. and Lux H. D. (1981) The time courses of intracellular free calcium and related electrical effects after injection of CaCl, into neurons of the snail, Helix pomatia. Pfliigers Arch. ges. Physiol. 391, 242-251. Juel C. (1982) Evidence for the existence of dopaminergic autoreceptors in Helix pomatia. Camp. Biochem Physiol. 73C, 157-160. Juel C. (1983) Pre- and postsynaptic effects of dopamine antagonists on dopaminergic synaptic transmission in Helix pomatia. Camp. Biochem. Physiol. 76C, 203-208. Kononenko N. I. and Mironov S. L. (1981) Effect of intracellular injection of cyclic AMP on electrical characteristics of identified neurons in Helix pomatia. Neurophysiology 12, 332-339. Kostyuk P. G. and Krishtal 0. A. (1977) Separation of sodium and calcium currents in the somatic membrane of molluscan neurones. J. Physiol. 270, 545-568. Lambert J. D. C. (1975) A long lasting hyperpolarization evoked in an identified neurone of Helix porn&a. Brain Res. 87, 118-122. Levitan I. B. (1978) Modulation of neuronal activity by peptides and neurotransmitters: possible role of cyclic nucleotides. J. Physiol. (Paris) 74, 521-525. Levitan I. B. and Norman J. (1980) Different effects of CAMP and cGMP derivatives on the activity of an identified neuron: biochemical and electrophysiological analysis. Brain Res. 187, 415429. Meech R. W. and Standen N. B. (1975) Potassium activation in Helix aspersa neurones under voltage clamp: a component mediated by calcium influx. J. Physiol. 249, 21 l-239. Parnas I., Armstrong D. and Strumwasser F. (1974) Prolonged excitatory and inhibitory synaptic modulation of a bursting pacemaker neuron. J. Neurophysiol. 37, 594608. Parnas I. and Strumwasser F. (1974) Mechanisms of longlasting inhibition of a bursting pacemaker neuron. J. Neurophysiol. 37, 609-620. Pinsker H. and Kandel E. R. (1969) Synaptic activation of an electrogenic sodium pump. Science 163, 931-933. Sakharov D. A. and Salanki J. (1969) Physiological and pharmacololgical identification of neurones in the central nervous system of Helix pornaria. Acta Physiol. Hung. 35, 19-30. Standen N. B. (1981) Ca channel inactivation by intracellular Ca injection into Helix neurones. Nature, Lond. 293, 158-159. Treistman S. N. and Levitan I. B. (1976) Intraneuronal guanylyl-imidodiphosphate injection mimics long term synaptic hyperpolarization in Aplysia. Proc. natn. Acad. Sci. USA 73, 46894692. Wilson W. A. and Wachtel H. (1978) Prolonged inhibition in burst firing neurons: synaptic inactivation of the slow regenerative inward current. Science 202, 772-775.