Strychnine interactions with acetylcholine, dopamine and serotonin receptors inAplysia neurons

Brain Research, 65 (1974) 109-126 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

109

STRYCHNINE INTERACTIONS WITH ACETYLCHOLINE, DOPAMINE AND SEROTONIN RECEPTORS IN APLYSIA NEURONS

DONALD S. FABER* kND MANFRED R. KLEE Max-Planck-Institut fiir Hirnforschung, Neurobiologische Abteilung, Frankfurt/ M-Niederrad ( G.F.R.) and Neurosensory Laboratory, Department of Physiology, State University of New York at Buffalo, Buffalo, N.Y. (U.S.A.) (Accepted June 10th, 1973)

SUMMARY The effects of strychnine on synaptic transmission in the abdominal ganglion of Aplysia californica and on the responses of individual neurons to iontophoretic application of acetylcholine, dopamine and serotonin were studied using conventional techniques of intraceUular recording. Strychnine inhibited all classical (relatively shortlasting) excitatory and inhibitory postsynaptic potentials as well as the various sodiumand chloride-dependent phoresis responses. Only the potassium-dependent inhibitions of prolonged duration, activated in some cells transsynaptically or by dopamine or acetylcholine application, were not antagonized; sometimes these inhibitions were enhanced. Log-dose-response curves indicated that for each of the 3 drugs the depolarizing responses were more sensitive to strychnine than were the hyperpolarizing responses. Also, a given strychnine concentration generally inhibited the serotonin and dopamine responses to a greater extent than the acetylcholine responses. The antagonism by strychnine of the N a +- and Cl--dependent phoresis responses - - similar to the action of curare on these receptors - - is apparently due to selective interactions with some membrane receptors for the applied drugs. Finally, the limitations to the analysis of the dose-response curves generated by the iontophoretic technique are briefly discussed. It is suggested that the strychnine inhibitions of the acetylcholine and dopamine depolarizing responses might be due to a competitive inhibitory action, whereas in respect to the changes in their doseresponse curves, the chloride-dependent A C h - H response and the 5-HT-D and H responses o f these receptors seem to be blocked by a non-competitive action of the drug.

* Present address: Department of Physiology, Medical Center, University of Cincinnati, Cincinnati, Ohio, U.S.A.

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INTRODUCTION

One aspect of the convulsant properties of strychnine is its specific blockage of glycine-mediated postsynaptic inhibitions in the mammalian spinal c0rd15,16; while others are its effects, possibly at slightly higher concentrations, on neuronal excitability and different higher level inhibitory processes. The latter effects are often referred to as the 'non-specific' actions of strychnine 14,15, a distinction that has proved useful in the identification of glycine as the transmitter at the inhibitory synapses on the spinal motoneurons and for the differentiation between the actions of glycine and GABA16,17. However, such a distinction may be misleading when applied to the convulsant action of strychnine 15,39, especially with reference to higher level centers such as the cerebral cortex which often form the foci for strychnine seizures despite indications that glycine is not a transmitter at these levels and that the IPSPs of these neurons are unchanged by strychnine 2,12,15,33. This action may involve both alterations in excitability such as have been reported for some isolated neuronal preparations ~,~7,4~,59 and effects on synaptic transmission. The second point is related to the reports that, in the cerebral cortex, strychnine blocks the depressant actions of acetylcholine (ACh), serotonin (5-HT) and noradrenaline (this last contrary to the effect in the spinal cord 6) and of stimulation of the lateral hypothalamus and the reticular formation 46, as well as causing the reversal of the antidromically-activated inhibitory postsynaptic potentials (IPSPs) in the cerebral cortex, whereas IPSPs due to thalamic and caudate stimulation are unchanged 34,as,52. The mechanism(s) underlying these strychnine effects are not yet understood. A somewhat similar situation exists in the isolated abdominal ganglion of Aplysia californica where strychnine (0.1-1 mM) induces convulsive-like multiple discharges, and at least part of this action is due to an increase in membrane excitability 36-~8. However, over basically the same concentration range (0.01-1.0 raM) strychnine also blocks most excitatory and inhibitory PSPs in the ganglion and, when at the highest concentration, the antidromic invasion of most neurons 35,3s. Consequently, to determine the mechanism by which strychnine interferes with synaptic transmission in the ganglion and to ascertain whether its site of action is pre- or postsynaptic 42, we investigated its effect on the responses of identified neurons to iontophoretically applied ACh, 5-HT and dopamine (DA). We chose this isolated preparation because of its experimental advantages in comparison with the mammalian CNS and because, as described above, the multiple effects of strychnine are observed in both types of preparation. Brief reports of some of the results have appeared elsewhere 2°,~5,38. MATERIAL AND METHODS

The abdominal ganglion of Aplysia californica (Pacific Bio-Marine Supply Co., Venice, Calif., U.S.A.) was dissected free from the animal and fixed in a chamber continuously perfused with artificial sea water (Instant Ocean, Wycliffe, O hio,U. S.A.) maintained at pH 8.0 and 18 °C. The nerves of the ganglion were each placed on bipolar stimulating electrodes for antidromic and orthodromic stimulation of the

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neurons being investigated. The system adopted for the identification of the neurons was that of Frazier et al. ~1 and utilized the criteria of location, size, response to ACh, nature of spontaneous activity and responses to nerve stimulation. The recordings were intracellular, a cell being penetrated by one or two microelectrodes. With a single electrode, a standard bridge circuit z7 enabled the simultaneous passage of transmembrane currents and membrane potential measurements, whereas with double electrodes one electrode was used for current injection and the other for voltage recordings. Input resistance of the cells was measured by applying ramps or constant trans-membrane current pulses. The electrodes were filled with 0.6 M K~SO4 and had a resistance of 5-15 Mr2. Anodal current pulses of 0.1-2.0 sec duration were used for the iontophoretic ejection of ACh (10 ~), 5-HT (2.5 ~o) or dopamine (10 ~ ) from low resistance microelectrodes positioned on or near the cell surface, and leakage from the pipet tip was prevented by appropriate holding currents. Constant phoresis currents were attained by placing 100 M ~ in series with the phoresis electrode, and phoresis current was measured between the preparation bath and ground by a current-to-voltage converting operational amplifier. When the responses of a neuron to two agents were studied at the same time, either two independent phoresis electrodes or double-barreled 'theta' electrodes were used, with no differences being found between the two techniques. Log-dose-response curves were obtained by varying the phoresis current amplitude and maintaining its duration constant. Strychnine was added as its salt, strychnine sulfate, to the perfusate. The outflow from the chamber was suction-controlled, and a stopcock situated on the inlet side of the perfusing system permitted rapid shifting between the control solution and solutions with different strychnine concentrations. Dose-response curves were made in the order of increasing strychnine concentration and only after enough time had elapsed for a complete solution exchange and a 'steady-state' condition to have been reached (7-10 min). Most dose-effect curves were derived from cumulative strychnine concentrations, i.e., without washing between concentrations, which appreciably shortened the time required for obtaining curves with 6 concentrations. RESULTS

Characteristics of responses to ACh, 5 - H T and DA The analysis of the interactions of a drug such as strychnine with the receptors for ACh, DA and 5-HT in Aplysia neurons is somewhat complicated because each agent may be either depolarizing (D) or hyperpolarizing (H), depending upon the cell under investigation. Furthermore, the different responses appear to involve a specific increase in the membrane permeability for a different ion. In this section we present the basic characteristics of these responses which are pertinent to our study. Fig. 1 contains examples of the responses of 3 cells to ACh and DA, and illustrates that both may produce either D or H responses with no correlation between the nature of the responses of a cell to the two. That is, they may both be excitatory

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L- 7, ACh: D, DA: D

A1 DA

2 CoL: 20 mY, 5 sec R-B, ACh: D, DA: H

B L-4, ACh: H, DA: H

c Cal.: 10 mV, 10 sec Fig. 1. The responses of 3 different neurons to acetylcholine (ACh) and dopamine (DA). Ax, A2: neuron L-7 depolarized (D) by both ACh and DA. The cell was hyperpolarized to --85 mV to unmask the full depolarizing responses. Each drug was applied at 40-sec intervals. In A1 they were applied alternately, and the DA response rapidly desensitized while the ACh response remained constant. In A2 only DA was applied, and the same desensitization occurred. Calibration pulse at the end of As pertains to A1 as well. B is an example of a cell (R-B) depolarized by ACh and hyperpolarized (H) by DA, and C illustrates byperpolarization of another cell (L-4) by both. In each example, the upper record is the intracellular recording and the middle and lower records measure the ACh and DA phoresis currents, respectively. The abbreviations used apply to all subsequent figures.

or i n h i b i t o r y or m a y have o p p o s i n g effects. T h e cell L-7 in Fig. 1A shows an a l m o s t pure d e p o l a r i z i n g response to A C h during artificial h y p e r p o l a r i z a t i o n o f its m e m b r a n e p o t e n t i a l to - - 85 m V a n d no desensitization when the intervals between the A C h pulses were 40 sec57; the effect o f strychnine on the L - 7 - A C h - D response was the same as its effect on the D responses o f o t h e r cells, i.e. R-15, L-9, R-B g r o u p (see Figs. 5A, 3B and 3C). The records show t h a t there was no interaction between the D responses o f this cell to A C h and D A . I n A1 the two were given alternately at intervals o f 20 sec, and the a m p l i t u d e o f the A C h response r e m a i n e d c o n s t a n t while the d o p a m i n e response r a p i d l y decreases until it was a l m o s t c o m p l e t e l y a b s e n t after the third application. A z d e m o n s t r a t e s t h a t this r a p i d desensitization is a p r o p e r t y o f the d o p a m i n e receptor alone and n o t related to an interaction between the two receptors. This desensitization, described previously b y A s c h e r et al. 4, is a p r o p e r t y o f b o t h D a n d H responses to D A , a n d necessitated using intervals o f a p p r o x i m a t e l y 3 min between successive a p p l i c a t i o n s o f D A 3. W e f o u n d no obvious c o r r e l a t i o n between the nature of the 5-HT responses a n d A C h or D A responses, a n d no interaction between 5-HT a n d A C h responses. I n c o n t r a s t to the D A responses, however, the 5-HT responses showed less desen-

STRYCHNINE INTERACTIONS W I T H

Aplysia

1 13

RECEPTORS

TABLE I RESPONSESOr Aplysia (A), Helix ( H ) AND Cryptomphallus (C) NEURONS TO PHORESIS OF ACETYLCHOL1NE, DOPAMINE AND SEROTONIN AND THEIR ASSOCIATED CONDUCTANCEINCREASESTO CERTAIN IONS

Depolarizing (D) responses H y p e r p o l a r i z i n g (H) responses

ACh

DA

5-HT

A: C: A: C:

?

C: Na + (22)

A: K + (3)

H: C1 /and K + (22)

Na + (7,38,51) Na +/and CI- (9) C1- /and K + (7,29) C1- (8)

sitization; the H responses could be repeated at 30 sec intervals whereas the D responses required 2 or 3 priming stimuli and then a repetition interval of 30-60 sec to maintain stability. As stated above, the responses each involve an increase in membrane conductance to a specific ion, i.e. in gK+, g~a + or gcl-. Table I summarizes the specific ionic conductance increases involved in the generation of the different responses as well as related responses in Helix and Cryptomphallus. The C1-- and Na+-dependent ACh responses of Aplysia have been well established by Sato et al. 51 and Blankenship et al. 7, and we also reported a N a ÷ dependence of the D responses of the D I N I and D I L D A cells as. A m o n g the 5-HT responses, the D response might have been due to an increase in either gel- or gNa+, with Gerschenfeld's results on Helix aspera suggesting the latter e2. On the other hand, the H responses we have investigated are presumably due to an increase in gcl- since we have found they have the same equilibrium potential and relationship to changes in the extracellular chloride concentration as the C1-dependent A C h - H responses of the same cells. The K÷-dependent H responses evoked by ACh and D A deserve special mention in that they are of prolonged duration, mimicking similar long-lasting postsynaptic potentials (ILD) in cells R-B and R-15 in the case of D A 3 and in the cells of the L-2-L-6 group in the case of A C h a2,47. Although the K ÷ dependency of the A C h responses has been clearly established, they were initially attributed to activation of an electrogenic N a + p u m p 47; and activation of a regenerative current source has been recently implicated in their prolonged duration 58. Furthermore, we have reported 19 that the K+-dependent prolonged inhibitions are found in cells with pronounced anomalous rectification 28 and subthreshold K + activation 53, two properties of bursting pacemaker neurons. Finally, the A C h C1-- and K÷-dependent H responses are mediated by pharmacologically distinct receptors, the former being blocked by both curare and atropine 47 whereas the latter are resistant to these agents and are blocked by T E A and methylxylocholine al. The report by Chiarandini et al. 1° that 30 m M CuSO4 selectively blocks chloride permeability mechanisms in another mollusc, Cryptomphallus aspera, led us to test its action on these two-component cholinergic inhibitions. Unfortunately it blocks both components equally in Aplysia.

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Fig. 2. Effect of strychnine on EPSPs and IPSPs. A: simultaneous intracellular recordings of the EPSP in R-2 (upper) and the IPSP in L-11 (lower) evoked by stimulation of the right connective. Stimulus occurred at sweep onset. A1, control; A 2, 4 min 0.03 m M strychnine; A 3, 9 min strychnine; A4, 7 min wash (W) with control solution. The calibration pulse is near the end of each sweep; its baseline is the L-11 and the R-2 record as well (less gain for the recording from R-2). B: effect of 3 m M strychnine on the same EPSP in another R-2. B1, control; B2, 2 rain strychnine; Bs, 5 min strychnine; B4, 35 min wash. Note that the calibration pulse precedes the EPSPs. All records are the superposition of 3 or more traces. In A~ one EPSP triggers a spike in R-2.

Actions of strychnine (1) Synaptic transmission In a concentration of 1 mM, strychnine blocked all EPSPs and all C1--dependent IPSPs in the ganglion, sparing only the long-lasting K+-dependent IPSPs, especially the ILDs in R-15. There were different sensitivities of the PSPs to strychnine, the most sensitive being blocked by a concentration in the range 0.01-0.03 mM. An example of this differential effect is shown in Fig. 2A where the IPSP evoked in L-11 by stimulation of the right connective is blocked by 0.03 m M strychnine, whereas the EPSP in R-2 produced by the same stimulation is relatively unchanged. Fig. 2B shows that this EPSP in another R-2 is completely blocked by 3 m M strychnine. This difference in sensitivity did not relate to IPSPs vs. EPSPs, but presumably to the transmitters involved. (2) D responses All D responses investigated were reversibly reduced by strychnine, the responses to the dopamine and 5-HT being more sensitive than the ACh responses. Examples of this blocking action are shown in Fig. 3 for ACh and 5-HT responses, respectively. In A is the control response to ACh of an R-B neuron that had been hyperpolarized to a membrane potential of --80 mV to allow measurement of the depolarizing response with minimal contamination from evoked spikes. After 5 min exposure to 1 m M strychnine the response was reduced by approximately 80 ~ (B), but a doubling

STRYCHNINE INTERACTIONS WITH

Aplysia RECEPTORS

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Fig. 3. Effect of strychnine on ACh- and 5-HT-D responses of R-B neurons. B :strychnine inhibition of ACh-D responses. Intracellular recordings from an R-B neuron hyperpolarized to - - 8 0 mV by an applied trans-membrane DC current. A, control; B, starts after 5 min exposure to 1 m M strychnine, whereas C and D start after 2 and 7 rain of the washing period, respectively. Arrows indicate onset of the phoresis pulses. Note that in one case in B two pulses in succession were applied. E, F: antagonism of the 5-HT-D response of another R-B neuron, hyperpolarized to --90 mV by applied trans-mem-

brahe current. E, control; F, starts 6 rain after onset of 0.01 mM strychnine. In each record the phoresis current magnitude was reduced stepwise from a maximum of 1.6/~A (first responses) to 0.2 #A (last responses). The sequence was 1.6-1.4-1.2-1.0-0.8-0.6-0.4-0.34).2 #A. of the phoresis current (double arrows) still produced a response comparable to the control. C and D illustrate the gradual recovery of the response during washing. Figs. 3 and 4 demonstrate the manner in which the dose-response curves of Figs. 5 and 7 were generated. Fig. 3E shows that 0.01 m M strychnine produced a 60 % reduction of the D responses to 5-HT of an R-B neuron (E vs. F), the record in F starting 6 min after the introduction of strychnine into the bathing medium. When the strychnine concentration was increased to 0.1 m M (not shown), the response to the 1.6/~A 5-HT application was only 1.5 mV, i.e. 5 % of the control, but slightly larger responses could be evoked by increasing the phoresis current.

(3) Cl--dependent H responses We investigated the effects of strychnine on Cl--dependent H responses to only

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1.8 2.0 ,uA Fig. 4. Differential sensitivity of 5-HT and ACh-H responses to strychnine. Each record illustrates the response of L-11 to the successive phoretic applications of 5-HT (open symbols) and ACh (shaded symbols) for two phoresis current intensities. A is the control and B was taken 7 min after the onset of perfusion with 0.01 mM strychnine. The response to 5-HT is strongly reduced, while that to ACh is minimally effected. Three minutes later the strychnine concentration was raised to 0.1 mM, and the records in C are from after 6 min exposure to that concentration. Note that two successive 5-HT pulses were applied (double arrows) and the response was still minimal. A single application was ineffective. At this concentration partial reduction of the ACh response is seen. D, recovery of both responses after washing for 15 rain in strychnine-free solution. The duration of the 5-HT and ACh phoresis currents was always 200 msec, their onset being indicated by arrows. The control responses illustrated were relatively maximal, i.e., from the upper regions of the dose-response curves.

A C h and 5-HT, since we have not e n c o u n t e r e d c o m p a r a b l e responses to D A . These H responses were also blocked by strychnine, the 5 - H T responses again being the m o s t sensitive. The c o m p a r a t i v e characteristics o f this action are shown in Fig. 4 for cell L-I1 which gave H responses to b o t h agents. Fig. 4B shows that the 5 - H T response was strongly reduced c o m p a r e d to the c o n t r o l (Fig. 4A) by 0.01 m M strychnine, a l t h o u g h the A C h response was not influenced. Fig. 4C shows, however, a significant r e d u c t i o n in the A C h response after the strychnine c o n c e n t r a t i o n had been raised 10-fold, while even a double 5 - H T application had but a m i n i m a l effect. The

I

STRYCHNINE INTERACTIONSWITH Aplysia RECEPTORS

117

A C h - H response of this a n d other cells was further reduced when the strychnine c o n c e n t r a t i o n was increased to between 0.5 a n d 1.0 m M , as shown in the d o s e response curves of Fig, 5b. Finally, Fig. 4D demonstrates the reversibility of these effects. Generally, the differential sensitivity of the two responses to strychnine was n o t as p r o n o u n c e d as i n the example in Fig. 4, which has been chosen to d e m o n s t r a t e t h a t this differential effect was f o u n d when the strychnine effect o n two different receptors of one cell were studied at the same time.

(4) Mechanism of reductions of the phoresis responses I n these a n d parallel studies of the effects of strychnine o n m e m b r a n e properties of Aplysia neurons, we f o u n d only m i n i m a l effects o n m e m b r a n e potential a n d resis-

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Fig. 5. Log-dose-response curves of the strychnine interactions with the ACh-D and H responses (a and b), the dopamine-D and H responses (c and d) and the 5-HT-D and H responses (e and f). The response amplitudes are each plotted as fractions of the control maximal response (1.0) vs. log phoresis current for the control (O) and a series of different strychnine concentrations. Throughout each experiment the phoresis current duration remained constant. Since it varied from one experiment to the next, and because of technical variability, no significance is attached to the differences in sensitivities of the control responses to the phoresis currents. In b the ACh-H response was the C1-dependent one. Note that only the K+-dependent dopamine-H response is not antagonized by strychnine and that the minimal strychnine concentrations necessary to demonstrate the antagonisms do not exceed 0.01-0.05 mM.

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tance. Specifically, strychnine concentrations in the range 0.1-3.0 m M produced at most 15~o reduction or increase in membrane resistance and often none at all ( < 4- 5 ~). None of the blocking actions described above could be due to such minimal effects. However, when resistance changes did occur, the measured amplitudes of the phoresis responses were appropriately corrected to allow quantitative comparison of the log-dose-response curves. Since we found no evidence to support the concept that strychnine alters the equilibrium potentials for the ions involved, the blocking effects were presumably due to selective interactions of strychnine with the membrane receptors for ACh, 5-HT and DA. We also found no evidence that strychnine specifically increases gel-, as apparently it does in the leech 49. The dose-response curves in Fig. 5, in addition to quantifying the different actions of strychnine, are therefore related to the question of the nature of these drug-receptor interactions. First, with reference to the comparative sensitivities of the receptors to strychnine, inspection of Fig. 5a-f, reveals that the D responses were more sensitive than H responses for every transmitter. This is shown by the increase in phoresis current (A log I) necessary after the addition of 0.1 m M strychnine to produce a response equal to 50 ~ of the control maximum. On the basis of this criterion, both the ACh and 5-HT Cl--dependent H responses were only about 40-50 ~ as sensitive to strychnine as were the D responses. The same criterion also shows that the DA- and 5-HT-D responses were more sensitive to strychnine than were the 5-HT-H or the ACh-D responses. Expressed in other terms, the blocking effect of 0.05 m M strychnine on the DA-D receptors seems equal to that of 0.5 m M on the ACh-D receptors. Although there are many points to be considered before we draw conclusions on the nature of the drug's action from dose-response curves such as those in Fig. 5 (see Discussion), the results suggest that in 2 of the 5 blocking actions strychnine acted competitively with the transmitter. These are the ACh-D and the DA-D responses. This conclusion is based on the parallel shift (to the right) of the dose-response curves after the application of strychnine, which reduced the sensitivity to the transmitter by a factor of 10. An increased phoresis current could completely compensate here for the reduced responsiveness under the influence of strychnine. In contrast, the dose-response curves for the ACh-H (C1-) and the 5-HT-D and I-I responses show a more complex change.

(5) K+-dependent phoresis responses to DA and ACh and prolonged IPSP (ILD) The DA-H response was K+-dependent (see Table I). As shown in Fig. 5d, this response was only slightly affected. It had a minimal decrease in 0.05 m M strychnine, which disappeared when the strychnine concentration was increased to 0.5 mM, and reappeared later in 2.5 m M strychnine. Similar behavior was found in several experiments. These results correlate with our observation that the ILD in cell R-15, which can be mimicked in many respects by application of DA 3, was also affected only slightly by strychnine in concentrations of up to 1-2.5 mM. The action of strychnine on the K +- and C1--dependent ACh-H responses of cells L-2-L-6 was also peculiar and completely different from the 6 curves of Fig. 5. These ACh responses were increased by strychnine, as shown in Fig. 6. Strychnine

STRYCHNINE INTERACTIONS WITH Aplysia RECEPTORS

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Fig. 6. Strychnine and high calcium enhancements of the prolonged cholinergic K+-dependent hyperpolarizations. A-E: effects of strychnine and 8 × (Ca2+)0 on the ACh-H response of L-3. A, control; B, enhanced response after 7 min perfusion with 1.0 mM strychnine added; C, recovery after 12 min washing; D, enhancement produced by raising extracellular calcium concentration to 8 times control level, i.e. to 88 mM (7 min perfusion); E, in the presence of 8 × (Ca~+)0, the effect of 1.0 mM strychnine is even more pronounced than in B. The phoresis current was the same in each record, and the arrows (horizontal bars) indicate the current onset. F, G: similar effects of strychnine and the ACh response of L-2 (upper cell, high gain with action potentials truncated) and on its postsynaptic inhibition by a burst of impulses in interneuron L-10 (lower cell, low gain records). F, control; G, 8 min 0.1 mM strychnine. In the beginning of each segment a depolarizing transmembrane current pulse (current recorded in second traces from bottom) activates L-10 and produces a summated IPSP in L-2; at the end of each segment iontophoretically applied ACh (phoresis current shown in lowest traces and onset labeled with arrows) to the soma of L-2 produces a comparable hyperpolarizing response. Both responses are enhanced by strychnine, although the effect on the ACh response is the most pronounced.

especially increased the d u r a t i o n o f the responses (Fig. 6B), b u t also increased the a m p l i t u d e w h e n it r e d u c e d the resting potential. In the instances w h e n the response e n h a n c e m e n t occurred, it occurred with all doses o f strychnine an d was m o s t significant for the s u b m a x i m a l A C h responses, i.e. in the l o w er end o f the d o s e - r e s p o n s e curves. I n the e x a m p l e s h o w n in Fig. 6 the e n h a n c e m e n t o f the A C h response was

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reversed by perfusing the ganglion with strychnine-free solution (Fig. 6C); occasionally, however, the responses initially disappeared during the wash period and only recovered after 10 min or more washing. A similar strychnine- or curare-induced increase in the comparable K+-dependent ACh-H responses of a group of cells in the pleural ganglion was initially described by Kehoe 29. Since some aspects of the strychnine effects on excitability are comparable to those of reduced extracellular calcium 88, we tested the effects of different calcium concentrations on these H responses and the strychnine effect. Calcium reduction was of minimal consequence whereas, surprisingly, raising the calcium concentration 8fold produced essentially the same effect as did strychnine alone (Fig. 6D). The subsequent addition of strychnine to the bathing medium produced a further increase in the response (Fig. 6E). It is not possible to evoke the comparable long-lasting cholinergic IPSPs by nerve stimulation; however, they can be produced by direct intracellular stimulation of an identified interneuron, L-1057. Although Waziri 6° has demonstrated that activation of L-10 produces not only monosynaptic C1-- and K+-dependent IPSPs in L-2-L-6, but also disynaptic effects, it is nevertheless possible to compare qualitatively the effects of strychnine on the long-lasting IPSPs with its effects on the ACh responses. Fig. 6F illustrates one case in which such a study was made: both responses were enhanced by strychnine (Fig. 6G), the increase in the ACh response being more pronounced. It is possible that the increase in the K+-dependent H responses, induced synaptically29, 31 or by ACh, is simply a manifestation of the strychnine inhibition of the C1--dependent responses with which they are generally mixed. That is, when the C1-dependent responses and the associated increase in membrane conductance are blocked there will be less shunting of the K+-dependent responses and a greater membrane potential change. This hypothesis could not account for effects as large as those shown in Fig. 6F, particularly since the enhanced responses were associated with increased and prolonged membrane conductance increases. Furthermore, the effect occurred in cells where, before the application of strychnine, the majority of the H responses recorded at different membrane potentials in the range of -- 45 to -- 1 l0 mV appeared to consist of only the K+-dependent component (in contrast with results from cells in the pleural ganglion, where the two components could be detected easily, using the same technique), a distinct possibility since we do not excise the connective tissue capsule surrounding the ganglion (Kehoe, personal communication). The equilibrium potential of this composite response should lie between those of the two components: E c l - = --65, EK+ = --85 mV, (--71 mV in ref. 40, see also refs. 32 and 47) if both are actually activated and should shift in the hyperpolarizing direction when the addition of strychnine blocks the CI -dependent component. However, we observed no such shift, suggesting that the increase in the K+-dependent responses is not simply due to the blockage of the C1--dependent responses. Fig. 7 demonstrates that the enhancement by strychnine of the K+-dependent responses were also produced by hexamethonium (Fig. 7b) and curare (Fig. 7c). The records for the dose-response curves were obtained from L-14, which is situated close

Aplysia RECEPTORS

STRYCHNINE INTERACTIONS WITH L-l/, 15

121

ACh:H

A~ STRYCHNINE 0,1 mM- o

B: HEXAMETHONIUH 104 WlV - o

C: CURARE / 10~ WIV- Z~

~,

1.0

a

0.5

/ SJ 02

10

100 200 G2

10

IOQ 200 02

~o ~ o % ~oo 2o.o

Fig. 7. Log-dose-response curves of the effects of strychnine (A), hexamethonium (B), and otubocurarine (C) on the ACh-H response of L-14. The curves were generated as in Fig. 5. Each application of drug was followed by a wash period of 30 min or more during which the responses returned to within 10 ~ of the original control (control of A) and stabilized.

to the cell L-5 of the L-2-L-6 cluster and apparently has the same type of ACh response. The effect of each antagonist was reversed by returning to the normal perfusate. That hexamethonium also produces this effect is further evidence that the effect is not merely due to a blockage of C1--dependent H responses since it has been shown that hexamethonium does not block those responses 58. In general, the similarity of the effects of strychnine and curare is consistent with the 'curare-like' action of the former; we have observed that curare also blocks the DA-D responses, and Gerschenfeld 22 has shown that it blocks 5-HT-D and H responses in the land snail. Finally, the enhancement of the K---dependent ACh-H response by strychnine does not appear to be a manifestation of an anticholine esterase action, as has been attributed to strychnine at the muscle endplate 4z, since it occurs in the presence of eserine (10-4 g/ml). DISCUSSION

Our results demonstrate that, in Aplysia neurons, strychnine blocks all 'classical' EPSPs and IPSPs as well as the Na ÷- and Cl--dependent responses of these neurons to iontophoretically applied ACh, 5-HT and DA, but has little effect on the K +dependent ones. The nature of the dose-response curves of the effects of strychnine on ACh-D and DA-D phoresis responses, the evidence for differing sensitivities of the responses to strychnine, the lack of correlated generalized changes in membrane properties z7, and the absence of an antagonism of the prolonged K+-dependent inhibitory processes all indicate that the blocking effects are due to a selective inter-

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action of strychnine with the postsynaptic membranes. Our results concerning the blocking action of strychnine on the ACh-D and D A - D response and the different action on the C1-/K+-dependent ACh-H response20,35, 3s were confirmed in recent papers by Kehoe 31 and Aseher 3. The classification of a drug action as selective implies that a certain membrane system is more sensitive to the drug than are other membranes. Further, it suggests that the drug combines with a specific membrane receptor, as apparently occurs between T T X and the sodium channels activated during the generation of the action potential44, 45. And it may also relate to the situation where a drug has more than one action, for example, T E A blocks selectively the delayed rectification currents of electrically excitable membranes24, ~ and certain cholinergic receptors of chemically excitable membrane patches (e.g. Kehoe30). Even although the latter effect occurs at a much lower TEA concentration than the former a0 both actions are 'selective' in that other effects of TEA on electrogenesis or postsynaptic receptors require appreciably higher concentrations. The situation with strychnine is somewhat the same in Aplysia; it has selective effects on both electrical and chemical excitabilitya6, 37, the chemically excitable membranes being more sensitive to strychnine than the electrically excitable regions. Different chemically excitable membrane patches are affected by similar strychnine concentrations. For heuristic reasons we like to assume that the effects of strychnine on the chemically excitable membrane patches result from its interaction with the receptors of the transmitters we have studied. At least it seems necessary to assume from the dose-response curves that strychnine specifically interacts with the receptors for the ACh-D and D A - D responses. Strychnine, then, must be assumed to act not only on the receptors for the glycine-mediated inhibition but also on the receptors for certain other transmitters. This concept may explain the 'curare-like' action of strychnine 1,5,23 which is extended in our study of molluscs, since D-tubocurarine (D-TC) in low concentration also blocks the N a +- and Cl--dependent ACh responses 56, the presumably CI-dependent dopamine responses 3 of Aplysia neurons and the Na~- and Cl--dependent 5-HT responses of Helix aspera 22, i.e. all the responses blocked by strychnine. In fact, both drugs appear to spare only the receptors responsible for the activation of prolonged inhibitions mediated by an increased g~+. It may be asked then, which of the strychnine actions relates to convulsions? In relatively low concentrations, strychnine apparently can be a specific tool for distinguishing mammalian glycine receptors 17, while its other numerous effects are manifested in overlapping concentration ranges, in both Aplysia and cat CNS, making it difficult to evaluate which effects are significant for its epileptogenic action. For example, subconvulsive doses are sufficient to block glycine-mediated inhibitions 11, 13,16. Therefore other mechanisms are required in addition for strychnine to produce convulsions, especially in those neuronal networks in which glycine-mediated IPSPs do not constitute the main inhibitory system. As we have discussed elsewhere 37 (e.g., Aplysia neurons), the effects of strychnine on the electrical excitability are more important for the generation of high-frequency discharges than its effects on chemical

STRYCHNINE INTERACTIONS WITH

.4plysia RECEPTORS

123

excitability. In other preparations this might also include the highly specific effect of strychnine on glycine receptors. In fact, in Aplysia neurons these abnormal discharges can be induced by 0.1 m M strychnine through an increased electrical excitability expressed by distinct changes in the I/V relationship which can be observed not only after a blockage of Na +- and C1--dependent EPSPs and IPSPs but also in cells that have been completely isolated from their synaptic input by axonal ligaturea7, as.

Mechanisms of antagonistic actions of strychnine While it is clear that the ACh, DA and 5-HT receptors are distinct from each other, the evidence that a compound such as strychnine or curare can, over a limited concentration range, block the activation of 5 of the 7 receptors we have studied, might be considered to suggest similarities in the receptors' structures, a possibility which should be further investigated. In reality, however, it is difficult to exclude completely the possibility that strychnine antagonizes the action of the agonistreceptor complexes rather than the association of the two. Nevertheless, if we operate under the assumption that strychnine is an inhibitor acting on the same receptors as the agonists, can the log-dose-response curves of Fig. 5a-c, e, f be used to distinguish between competitive and non-competitive inhibitory actions? Conclusions based on the log-dose-response curves used here must be tendered cautiously because of the additional theoretical and practical limitations inherent in the iontophoretic technique 26. Some of these limitations are (1) non-uniform distribution of the applied drug in the receptor region, particularly if the receptors are somewhat distant from the phoresis electrode, as some of the receptors studied in this paper 3 may be; (2) an upper limit to the available phoresis current; (3) no proof of a linear relationship between phoresis current amplitude and agonist concentration in the receptor region; and (4) the conductance change, measured by the voltage clamp technique, is linearly related to the receptor occupancy by the drug while the use of the depolarization as a measure of the drug effect, as in our experiments, can be rather unsatisfactory in some preparations 5°. Despite these limitations, the dose-response curves for the strychnine antagonisms of the ACh-D and DA-D responses can apparently be described in terms of reductions in the apparent affinities of the agonists for their receptors, i.e., as competitive inhibitions; however such a conclusion must be regarded as tentative.

The K+-dependent inhibitions and strychnine As described above (see Results) these responses, especially those evoked by ACh, are pharmacologically unique; the finding that strychnine either had no effect on these responses or enhanced them is but another example of this uniqueness. The same remark applies to the observation that raising the extraeellular Ca 2+ concentration increased the K+-dependent ACh-H responses and the effect of strychnine on the H response, but did not counteract the strychnine-related depression of the ACh-D response, since high calcium generally increases postsynaptic responses as a result of a presynaptic actionlS, 25 while having a minimal depressant 51,54 effect on the postsynaptic membrane.

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ACKNOWLEDGEMENT W e w o u l d like to t h a n k D r . W . - D . Heiss for his h e l p d u r i n g the initial experim e n t s , D r s . W. K . N o e l l a n d C. M . S m i t h for discussion, V. W a l k e r a n d M . D u e s m a n n for t e c h n i c a l assistance, a n d D r . R. H a s s l e r for his g e n e r o u s help. T h i s w o r k was p a r t i a l l y s u p p o r t e d by a g r a n t o f t h e Buswell F o u n d a t i o n o f SUNYAB.

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