Neurotransmitters in Invertebrates: Satellite Symposium of the 28th International Congress of Physiological Sciences, Veszprém, Hungary, 1980

R.

WELCOME ADDRESS

Katalin S. Rózsa,     Biological Research Institute of the Hungarian Academy of Sciences, Tihany, Hungary

Ladies and Gentlemen,

It is a great pleasure to greet you at the Satellite Symposium of the 28th International Congress of Physiological Sciences. Since the sections and symposia of the main congress did not afford an opportunity for gathering together those scientists who are interested in neurotransmitters of invertebrates, we decided to organize this precongress symposium on the basis of the Biological Research Institute of the Hungarian Academy of Sciences, with the help of an international organizing committee. I would like to thank them for their contributions in organizing the scientific program.

The topic of our Symposium is Neurotransmitters of Invertebrates. The neurotransmitters came into the limelight of investigation at the very early stage of the development of the physiological sciences and the results of this field contributed significantly to the understanding of the regulation of physiological processes.

Transmitter research in invertebrate animals has always been used as a model. Throughout the animal kingdom there is a great similarity in biochemical mechanisms and structural characteristics. It is well known that from the first appearance of a nervous system the discontinuity of the neurons is common in all organisms and the connection between the neurons is achieved mainly by neurotransmitters. The same is true for neuroeffector relations. At the same time, the first neurotransmitters were identified more than 60 years ago and only about half a dozen of these original molecules have been described as having properties of mediators. Among them only acetylcholine, noradrenaline, dopamine, 5-hydroxytryptamine and several amino acids function as neurotransmitters throughout the animal world. However, the belief is widespread that more neurotransmitters and neuromodulators must exist. This point of view seems to be justified by looking at the list of new putative transmitters, including amines /octopamine, histamine/ amino acids /L-glutamate, L-aspartate, etc./ and peptides /proctoline, FMRF-amid, opioid peptides/.

All the known neurotransmitters seem to be simple in structure, the peptides which are supposed to function as neurotransmitters belong also to substances with low molecular weight. The neurotransmitters have a wide distribution in the organisms and they function as a chain in the conduction of information using a synaptic machinery including the metabolism, storage and liberation mechanisms. Most of the synaptic substances play a very extensive role in the neural regulation, for example, the same agent can have inhibitory /ACh at the heart/ or excitatory /ACh at CNS/ influences, depending on the receptors of the target cell. The same substance can have the role of neurotransmitter or of hormone /catecholamines/.

Studies on invertebrates contributed significantly to the discovery or postulation of new transmitters. It was found that some choline, hydroxytryptamine and catecholamine derivatives may have higher synaptic efficiency in certain invertebrates than those which are accepted in vertebrates as neurotransmitters. For instance, 4-hydroxytryptamine discovered in the salivary gland of Cephalopoda and 6-hydroxytryptamine in the heart of Crustacea are more effective than 5HT. Dopamine proved to be more potent than noradrenaline in the heart of many invertebrates.

Due to the number of neurotransmitters the question of their specificity attracted special attention. In reference to the effect of the same transmitter on changes in ion permeability of the postsynaptic membranes can vary in various synapses, depending on the local circumstances, determining the specific effect of the given transmitter. On the postsynaptic membrane the effect of the transmitter appears always in a complex form, it can alter the membrane permeability for several ions and it can be blocked in different organisms with different inhibitors. All neurotransmitters can cause inhibition or excitation depending on the nature of their site of action. It is possible to study the nature of these receptors by using specific inhibitors and using immunological methods to identify them. It is now commonly accepted that multiple receptors exist for all the neurotransmitters regulating various ion permeabilities. Three types of acetylcholine receptors have been characterized pharmacologically, controlling increase of Na+, K+, or Cl− conductance. Five serotonin receptors are known: three types are connected with conductance increase /Na+, K+, Cl− / and two with conductance decrease /for K+, and Na+/. It would be important to study the relation between the pharmacological properties of various receptors and their biochemical structure.

The basic mechanisms of neurotransmission proved to be the same, nevertheless some variation can be found in invertebrate phyla. For instance, considerable differences can be demonstrated in sensitivity to neurotransmitters following the phylogenetic order. This general rule can be used for developing a new trend in the insecticide research. It is well known that all the insecticides used today disturb the cholinergic mechanisms by inhibiting acetylcholinesterase, at the same time the monoaminergic mechanisms are poorly understood in the insects, although they could also be useful for pesticide research.

In the investigations of peptide mechanisms the invertebrate preparations proved also to be useful. Two neuropeptides were determined to be neurotransmitters, proctolin and FMRF amide which were isolated from insects and molluscs, respectively. The cardio-active peptides were also detected at Crustacea and Insecta. Peptidergic neurons are distributed throughout the animal kingdom, but their role is poorly understood. Compared to other transmitters the peptides have long-lasting effects /minute range/ and more often they can play the role of modulators. Some peptides can initiate bursting pacemaker activity at the single cell level, as was demonstrated first on molluscan neurons. These effects are unlike those caused by conventional neurotransmitters and may be responsible for new forms of modulation of electrical activity.

The peptides can be modulatory substances, but it must be taken into consideration that the properties of neuromodulators are not well defined. Opioid mechanisms have now been demonstrated in invertebrates. These substances have assigned modulatory roles in the mammalian CNS. Neuromodulators are substances that alter the input-output relations of the neurons. Modulator actions can take place synaptically and at nonsynaptic sites. The modulatory effect of the peptides seems to belong to the latter category. The peptides represent promising molecules as neurotransmitters or neuromodulators.

It is now commonly accepted that postsynaptic effect of neurotransmitters is mediated by a second messenger, e.g. cyclic nucleotides and Ca²+ –ions. Interaction of the transmitters with a receptor on the cell membrane initiate intracellularly messages that result in the formation of second messengers. This system seems to work also throughout the animal world.

All the results in neurotransmitter researches proved that the nervous system from the very primitive invertebrates use various molecules for transmitting the information and regulating physiological processes. This underlines the importance of chemical differentiation. The fact that one cell can use more than one transmitter creates possibility for modulation and plasticity of the synaptic function. Also the cooperation and simultaneous action of several transmitters in one pathway seems to be a very important aspect to be investigated.

During the next three days many of these questions will be discussed from various points of view. The number of papers and participants is favourable for unlimited discussions and for establishing personal contacts between the participants both in and outside of the lecture-room. I wish that the symposium – besides informing on newest results – help to clear up recent problems arising in invertebrate neurotransmitters, which will lead certainly to better understanding of the many sided and complex regulatory role of neurotransmitters in the whole animal kingdom.

Cholinergic mechanisms

QUANTAL RELEASE OF ACETYLCHOLINE FOLLOWING INJECTION OF ACETYLCHOLINESTERASE INTO THE PRESYNAPTIC NEURONE

L. Tauc, G. Baux and M. Simonneau,     Laboratoire de Neurobiologie Cellulaire, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France

Publisher Summary

The study of synaptic transmission in central neurons is restricted by the difficulty of recording individual miniature postsynaptic potentials (PSP) or currents of known presynaptic origin because of the multiplicity of inputs to the postsynaptic cell. The lack of quantal analysis severely limits the usefulness of preparations such as the cholinergic neuronal couples in the buccal ganglion of Aplysia, which is otherwise exceptionally suitable for experimentation. This chapter presents an experiment that uses quantal analysis to study the modification of quantal parameters under an imposed experimental condition. This method can be successfully applied for the study of the mechanism of transmitter release at synapses that are close to the cell body of the presynaptic neuron.

INTRODUCTION

The study of synaptic transmission in central neurones is restricted by the difficulty of recording individual miniature postsynaptic potentials /PSP/ or currents /PSC/ of known presynaptic origin because of the multiplicity of inputs to the postsynaptic cell. The lack of quantal analysis severely limits the usefulness of such preparations as the cholinergic neuronal couple in the buccal ganglion of Aplysia, which is otherwise exceptionally suitable for experimentation. For example, both pre- and postsynaptic neurones can be easily identified and substances of very high molecular weight can be injected into the presynaptic neurones. These agents can then migrate to the not distant synaptic terminals and eventually affect the transmission process, as can be monitored from the modification of the postsynaptic response. For instance acetylcholinesterase and nduraminidase, injected presynaptically, are thus shown to block synaptic transmission /Tauc and Hinzen 1974, Tauc et al. 1974/.

This type of experimentation can furnish information about the mechanism of synaptic function. To extend the value of such information, we have developed a method described here, which enables quantal analysis at this synapse. We could not use failure statistical analysis already tried on another Aplysia synapse /Castellucci and Kandel 1974/ as we needed to obtain quantal parameters even if our experimental conditions interfered with the conducted spike.

The method is based on the observation that prolonged depolarization of the presynaptic neurone produces a continuous release of the transmitter /Baux et al. 1978/. Both pre- and postsynaptic neurones were clamped in the presence of tetrodotoxin /TTX/. Statistical fluctuation analysis of the postsynaptic current response to prolonged presynaptic depolarization was used to determine the size of the miniature postsynaptic current /MPSC/ and its decay time. Since the transmitter is known, we could compare these characteristics to those of acetylcholine /ACh/ operated channels on the same postsynaptic cell and estimate the number of quanta released by a single spike, as well as the number of postsynaptic channels opened by a single quantum /Simonneau et al. 1980a, Simonneau et al. 1980b/.

MATERIAL AND METHODS

Experiments were performed on the buccal ganglion of Aplysia californica. The postsynaptic neurones and the two presynaptic cholinergic interneurones from which they receive afferents are all of about 200 microns in diameter and easily identified. The postsynaptic response is inhibitory, Cl− dependent and with a normal reversal potential of –60 mV. ACh receptors are also present on the cell body and show no desensitization to injected ACh.

One presynaptic and one postsynaptic neurones were each penetrated by two separate KCl filled microelectrodes and both cells were simultaneously voltage clamped /schema fig. 1B/. Unless indicated otherwise, the holding potential of the presynaptic neurone was –50 mV, that of the postsynaptic neurone –80 mV; the duration of the presynaptic pulse was 3 sec. 10−4M tetrodotoxin /TTX/ was added to block sodium channels. Experiments were performed at room temperature. The data were recorded by a Nicolet /1090 A/ digital oscilloscope and analyzed on an HP 9825 A calculator.

Fig. 1 A-Postsynaptic current responses obtained with sustained depolarization of the presynaptic neurone. Both cells were voltage clamped. Upper traces represent low gain DC recordings of the membrane current and lower traces high gain recordings of the fluctuations of the membrane current. The presynaptic neurone held at –50 mV was transiently depolarized to zero membrane potential. Holding potentials of the postsynaptic cell are indicated. The small curare resistant inward current is partly attributed to electrotonic coupling between the two cells and partly to K+ accumulation induced by the presynaptic current /unpublished data/. Calibrations: current, 10 nA for DC and 1 nA for AC recordings, time 1 sec. B-Schematic representation of the recording circuit. One of the two presynaptic neurones and the postsynaptic neurone were simultaneously voltage clamped. i1 is measured in the current loop of the presynaptic cell, the virtual ground gives the sum i2 of both pre and postsynaptic currents; the presynaptic current is thus given subtracting i1 from i2. /From Simonneau et al. 1980b/.

Determining quantal parameters

A long depolarizing pulse applied to the presynaptic neurone evokes a sustained postsynaptic response on top of which typical fluctuations /noise/ are apparent /Fig. 1A/. At a holding potential of -20 mV, the postsynaptic current response to a presynaptic depolarization to θ mV is outward corresponding to a Cl− influx. At -42 mV, the reversal postsynaptic potential of this cell, the same presynaptic depolarization no longer induces a postsynaptic current. At -80 mV, the current response is reversed, corresponding to a Cl− efflux. The amplitude of the noise follows that of the current. The current and noise are similarly blocked by curare or when cobalt is added to, and Ca++ removed from, the bath. This clearly confirms that the postsynaptic response is evoked by the release of acetylcholine by the presynaptic neurone.

where V is the postsynaptic membrane potential and Veq the equilibrium potential of the postsynaptic response. The decay time constant /τmin/ can be determined from the power spectrum of the noise using the fast Fourier transform.

Moreover, because the transmitter at this synapse is known, it is possible to inject acetylcholine onto the surface of the presynaptic cell and get information on the elementary conductance / rch / and the life time /τch/ of the ACh activated channels and compare them with r min and τmin. The response produced by ionophoretic injection of ACh /Fig. 2/ can be submitted to a similar statistical analysis as stated above, except that the relation determining rch has to take into account that a channel opens and closes suddenly whereas a MPSC has an exponential decay. Thus rch = E² /I/V-Veq/.

Fig. 2 A-Somatic acetylcholine activated chloride channel noise in the postsynaptic neurone voltage clamped at –100 mV for two different intensities of ACh ionophoretic application. Upper traces, low gain DC current recordings; lower traces, high gain AC recordings. Calibrations: DC 20 nA, AC 0.5 nA, time 2 sec. The chloride reversal potential ECl was –35 mV. B-Power density spectrum of current noise produced by a steady ionophoretic application of acetylcholine to a voltage-clamped postsynaptic cell in the buccal ganglion of Aplysia. The holding potential was –100 mV, 13 spectra of 1000 points were averaged. The background noise was not subtracted but its contribution was less than 10% for the whole frequency range. The experimental points were tentatively fitted by a double Lorentzian of the form S /f/: S /o/1+/f/fC1+f/fC2/². The half frequency fC corresponds to a mean channel duration τch = 1/2 fC. In this cell, fC1 = 14 Hz and fC2 = 100 Hz corresponding to 11.4 and 1.6 ms, respectively. A single Lorentzian is also shown with fC = 16.5 Hz corresponding to τch= 9.6 ms and indicated by an arrow In this experiment S/0/ = 508.8×10−24A²s. Neither the single nor the double Lorentzian fit in a satisfactory manner. However, in both cases the deduced lifetime for long life channels /10 ms/ would be that expected if a given channel opened only once when a single ACh quantum is released. /From Simonneau et al. 1980b/.

The mean values obtained for the conductances and the decay time constant are:

PSC represents the evoked current response to a presynaptic spike.

It can thus be estimated that at this synapse one presynaptic spike releases about 180 qunanta, each opening 500 chloride channels. The above values were obtained at room temperature, was shown to be temperature dependent with Q10 = 2.7. The similar values obtained for τch and τmin indicate that a given channel opens only once when a single ACh quantum is released.

The values for rmin were calculated from responses obtained with presynaptic depolarization to less than θ mV absolute membrane potential. Plotting variance against the mean current, one would expect a linear relationship. However, as shown in fig. 3, there is a clear break in the slope at a mean current level corresponding to a response produced at θ mV presynaptic membrane potential and above. Assuming that Campbell’s theorem is valid for all responses this break may express a real decrease in size of the MPSC released by strong influx of Ca++ in the terminal /a sort of subminiature postsynaptic response, as described at the neuromuscular junction by Kriebel and Gross /1974// or the decrease of the size of the quanta is only apparent, due to addition of a non quantal component in the release of transmitter. The non quantal release of ACh at rest was shown at the neuromuscular junction /Katz and Miledi 1978/. Perhaps our results indicate the existence of a voltage and calcium dependent non quantal release of acetylcholine. Such a possibility was considered for a non synaptic release of acetylcholine by Johnson and Pilar /1980/.

Fig. 3 Relation between mean postsynaptic current I and its variance E². Using values of I below 4.5 nA, the calculated imin was 72 pA and rmin = 1890 pS. For mean currents greater than 4.5 nA a break appears in the slope. /from Simonneau et al. 1980b/.

Quantal size after AChE injection

Acetylcholinesterase /AChE/ was shown to block synaptic transmission when injected into the presynaptic cholinergic interneurone /Tauc et al. 1974, Tauc 1977, Tauc and Baux 1980/ possibly by hydrolysing the free cytoplasmic ACh, whereas the vesicular ACh was not affected. Using the above described technique we were able to determine that the depression of the postsynaptic response by injected AChE results in the diminution of the quantal content of the response, but that the size of the quantum remains constant /Simonneau et al. 1980/ /Fig. 4/. This indicates /assuming that AChE does not interfere directly or indirectly with the transmitter release mechanism/ that the quantal size is independent of the cytoplasmic ACh concentration and that ACh is apparently not released through a simple ACh channel in which ACh will move following its electrochemical or concentration gradient. More likely, to explain the constancy of quantal events /in case we discard the vesicular hypothesis/, the release might be elicited by a membrane structure which binds ACh to saturation, a sort of a carrier mechanism or restricted or saturated gates /Vesigate, Tauc 1979/.

Fig. 4 Depression of postsynaptic current responses obtained with sustained depolarization 13 sec/ of the presynaptic neurone after the injection of acetylcholinesterase into the presynaptic neurone. The response at 52 min can be considered as control. DC and AC recordings as in 1A. The fluctuation analysis show that the depression of the response results from the reduction of the quantal content, whereas the size of elementary quanta is unchanged.

In addition, the constancy of the size of the quanta whereas the quantal content decreases, suggests that the quantity of transmitter released could /in the normal situation/ be somehow related or perhaps even regulated by the concentration of cytoplasmic ACh.

CONCLUSION

The example shown above using quantal analysis to study the modification of quantal parameters under an imposed experimental condition, shows that this method can be successfully applied for the study of the mechanism of transmitter release at synapses which are close to the cell-body of the presynaptic neurone. The cholinergic couples in the buccal ganglion of Aplysia are of course exceptionally suitable especially since because of the size of the cell bodies it is possible to manipulate the transmitter release mechanisms by injecting appropriate molecules, which can be transported to the terminal. Further experiments in this direction are in progress.

REFERENCES

Baux, G., Simonneau, M., Tauc, L. Brain Res. 1978; 152:633–638.

Castellucci, V.F., Kandel, E.R. Proc. Nat. Acad. Sci. 1974; 71:5004–5008.

Johnson, A.D., Pilar, G. J. Physiol.(Lond.). 1980; 299:605–619.

Katz, B., Miledi, R. Proc. R. Soc. Lond. B. 1977; 196:59–72.

Kriebel, M., Gross, C. J. Gen. Physiol. 1974; 64:85–103.

Simonneau, M., Baux, G., Tauc, L. Quantal analysis of transmitter release at an identified synapse. In: Taxi J., ed. Ontogenesis and functional mechanisms of peripheral synapses. Elsevier; 1980:179–189.

Simonneau, M., Tauc, L., Baux, G. Proc. Natl. Acad. Sci. USA. 1980; 77:1661–1665.

Tauc, L. Transmitter release at cholinergic synapses. In: Cottrell G.A., Usherwood P.N.R., eds. Synapses. London: Blackie Glasgow; 1977:64–78.

Tauc, L. Biochem. Pharmac. 1979; 27:3493–3498.

Tauc, L., Baux, G. Libération du transmetteur synaptique: examen critique de la théorie vésiculaire. In: La transmission neuromusculaire. Masson, Paris: Fondation Singer-Polignac, ed; 1980:121–127.

Tauc, L., Hinzen, D.H. Brain Res. 1974; 80:340–344.

DISCUSSION

SALÁNKI, J.: What happens if you inject ACh or ACh-precursor into the presynaptic cell? Do you have an opposite effect to AChE-injection?

Does AChE break down only extravesicular ACh or does it have effects also on the number of presynaptic vesicles?

TAUC, L.: Injection of ACh increases quantal content of the response without changing the size of the quanta. The increase in quantal content is a complex phenomenon which I cannot discuss for the moment.

In in vitro studies it was shown that AChE does not hydrolyse ACh in the vesicles. This in fact is the basis of identification of vesicular ACh content. When we injected AChE and analyzed the terminals in EM, the number of vesicles did not seem to be changed, they were clear and the histochemically revealed AChE was not in the vesicles.

WITTE, O.: Could you block the action potentials of many investigated cells with TTX, and what concentration of TTX did you use?

TAUC, L.: In Aplysia ganglia there are cells which show spikes either purely sodium dependent, or more or less sodium and calcium spikes. Active sodium channels can be blocked with TTX, at a concentration 10−4 w/v. Cells can survive for many hours with TTX and the effect is rapidly reversible with washing. TTX has no action on active calcium channels.

ELEKES, K.: What about the role of cytoplasmic ACh?

What about the relation between the release of the vesicular and non-vesicular ACh? According to Israel and co-workers vesicular hypothesis is already not valid at the cholinergic synapses of Torpedo electric organ.

TAUC, L.: Acetylcholine is synthetized in the cytoplasm and then it is eventually incorporated into the vesicles by a process so far unknown. In a non-vesicular release ACh would not need to go through vesicle incorporation. Israel and his group have shown that during intense stimulation of the electric organ of Torpedo the only compartment running over was the cytoplasmic ACh compartment, the vesicular compartment remained inert, then apparently the ACh released could not come from the vesicular compartment. This is exactly in line with our concept of release.

USHERWOOD, P.N.R.: Is the change in the variance /mean relationship for the transmitter noise with presynaptic membrane potential time dependent?

TAUC, L.: Given the nature of the analysis it is not possible to analyze segments of noise of less than 0.5 sec duration.

EFFECTS OF ACETYLCHOLINE ON IDENTIFIED NEURONS OF HELIX POMATIA

O. Witte and E.-J. Speckmann,     University of Münster, Institute of Physiology, Westring 6, D-4400 Münster, FRG

Publisher Summary

In some molluscan neurons, acetylcholine (Ach) elicits depolarization. The ionic mechanisms underlying this depolarization are not completely understood as of now. The analysis of the depolarizing ACh response is complicated mainly for two reasons. On one hand, it is difficult to determine the actual reversal potential of the Ach reaction, mainly because a strong delayed rectification is often found in molluscan neurons. On the other hand, the investigation of the mechanisms underlying the ACh depolarization is complicated by the possibility that the depolarizing ACh response may be mixed with a hyperpolarizing one. This chapter presents an investigation to gather information on the ionic mechanism of the depolarizing Ach response. For this purpose, the reversal potential of the depolarization was measured in the first series of experiments applying special techniques. In the second series of experiments, it was tested whether pharmacologically different types of responses were involved in the ACh-induced depolarizations of the investigated cells. In the first series of experiments, it was found that it is possible to polarize the neurons to positive membrane potentials for a period long enough to record the ACh response when the cells were isolated and thus, the membrane resistance was increased. In the second series of experiments, it was tested whether the ACh-induced depolarization of the investigated neurons was mixed with a hyperpolarizing ACh response.

INTRODUCTION

In some molluscan neurons acetylcholine /ACh/ elicits a depolarization. The ionic mechanisms underlying this depolarization are not completely understood as yet.

The analysis of the depolarizing ACh response is complicated mainly for two reasons. On the one hand, it is difficult to determine the actual reversal potential of this ACh reaction, mainly because a strong delayed rectification is often found in molluscan neurons. The reversal potentials reported in literature were obtained by extrapolation. They ranged from -20 to 0 mV /Blankenship et al. 1971, Chiarandini et al. 1967, Kerkut and Meech 1966/. From single channel analysis Ascher et al. /1978/ estimated a reversal of the depolarizing ACh response at +20 mV. On the other hand, the investigation of the mechanisms underlying the ACh depolarization is complicated by the possibility that the depolarizing ACh response may be mixed with a hyperpolarizing one. Chiarandini et al. /1967/ and Ascher et al. /1972/, for instance, described a monophasic ACh-induced depolarization which was due to the simultaneous activation of a depolarizing and a pharmacologically distinct hyperpolarizing ACh response, the latter being mediated by an increase in Cl− conductance.

The aim of the present investigation was to get further information on the ionic mechanism of the depolarizing ACh response. For this purpose the reversal potential of the depolarization was measured in a first series of experiments applying special techniques. In a second one it was tested whether pharmacologically different types of responses were involved in the ACh-induced depolarizations of the investigated cells.

METHODS

The experiments were performed on the identified neurons Bl and B3 of the lateral part of the buccal ganglion of Helix pomatia /Schulze et al. 1975/. Some non-identified cells located in the medial part of the buccal ganglion were also investigated. In contrast to the identified cells these neurons responded with a hyperpolarization to ACh. In some experiments on the identified cells the neurons were isolated at the axon by a small thread. This procedure prevented synaptic contributions to the reaction and led to a remarkable increase in membrane resistance.

The experimental chamber had a volume of 3 ml and was continuously perfused with saline at a rate of 15 ml/min. The control Ringer solution consisted of NaCl 130, KCl 4.5, CaCl2 9 and tris 5 mmol/l. The pH was adjusted to 7.4 with HCl. The experiments were carried out at bath temperatures between 18 and 22 °C. The microelectrodes were filled with 0.6 mol/l K2SO4. ACh was applied iontophoretically or by pressure ejection.

The recording and voltage clamp techniques applied have been described in detail elsewhere /Speckmann and Caspers 1978/.

RESULTS AND DISCUSSION

In a first series of experiments the reversal potential of the ACh-induced depolarization was measured in current and in voltage clamp experiments /Figs 1 and 2/. It was found to be possible to polarize the neurons to positive membrane potentials for a period long enough to record the ACh response when the cells were isolated and thus the membrane resistance was increased. Also some non-isolated cells could be polarized to positive membrane potentials provided that part of the current underlying the delayed rectification was inactivated by a preceding depolarization. The described procedures did usually not alter the cell functions. Applying the above-mentioned techniques the reversal potential as determined by interpolation was -2.6 ± 8.5 mV /mean ± SD, n = 56/.

In a second series of experiments it was tested whether the ACh-induced depolarization of the investigated neurons was mixed with a hyperpolarizing ACh response. This problem arose from the negative value of the reversal potential measured in the first series and from findings in literature which showed two pharmacologically different types of ACh—induced hyperpolarizations to be present in some molluscan neurons /Gerschenfeld 1973, Kehoe 1972a, Tauc and Gerschenfeld 1961/. These hyperpolarizing responses were reported to be due to changes in Cl− and K+ conductance, respectively.

The presence of a Cl− response was tested by ion substitution and blocking experiments. As shown in part B and F on Fig. 1 the reversal potential of the depolarizing ACh response was neither affected by substitution of propionate for Cl− nor by adding anthracene-9-COOH to the bath fluid. The latter substance was shown to suppress the Cl− conductance in rat muscle /Palade and Barchi 1977/. Since the described experimental procedures were found not to alter the equilibrium potential of the ACh depolarization, they were for comparison also applied in neurons located in the medial part of the buccal ganglion, which responded to ACh with a rapid hyperpolarization. The reversal potential of the ACh response of these cells usually ranged from -50 to -60 mV. It moved towards more positive membrane potentials when part of the Cl− in the bath solution was replaced by propionate, but it was neither affected by replacing all Na+ by tris nor by adding 0.1 mmol/l arecoline. According to Kehoe /1972b/ the last substance should totally block a K+-mediated hyperpolarizing ACh response in this concentration. Anthracene-9-COOH, however, in a concentration of 0.5 mmol/l totally and reversibly inhibited this ACh hyperpolarization. From these experiments the conclusion may be drawn that the ACh-induced depolarization in the identified cells is not mixed with a Cl− -mediated hyperpolarizing