Electrophysiological effects of a neurotoxin extracted from the skin of the Australian frog Pseudophryne coriacea

Camp. Biochem. Physiol. Vol. 103C, No. 2, pp. 291-297, 1992 0306~4492/92 $5.00 + 0.00 Printed in Great Britain 0 1992 Pergamon Press Ltd

ELECTROPHYSIOLOGICAL EFFECTS OF A NEUROTOXIN EXTRACTED FROM THE SKIN OF THE AUSTRALIAN

FROG PSEUDOPHRYNE CORIACEA

0. SACCHI, R. BARDONI, P. C. MAGHERINI* and 0. BELLUZZI

Istituto di Fisiologia Generale dell’Universita, Via L. Borsari 46, I-44100 Ferrara, Italy (Tel. 0532-207410); *Istituto di Fisiologia Umana dell’Universit& Via Campi 287, I-41 100 Modena,

Italy (Tel. 059-366486)

(Received 24 March 1992; accepted for publication 1 May 1992)

Abstract-l. The electrophysiological effects of a pumiliotoxin-B-like alkaloid extracted from the skin of the Australian frog Pseudophryine coriacea (PsC) have been studied in rat superior cervical ganglia at 37°C.

2. PsC (50mg/ml) elicits a broadening of the evoked compound action potential and, at rest, the appearance of spontaneous spike discharge at l&20 Hz. Action potentials presumably originate far away from the soma, which is invaded in a typical IS-SD sequence.

3. The toxin effect is not related to any direct action on the preganglionic fibers of the sympathetic trunk, and does not involve synaptic mechanisms.

4. Two-electrode voltage-clamp experiments showed that the main properties of the major voltage- dependent ionic currents are apparently unaffected by the toxin, while the cell input resistance is considerably reduced.

5. The data are consistent with the hypothesis that PsC elicits a cationic permeability increase generating a pacemaker current in a region close to the cell soma.

INTRODUCTION

The skin secretion of several Australian frogs of the genus Pseudophryne (Myobatrachidae) contains many compounds of biological interest, among others: biogenic amines, especially 5-hydroxy- tryptamine (5-HT), and active peptides of the caerulein, bombesin and tachykinin families (Roseghini et al., 1986). In the course of an extensive screening for active peptides in the skin of more than one hundred species of frogs from Australia and Papua New Guinea, Erspamer et al. (1984) have found that skin extracts of Ps. coriacea, Ps. corrobor- ree and Ps. nicholsi exhibited pharmacological activities similar to those of pumiliotoxin-B (PTX-B), one of the several alkaloids obtained from the skin secretions of the Panamanian frog Dendrobates pumilio (Daly and Myers, 1967; Daly et al., 1980).

Very little is known concerning the action of skin extracts of Ps. coriucea (PsC) on excitable mem- branes, while the analogous compound PTX-B has been extensively studied in different preparations, including skeletal muscle (Albuquerque et al., 1981; Rao et al., 1987), smooth muscle and cardiac tissue (Mensah-Dwumah and Daly, 1978), neuroblastoma cells and brain synaptoneurosomes (Daly et al., 1990). The effects described include: appearance of epileptiform-like discharges in guinea-pig hippocampal neurons (Sheridan et al., 1991), increase in neurotransmitter release (Rao et al., 1987), potentiation of muscle twitch upon direct stimulation of muscle fiber (Albuquerque et al., 1981; Mensah-Dwumah and Daly, 1978; Rao et al., 1987). These effects, however, are still not understood on the basis of a common

molecular mechanism: most of them are explained by a Na+ conductance increase (Daly et al., 1990), and recent reports strongly support this view (Schofield et al., 1988; Sheridan et al., 1991), but alternative or additional mechanisms are possibly involved, including activation of Ca 2+ channels (Albuquerque et al., 1981; Rao et al., 1987) and stimulation of phosphoinositide breakdown (Daly et al., 1987, 1988).

PsC extracts have been tested in smooth and skeletal muscle (Erspamer et al., 1985; Falconieri Erspamer et al., 1986; Re et al., 1991), and cardio- vascular system (Falconieri Erspamer et al., 1989). The spectrum of the toxin action proved to be rather broad, since increase in neurotransmitter release (Erspamer et al., 1985), effects on systemic blood pressure (Falconieri Erspamer et al., 1989) and potentiation of muscle twitch upon direct stimulation of the fiber (Falconieri Erspamer et al., 1986) have been described.

The present study was designed to obtain insights into the action of PsC on the rat sympathetic neuron. This preparation appears to be well suited for the study of neurotropic agents since the mechanisms underlying electrical activity have been more completely characterized here than in any other mammalian neuron (Belluzzi and Sacchi, 1991). The toxin was tested on the excitability of the principal postganglionic neurons, on the conduction mechanism in the preganglionic fibers and on synaptic transmission between pre- and postganglionic cells. PsC proved to be very effective in inducing spontaneous repetitive firing with a mechanism which does not involve either synaptic transmission, somatic voltage-dependent currents or nerve conduction.

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6

I 15ms

0.1 mV

Fig. 1. Compound action potentials (upper tracings) and resting activity (lower tracings, higher gain) recorded from the internal carotid nerve in an isolated rat superior cervical ganglion (SCG) at 37°C. The compound action potential was evoked by supramaximal stimulation of the sympathetic trunk at 0.5 Hz.

A, control; B, 10 min after bath addition of PsC (50 pg/ml); C, after 25 min wash-out.

MATERIALS AND METHODS

Preparation and solutions

Wistar rats (IO&l20 g body weight) were anesthetized with ethyl-urethane. The right superior cervical ganglion was dissected together with a tract of the cervical sympathetic trunk and a segment of the internal carotid nerve. The ganglia were superfused in a small chamber with continu- ously flowing Krebs solution at 37”C, gassed with a mixture of 95% 0, and 5% CO, and adjusted to pH 7.3. The composition of the solution was as follows (mM): NaCl, 136; KCl, 5.6; CaCl,, 2.2; MgCl,, 1.2; NaH,PO,, 1.2; NaHCO,, 14.3; glucose, 5.5.

Skin extracts

The skin extract used in this study was kindly supplied by Dr V. Erspamer. It was a semi-purified extract of Ps. coriacea skin (Erspamer et al., 1985) obtained by passage of the crude methanol extract of the dried skins through an alkaline alumina column and then through a Bioge14 column. High pressure liquid chromatography shows a single peak of activity in several test preparations thus demonstrating that the material is free of biologically active contaminants. Doses of the extract, expressed in pg, refer to the weight of dried skin from which the semi-purified extract was obtained. On the basis of spectral analysis, J. W. Daly (personal communication to Dr V. Erspamer) has calculated that the biologically active pumiliotoxin-B- like alkaloid complex occurs in the skin of Ps. coriacea at the concentration of about 10 mg per g dried skin. Because of some unexplained quantitative discrepancies between spectral analysis and bioassay, however, concentrations in this paper are continue to refer to dried skin and not to pure pumiliotoxin-B.

Extracellular recordings

The ganglion cells were activated orthodromically by stimulating the sympathetic trunk which had been gently sucked into a glass pipette, fitted with silver/silver chloride electrodes. Postsynaptic potentials were recorded by sucking the cut end of the internal carotid nerve into a similar fluid filled recording electrode. Supramaximal square wave pulses of 0.3 msec. duration were applied, via an isolation unit, to the preganglionic fibers at frequencies ranging from 1 to

lO/sec. The same procedure was employed when recording the compound action potential from the preganglionic nerve fibers. A 6-8 nun segment of the cervical sympathetic nerve was dissected and removed from the clavicle up to the point where it joins the ganglion; stimulation was then applied to the caudal end of the nerve trunk.

Intracellular recordings

Intracellular recordings were obtained from superficial principal neurons via conventional glass microelectrodes of 40-60 MD resistance, tilled with neutralized 4M-potassium acetate. When required, current was applied through the same recording electrode by using a bridge circuit.

Voltage-clamp experiments

The methods used are described in detail in previous studies from this laboratory (Belluxxi et al., 1985). Individ- ual sympathetic neurons, viewed with Nomarski optics at 500 x magnification, were impaled with two independent microelectrodes and voltage clamped within 2 hr after sur- gery. Microelectrodes tilled with 4M-potassium acetate and with resistances of 25-30MR were used. Presentation of voltage pulses and collection of data was via an M24 Olivetti personal computer. Current traces were digitized at 5-10 KHz using a custom-made 15-bit analog-todigital converter (Analog Devices DASl153) and stored on disc for analysis.

RESULTS

Extracellular recordings

In a first group of experiments the sympathetic trunk was supramaximally stimulated at O.S/sec while the postganglionic compound action potential was recorded with a suction pipette from the internal carotid nerve. The stimulating electrode was placed sufficiently close to the ganglion body to evoke a relatively synchronous action potential (Fig. 1A) despite the different conduction velocities of the unmyelinated fibers present in the sympathetic trunk.

PsC (50 pg/ml) clearly induces a modification of the evoked response (Fig. lB, upper trace). While the

293 Neurotoxin-evoked autorhythmicity

3 C

Fig. 2. Compound action potentials (upper tracings) and resting activity (lower tracings) recerded from the internal carotid nerve in an isolated rat 8CG. A, control in normal saline; B, control in 0.2 mM Ca2+;

C, 5 min after PsC (SO ag/ml) treatment in low-Ca2+ saline.

peak amplitude of the initial response was not significantly affected, in~c~tin~ that the total number of neurons activated by synaptic stimulation was presumably mchannged, an evident second peak of smaller amplitude appeared which broadened the shape of the compound potential, suggesting that a distinct alteration in the firing modalities had occurred. Fu~he~ore, in the absence of any preganglionic stimulation, ext~a~llular recordings from the ~stgan~ionic fibers revealed that the toxin evoked an intense low-voltage as~c~ono~ discharge (Fig. IB, lower trace), indicating that a main~n~ spontaneous activity was originated somewhere in the preparation. Both PsC effects developed in 7-15 min and proved to be fully reversible since they completely disappeared within 5 min of washout (Fig. IC). The apparent toxin threshold concentration for distinct electrophysiological effects was around 20 @g/ml, while the full action developed at 50 lug/ml without any additional effects up to 100 pg/ml. In order to test whether the toxin could act presynaptically by increasing the evoked release of ACh, the sympathetic trunk was stimulated in plain Krebs solution (Pig. 2A) or in a modified saline containing 0.2 mM Ca*+ (Fig. 2B) and the postganglionic activity was recorded from the internal carotid nerve.

As expected, the strong reduction in Ca”+ ions of the perfusing solution markedly i~ibit~ the release of ~uro~ansmitter so that the postganglionic compound action potential was virtually abolished. Wnder these conditions the bath perfusion of PsC (50 &ml) was unable to restore. the depressed synaptic transmission to any extent, although the appearance of the rich asynchronous sp~n~neous activity induced by PsC could still be observed at the postsynaptic level in the absence of any stimulation (Fig. 2C). The p~g~~io~c ~myelinat~ fibers of the sympathetic trunk could be a possible target for the toxin action. The previous observation that the spontaneous activity induced by PsC is still evident under conditions that severely depress the synapse does not rule out this possibility since it has been demon- strated that a group of preganglionic nerve fibers crosses the ganglion, giving rise to collateral twigs, and thereafter projects into the internal carotid nerve without any synaptic interruption (Sacchi and Rossi, 1981). Therefore, experiments have been performed in which stimulation and extracellular recording were obtained directly from the same sympathetic trunk (Fig. 3).

The PsC did not significantly modify the elcctri- tally evoked compound action potential (upper row), nor induced spontaneous activity in the preganglionic

Fig. 3. Compound action potentials (upper tracings) and resting activity (lower tracings) in a rat sympathetic trunk. A, control in normal saline; B, 10 min after bath addition of PsC (SO &ml); C, after

20min wash-out with normal saline. Stimulation and recording from the same nerve segment.

294 0. SACCHI et al.

I 2OmV

c 200 ms

L

20 mV

100 ms

c

Fig. 4. Intracellular recordings from rat sympathetic neurons during PsC (50 pg/ml) treatment. A, B, recordings of spontaneous activity from the same neuron (SD potentials in A, IS potentials in B). C, autorhythmic activity in a neuron and its block following membrane hyperpolarization. Note that the

synaptic activation of the cell is apparently normal.

nerves typically observed in the intact preparation. Taken together, these experiments strongly suggest that the apparent site of the toxin action is postsynaptic and rule out any direct effect on either the fiber conduction mechanism or synaptic transmission.

Intracellular recordings

Intracellular recordings from the soma of sympathetic neurons clearly revealed the induction of autorhythmic spike activity at 10-20 Hz (Fig. 4) following PsC (50 pg/ml) treatment.

It should be pointed out that under physiological conditions the mammalian sympathetic neurons never exhibit spontaneous spiking at any natural resting potential. Close examination of the traces (Fig. 4A, B) revealed that the action potentials originated far away from the soma, and invaded the soma antidromically in a typical IS-SD sequence. This was inferred by comparing the steep early rise of the action potential with the IS potentials recorded in isolation shortly afterwards in the same cell and in the same ionic medium (Fig. 4B). Probably because of a more favorable location of the micro-electrode rela- tive to the site of spike genesis, traces of pace-maker activity were occasionally recorded in some cells, as illustrated in Fig. 4C. The trace, in fact, displays here some instability of the membrane potential level during the interspike interval and a distinct prepotential preceding the rise of the IS-SD spike can be recognized. When the soma of the neuron is

hyperpolarized by the injection of outward current through the recording electrode, the IS spike potential is no longer followed by the SD action potential; as the membrane potential is further shifted towards negativity, the spontaneous IS potentials decrease in frequency and eventually disappear. The neuron, however, still maintains normal excitability and a conventional action potential can be evoked synaptically by supramaximal stimulation of the sympathetic trunk. It will be noted in the records shown in Fig. 4, and this was systematically observed during the toxin action, the complete absence of spontaneous synaptic potentials (mEPSPs), which on the other hand would be easily discernible in the tracings because of the high input impedance of the sympathetic neuron. In some neurons the autorhythmicity does not arise spontaneously following the PsC treatment, but has to be elicited by short trains of action potentials evoked synaptically or by direct cell stimulation.

This effect is illustrated in Fig. SA, where a series of eight supramaximal shocks delivered to the sympathetic trunk is sufficient to trigger a repetitive discharge in a previously silent neuron. Isolated spontaneous action potentials arise within the interspike intervals during the synaptically driven activity; thereafter a self-sustained repetitive discharge is initiated in the cell. It should be noted that the start of spontaneous firing is not associated with any shift in membrane potential level detectable by the microelectrode. Antidromic stimulation was similarly effective

Neurotoxin-evoked autorhythmicity 295

20 mV L- L-

20mV

0.4 s IOOms

Fig. 5. Spontaneous firing in sympathetic neurons in the presence of PsC (50 pg/ml). A, autorhythmic activity is initiated by a train of eight supramaximal stimuli applied to the sympathetic trunk. B, the spontaneous discharge of IS potentials is interrupted by a synaptically driven action potential. Horizontal

bars show the constancy of the firing period before and after the externally evoked spike.

in making “idle” neurons autorhythmic. This observation confirms the negligible involvement of presynaptic mechanisms in sustaining the toxin action. In Fig. 5B a tentative explanation for the main toxin effect illustrated in Fig. 1B is presented. When the timing of a steady spontaneously repetitive firing is perturbed by an experimentally evoked action potential, the spontaneous spiking resumes at the very same frequency starting from the artificially induced event. In other words, the supramaximal synaptic stimulation shown in the figure elicits a synchronous activation of the whole neuronal pool eventually resetting a spontaneous rhythm which, randomly timed in the different neurons, now becomes for the first few milliseconds triggered by the externally driven action potential.

Voltage-clamp experiments

Ionic currents in the sympathetic neuron have been measured under two electrode voltage-clamp conditions in plain Krebs solution and during exposure to PsC (SOpg/ml) (Fig. 6).

The recordings reveal that the toxin has no drastic effect on the macrocurrents evoked in the neuron by depolarization. The only apparent result in this ex- periment is an increase in the I, peak amplitude which, however, is probably related to the improve- ment of the cell recording conditions frequently observed after the double impalement rather than to a specific toxin action. Since the I, is mainly related to the repolarization phase of the action potential (Belluzzi and Sacchi, 1985, 1991) this effect alone

PSC

Fig. 6. Ionic currents measured in a sympathetic neuron during voltage-clamp pulses in normal saline (control) and after 27 min treatment with PsC (50 pg/ml). A, the neuron was held at - 50 mV, conditioned for 1 set to - 100 mV to remove I, inactivation and tested at - 10 mV; B, same protocol as in A but

without conditioning.

296 0. SACCHI et al.

cannot account for the excitability modifications previously described. The more relevant currents, I,, and the delayed summated Ix, and IKca (and therefore presumably the underlying I&, are apparently unaffected by the PsC treatment. A pertinent observation is that the leakage appears to increase under the PsC action: the membrane resistance dropped from 11.6 MQ to 8.6 MS;1 in the cell shown in Fig. 6. This would indicate an increase in membrane permeability, occurring most likely in restricted and well defined domains of the neuron, capable of sustaining the maintained depolarization generating the autorhythmic activity. The development of IS potentials systematically preceding the SD cell activation (Figs 4 and 5A, B) would be in line with a remote increase in neuronal excitability.

DISCUSSiON

The experiments reported in this paper show that the skin extracts of Ps. coriacea induce spontaneous activity in the soma of rat sympathetic neurons. The occurrence of repetitive firing implies a drastic modi~cation of the cell excitability mechanisms since under physiological conditions the mammalian sympathetic neuron not only never exhibits spontaneous spiking, but also appears quite unable to fire repeti- tively. In fact, only a few dumped spikes can usually be evoked even during sustained membrane depolarization. The effect of the toxin is not due to a direct action on the unmyelinated fibers of the sympathetic trunk, and there is convincing evidence that PsC action does not involve synaptic mechanisms: the alkaloid did not increase the spontaneous EPSP frequency and was unable to restore the depression in synaptic transmission induced by low Ca2+. These negative findings are complemented by the evidence that the PsC site of action, while relatively proximal, is not in the cell soma, The kinetic properties of the major somatic voltage-dependent ionic currents are actually unaffected by the toxin, while the passive membrane properties measured at the somatic level are effectively modified; the cell input resistance, in fact, is considerably reduced indicating an increase in membrane permeability. This could be a pertinent observation since pacemaker currents belong to two broad types, depending on the way the final membrane depolarization is achieved: one group includes depolarizing inward currents, the other steady hyper- polarizing currents. In the latter case, depolarization is obtained by reducing the outward current com- ponent and this effect is thus associated with an increase in the overall membrane impedance (Zucker, 1988). The experimental observation that the membrane resistance is reduced suggests that the present pacemaker current belongs to the first type, therefore ruling out the possibility of a toxin modulation of a pure K+ conductance.

The appearance of repetitive action potential discharge during PTX-B action in response even to a single stimulus, without effects on resting membrane potential, has been observed also in amphibian sympathetic neurons (Schofield, 1988). There are, however, some aspects that can probably be explained on the basis of differences between preparations or the toxin type actually used: (i) PTX-B has also an

additional presynaptic effect on amphibian neurons, while neuronal somata exhibit repetitive spiking in response to a single stimulus even when isolated from the ganglion by enzymatic dispersion; (ii) in mammalian neurons PsC typically induces spontaneous firing in the absence of any triggering event; (iii) we failed to observe drastic changes in the Na current, while there is indirect evidence for the involvement of this channel in the amphibian neuron.

There is no unitary view to explain the effects of PTX-B or of PsC alkaloids in the different preparations. Rao et al. (1987) proposed that they could hinder the sodium inactivation process, thereby increasing Na + flux; Gusovsky et al. (1988) point out that PTX-B enhances the Na+ influx by interacting with a unique modulatory site of the voltage- dependent Na+ channel, which interacts with some scorpion toxin binding sites but is entirely different from the site at which batrachotoxin, veratridine, aconitine, tetrodotoxin, and saxitoxin bind. The hypothesis of an involvement of the Nat current receives support from a recent paper of Sheridan et al. (1991) showing that the PTX-B induces a 12 mV shift toward negative potentials of both the activation and inactivation Na + channel curves. Another possible mechanism for the PTX-B and PsC action is the control of Ca*+ movements: Albuquerque et al. (1981) suggested that PTX-B facilitates the release of Ca2+ from intracellular stores within the sarcoplasmic reticulum, and several reports indicating that these alkaloids stimulate the phosphoinositide breakdown would strengthen this view (Gusovsky et al., 1986; Daly et al., 1987, 1988). Falconieri Erspamer et al. (1986) provide further evidence suggesting that the voltage-dependent Ca*+ channels could also be directly regulated by PsC. We have indirect evidence that the pacemaker current induced by the toxin in the sympathetic neuron is sustained by an inward current; this is in line with several of the proposed mechanisms of action of the skin extracts.

The present experiments do not allow a precise understanding of the process initiating the spontaneous activity since no specific changes in the voltage-dependent currents at the somatic level have been observed. The voltage-clamp experiments failed to evidence any definite pacemaker current in the soma, while a few intracellular recordings, probably as a result of a favorable positioning of the microelectrode, occasionally revealed slowly depolarizing interspike potentials (Fig. 4C). These data, as well as the observation that IS potentials regularly precede the somatic spike, would suggest that the membrane modifications induced by PsC do not primarily occur at the soma level but rather in a restricted and well defined domain of the neuron, possibly in the functional axon hillock or the large dendrites from which the axon stems. It is interesting to observe that in other systems this is indeed the region from which the pacemaker activity is initiated (Adams and Benson, 1985). The repetitive end-plate potentials discharge detected at the mouse neuromus- cular junction in the presence of PsC could be ascribed to a similar mechanism occurring in the presynaptic nerves, but revealed in the muscle (Re et al., 1991).

Neurotoxin-evoked autorhythmicity 297

Acknowledgements-We are grateful to V. Erspamer (1st University of Rome, Italy) for encouragement and helpful comments on the manuscript and for continued supply of semipurified PsC extract. The work was supported by funds from MURST, Italy.

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Electrophysiological effects of a neurotoxin extracted from the skin of the Australian frog Pseudophryne coriacea