ABSTRACT
Picrotoxin, eserin, butyrylcholine and acetylcholine bring about increase of impulse discharges on the T large fibre in the cord. Picrotoxin gives conspicuous increase of impulse discharges in the response of the T large fibres to sound, while the excitatory effects of the latter three agents are not so conspicuous.
Such effects can be explained on the assumption that picrotoxin inhibits the inhibitory synapses, so that the T large fibre is fully activated by both tympanic nerves, while the latter three agents activate both the excitatory and inhibitory fibres.
GABA, γ-aminobutyrylcholine and D-tubocurarine act reversibly as inhibitors of the activity of the T large fibre. The response evoked in the T large fibre may be suppressed by the activities of the inhibitory interneurons activated by the tympanic nerve fibres.
After the application of picrotoxin solution to the prothoracic ganglion the threshold of the T large fibre near to a sound source rises while that of the opposite side falls. The inhibitory effect seems to be eliminated by the drug action and the T large fibre is activated by both the ipsilateral and the contralateral excitatory fibres.
The increased information about a source of sound which arises from the central inhibitory interaction is disturbed by the application of picrotoxin.
The conclusion that the T large fibre has excitatory synapses with the tympanic nerves, and inhibitory synapses with the inhibitory interneurons activated by the tympanic neurons, has been confirmed pharmacologically.
The inhibitory interneurons are activated not only by the natural activity of the tympanic nerve, but also by activity elicited electrically with square pulses.
INTRODUCTION
In the nerve cord of Gampsocleis buergeri (Tettigoniidae), the auditory T large fibre lying between the brain and the metathoracic ganglion was found electrophysio-logically by the present authors to receive not only an excitatory effect from the ipsilateral tympanic nerve at the prothoracic ganglion, but also inhibitory and weak excitatory effects from the contralateral one (Suga & Katsuki, 1961).
It was thus expected that the inhibitory effect was produced by inhibitory interneurons activated by the contralateral tympanic nerve. Recently the synaptic mechanism in lower animals has been revealed to some extent, and it may be very important to know in the present case what happens in these synapses. The pharmacological experiments with chemicals having specific actions on inhibitory or excitatory synapses are thought to be very profitable for the analysis of the central interaction between the tympanic nerves and the auditory T large fibres in the cord. The present paper will deal with the results which further elucidate the mechanism of interaction so far studied only by the electrophysiological technique.
The pharmacological agents used in the present work were picrotoxin, eserin, butyrylcholine, acetylcholine, GABA, γ-aminobutyrylcholine, D-tubocurarine, and strychnine. Those chemicals are known to act on synapses as follows. Picrotoxin is an excitant of excitatory synapses in the cat brain (Purpura, Girado & Grundfest, 1957). However, in crustacean stretch-receptors (Elliott & Florey, 1956; Bazemore, Elliott & Florey, 1956 ; Edwards & Kuffler, 1957), muscles (Van der Kloot, Robbins & Cooke, 1958; Grundfest, Reuben & Rickles, 1959; Reuben, Bergmann & Grundfest, 1959), and the central nervous system (Hichar, 1960), it is said to be an inhibitor of inhibitory synapses. Eserin suppresses the resolving action of cholinesterase on acetylcholine and enhances the acetylcholine response of a crayfish stretch-receptor (Wiersma, Furshpan & Florey, 1953). Butyrylcholine has a strong excitatory effect on a crayfish stretch-receptor and produces high-frequency discharge of impulses as acetylcholine does (Hagiwara, Kusano & Saito, 1960). GABA operates as an inhibitor on excitatory synapses in the cat brain (Purpura et al. 1957). However, in crustacean stretchreceptors (Bazemore, Elliott & Florey, 1956, 1957; Edwards & Kuffler, 1957, 1959; McLennan, 1957; Hagiwara et al. 1960) and muscle (McLennan, 1957; Grundfest et al. 1959; Reuben et al. 1959), GABA is an excitor of inhibitory synapses, γ-Aminobutyrylcholine has an effect on synapses similar to that of GABA in crayfish stretch-receptors (Hagiwara et al. 1960). In the cat brain D-tubocurarine an inactivator of excitatory and inhibitory synapses and brings about the inhibition of the excitation (Purpura & Grundfest, 1956; Grundfest, 1958). Metrazol and strychnine are excitants in the central nervous system of Mammalia. However, the former excites the excitatory synapses in the same way as picrotoxin does, while the latter inhibits selectively the inhibitory synapses (Purpura et al. 1957). In a crayfish stretch-receptor, metrazol has an effect similar to that of picrotoxin, but only in much higher concentrations. Strychnine has, however, no effect on it (Bazemore et al. 1956; Elliott & Florey, 1956). Acetylcholine, and D-tubocurarine also have no effect on lobster neuromuscular synapses (Grundfest et al. 1959).
It seems very interesting that one and the same chemical acts on the synapses of Mammalia and Crustacea in entirely different ways, as described above. From a viewpoint of comparative neuropharmacology it is also desirable to know what events are brought about on the insect synapses which have so far not been studied.
MATERIAL AND METHOD
The animal used was Gampsocleis buergeri. The insect was pinned on its back on a cork board and the ventral exoskeleton covering the thoracic nerve cord was then removed. The tracheae distributing along the nerve cord were also separated from the latter. The central nervous system and the peripheral nerves were cut so that there remained only the simple nervous system consisting of the pair of tympanic nerves, the prothoracic ganglion, and both the suboesophageal-prothoracic connectives. The operated insect was put in a sound-proofed room air-conditioned at about 24° C. and the physiological saline solution* was applied to the nerve in order to maintain the preparation at a good condition. Each of the suboesophageal-prothoracic connectives was hooked up in the air by a recording electrode of 200 μ. silver wire mounted on a micromanipulator. An indifferent electrode was provided by similar silver wire resting on wet cotton in the opened abdominal segments. In most cases the impulse discharges of the right and left auditory T large fibres (Suga & Katsuki, 1961) were displayed on the first and the second beams of an oscilloscope with three channels. A wave form of a sound stimulus or time signal was shown on the third beam alternatively according to need.
Several pharmacological agents in various concentrations (w/v of the physiological saline solution) were applied to the prothoracic ganglion. The change in the number of impulses in the T large fibres (post-synaptic fibres) was successively recorded on a running film after the application. The stimulating and the recording equipments used in the present work were the same as those described in the previous paper (Katsuki & Suga, 1960).
RESULTS
(1) Excitors
It was found that four drugs–picrotoxin, eserin, butyrylcholine and acetylcholine –brought about an increase of impulse discharges in the T large fibres. The effect of each drug, confirmed by several trials, will be described later. The T large fibre situated nearer to the sound source always discharged with more impulses than that of the opposite side. The difference in the intensity of a sound stimulating both tympanic organs was obviously recognized as the difference in the threshold and also in the number of impulses between a pair of T large fibres (Fig. 1 a).
(a) Picrotoxin
When 0·1% picrotoxin solution was applied to the prothoracic ganglion, impulse discharges of a pair of the T large fibres began to increase after about 6 min. from the application (b), and after 10 min. a train of impulses was observed not only on the left T large fibre, but also on the right as shown in Fig. 1 d. After 12 min. (e) after-discharges began to appear. The long-lasting train of impulses was suppressed by a tone burst following after about 18 min. (f). Such a suppression could be repeated for a few minutes after its appearance. In this concentration, the after-discharge became gradually shorter thereafter and the suppression became faint. After 26 min. the response pattern became stable (g). (The time signals are 10 msec.)
If one of the tympanic nerves was cut, many impulses of the ipsilateral T large fibre disappeared and a few impulses transmitted from the contralateral tympanic nerve always remained (h). The first impulse in the contralaterally evoked response delayed by about 8·0 msec, compared with that of the ipsilaterally evoked one (i). The time delay was 3·3 msec, in the shortest case. This result confirms the suggestion that the T large fibre is activated by some of the contralateral tympanic nerve fibres (Suga & Katsuki, 1961). When the material was washed with the physiological saline solution, the increased discharges began to decrease but the recovery was so slow that the number of impulses did not decrease to the original level even after 20 min. When 1 % picrotoxin solution was applied, many spontaneous discharges appeared in the T large fibres after 1 or 2 min. and then disappeared. The response to the tone burst also failed. But spontaneous discharges and the response reappeared after 5 or 6 min. Such a reappearance was always repeated twice or more. Finally a response with many impulses was found in some cases but not in other cases. With 0·01 % picrotoxin solution, the increase of impulse discharges was smaller compared with the case of 0·1 % picrotoxin.
(b) Eserin
Eserin applied to the prothoracic ganglion caused a remarkable increase of impulses in the T large fibre. The result shown in Fig. 2 A was obtained by the application of coco i % eserin solution to the prothoracic ganglion after increase of impulses in the right T large fibre by cutting the left tympanic nerve (a and b in Fig. 2 A). Impulse discharges in the T large fibre contralateral to the cut tympanic nerve began to increase in response to a sound stimulus in about 4 min. after the application and then the ipsilateral T large fibre was also brought into excitation with one spike, which was delayed by 3–5 msec, as compared with the first impulse of the contralateral one (c). The number of impulses became almost constant after about 17 min. Two or three impulses evoked contralaterally were found on the T large fibre on the same side as the cut tympanic nerve (d). The time signal at the bottom of d represents 10 msec. Among the contralaterally evoked impulses the first impulse was delayed by 2–3 msec, as compared with the ipsilaterally evoked one in the shortest case (e). (The time signal b i msec.) When eserin solution more highly concentrated than 0·0001 %, for example co 1 % eserin solution, was applied to the ganglion, after about 2 min. the impulses in the tonal response increased and also many spontaneous discharges were observed in the T large fibre. Both the T large fibres fired with trains of impulses after 3 min. and then ceased to fire completely. In contrast to picrotoxin, eserin did not cause any remarkable increase of impulses in the tonal response or any after-discharge.
This result may be well accounted for by the assumption that eserin is an anticholinesterase in the insect and that the activity of inhibitory neurons is also prolonged, so that the responses of the T large fibre are not augumented so prominently, whereas picrotoxin inhibits the inhibitory synapses between the T large fibre and inhibitory fibres, so that the T large fibre is fully activated by many tympanic neurons.
(c) Butyrylcholine
1% butyrylcholine solution was applied to the prothoracic ganglion connecting with both the tympanic nerves (a of column B in Fig. 2). When the number of impulses in the tonal response of the T large fibre was not very small, a conspicuous increase was observed after the application as shown in Fig. 2B. The impulse discharges in the response began to increase within 1 min. after the application (b), and spontaneous discharges were observed thereafter (c and d). The response became almost constant in 5 min. (e). No impulse evoked contralaterally by the activities of the tympanic nerves was found after cutting one of them. In many cases, however, when the number of impulses in a tonal response was very small, such an increase was not found after the application of 1 % butyrylcholine solution to the prothoracic ganglion with both tympanic nerves intact. When the solution was applied to the prothoracic ganglion after cutting one of the tympanic nerves, impulses in a tonal response in the contralateral T large fibre always increased, but the T large fibre on the cut side did not always fire. Such was not the case following the application of picrotoxin solution.
Those results can be explained as follows. The effect of butyrylcholine is influenced by the activity of the inhibitory synapse, namely, when the inhibitory effect from the opposite side is strong, the number of impulses in the T large fibre becomes less and the excitatory effect of butyrylcholine does not appear distinctly except when the solution is applied after cutting the contralateral tympanic nerve, because the inhibitory effect on the T large fibre from the tympanic nerve is eliminated by cutting it.
(d) Acetylcholine
The effect of acetylcholine was also excitatory even though it seemed to be slightly less effective than butyrylcholine. When 1 % acetylcholine solution was applied to the prothoracic ganglion after cutting one of the tympanic nerves, the number of impulse discharges in the contralateral T large fibre began to increase within 0*5 min. after the application. In 15 min. the contralateral impulses further increased and the ipsilateral T large fibre discharged an impulse. Several spontaneous discharges also appeared. But the number of impulses in the tonal response of the large fibre did not increase as with picrotoxin. The time delay between the impulses evoked contralaterally and ipsilaterally was 3·2 msec, in the shortest case. Washing with the physiological saline solution made the number of impulses decrease and restored the original response after 2 or 3 min.
(2) Inhibitors
Four drugs, GABA, γ-aminobutyrylcholine, D-tubocurarine and strychnine acted as inhibitors of electrical activity in the T large fibre.
(a) GABA
1 % GABA was applied to the prothoracic ganglion (Fig. 2 C, a). Impulse discharges in the T large fibres began to decrease after 1·5 min., and after 5 min. they disappeared completely (c). Washing the prothoracic ganglion with the physiological saline solution brought back the tonal response of the T large fibre. In many cases, within 1 min. after washing, the T large fibre discharged many impulses even after the cessation of a sound stimulus (d). Such after-discharges were often suppressed by tone bursts (e). But a train of after-discharges became shorter and shorter afterwards (f) and the original responses finally returned on the T large fibres. Reversibility was also complete when the response of the T large fibre to the tone burst was suppressed by the application of 10% GABA solution, 0·1 % GABA solution had almost no effect on the response of the T large fibre. Even if the number of impulses in a response slightly decreased at first, it soon returned to the original level.
(b) γ-Aminobutyrylcholine
1 % γ-aminobutyrylcholine suppressed the electrical activity of the T large fibre in the same way as GABA did and the reversibility of the effect was also complete (Fig. 2D), γ-Aminobutyrylcholine solution was applied after the increase of the number of impulses on the right T large fibre consequent upon cutting the left tympanic nerve (a and b). The enhanced response by elimination of the inhibitory effect on the T large fibre from the left tympanic nerve consisted of only five impulses within 0γ5 min. after the application (c). After 5 min. the response disappeared entirely (d). However, it returned after 3 min. washing with the physiological saline solution (e and f). The reversibility was the same even if 10% γ-aminobutyrylcholine solution was applied to the prothoracic ganglion.
(c) d-Tubocurarine chloride pentahydrate
If the prothoracic ganglion was treated with 0·03 % D-tubocurarine solution, impulse discharges in the T large fibre began to decrease within 3 min. after the application and all the tonal responses disappeared thereafter. 0·03% D-tubocurarine solution suppressed the increased discharges evoked by the application of 1 % acetylcholine solution, but did not cause them to disappear. But D-tubocurarine solution in higher concentration (0·3 %) caused them to disappear completely. The vanished impulses reappeared on the T large fibre after washing of the ganglion with the physiological saline solution.
(d) Strychnine
When the prothoracic ganglion was treated with 1 % strychnine solution, impulse discharges in the T large fibre decreased after about 1 min. and eventually disappeared, as after treatment with metrazol. After washing the prothoracic ganglion with the physiological saline solution the response to sound stimulation returned in the T large fibre within 9 min. after washing.
(3) Response frequency range and number of impulses after the treatment with picrotoxin
After the application of picrotoxin solution to the prothoracic ganglion the threshold of the tonal response of the T large fibre became higher and the latency became longer in spite of the remarkable increase of impulse discharges.
In the normal condition a large difference in the threshold between the two T large fibres was found for various sounds in the frequency range of the response as reported previously (Suga & Katsuki, 1961). However, it was noted that the difference became small after the application of picrotoxin solution to the prothoracic ganglion. One of the experiments is shown in Fig. 3 as an example. The response ranges of the T large fibres were measured before and after the application of 1 % picrotoxin solution. The threshold of the left T large fibre (near to a sound source) became higher than the original one , but that of the right T large fibre (far from the source) became lower than the original one . As that result, the difference in the threshold between the two fibres became small.
On the other hand, the increased information carried by the two T large fibres by reason of the central inhibitory interaction (Suga & Katsuki, 1961) was disturbed by the suppression of interaction following the application of picrotoxin solution. That is, the ratio between the number of impulses sent through one of the T large fibres and the number sent through the other became small (compare a and g in Fig. 1) and the number of impulses in both changed at the same rate with change of sound intensity. No increase in the number of impulses was found after cutting one of the tympanic nerves. These facts will be interpreted on the assumption that the T large fibres are fully activated by the ipsilateral and contralateral tympanic nerves after the suppression of the activity of inhibitory synapses by picrotoxin.
(4) Electrical stimulation
The left tympanic nerve was cut and its proximal end was hooked up in the air with a pair of silver wire electrodes. Long or short electric square pulses were given to the cut end of the tympanic nerve through these electrodes during application of a sound stimulus. The sound stimulus excited the right tympanic organ and the information was sent to the brain as a train of impulses through the right T large fibre only. The short square pulses (of a few milliseconds duration) applied to the left tympanic nerve gave rise to silent periods in a train of impulses on the right T large fibre, while the left T large fibre fired with one impulse for each square pulse. A negative square pulse of 100 msec, duration was applied to the left tympanic nerve at the same time as the sound stimulus. Almost all impulses in the right T large fibre were suppressed. On the other hand, the left T large fibre discharged an impulse at the onset of the square pulse and never discharged repetitively. However, if the right tympanic nerve was cut, the left T large fibre discharged with four or five impulses at the onset of the negative square pulse.
A positive square pulse of 100 msec, duration had no effect on the response to the tone burst on the right T large fibre even if it was applied to the left tympanic nerve at the same time as the tone burst. The left T large fibre discharged one impulse only, at the end of the square pulse. These experimental results described above further confirm the existence of the proposed innervation modus of the T large fibre by the tympanic nerve.
DISCUSSION
(1) Functional connexion between the tympanic nerve and the T large fibre
The effects of the excitatory agents are explained as follows. Picrotoxin, known as an inhibitor of the inhibitory synapses in a crustacean stretch-receptor, always brought about a remarkable increase in the number of impulses in the T large fibres. This fact accounted for the similar suppression of the inhibitory synapses between the auditory T large fibres and inhibitory interneurons. In contrast to picrotoxin, the agents eserin, butyrylcholine, and acetylcholine produced an excitatory effect on the synapses between the tympanic nerve and the T large fibre, and between the tympanic nerve and the inhibitory interneuron. The results showed that the change in the number of impulses after application of the pharmacological agents seemed to be always dominated by the activity of the inhibitory fibre in each specimen, that is, the increase of impulses was not so clear when the activity of the inhibitory fibre was high, but it was remarkable when it was low. However, when the agent was applied after cutting one of the tympanic nerves, i.e. after the elimination of the inhibitory action on the T large fibre from the opposite side, the T large fibre on the side opposite to the cut always discharged many more impulses, but the T large fibre on the same side did not.
Further, GABA (known as an excitor of the inhibitory synapses in Crustacea) strongly suppressed impulse discharges in the T large fibre, which may be due to the activation of the inhibitory synapse, though it is a fact that in the lobster GABA blocks the thoracic receptor which has no inhibitory synapses (Kuffler & Edwards, 1958). γ-Aminobutyrylcholine also extinguished impulse discharges on the T large fibres as GABA did.
Thus the pharmacological experiments have afforded further evidence of the central interaction between the tympanic nerves and the T large fibre which was originally discovered by the electrophysiological study of the central auditory mechanism, and have confirmed that the T large fibre has excitatory synapses with the pair of tympanic nerves and inhibitory synapses with the inhibitory interneurons activated by the tympanic neurons.
An anatomical study of these nerve nets has been made, but no definite configuration has so far been discerned. The time delay between the first impulse contralaterally evoked in the T large fibre and that ipsilaterally evoked must be due to the longer path from the contralateral tympanic nerve fibres to the T large fibre. The shortest time delay was found after the application of eserin solution to the prothoracic ganglion, being 2’3 msec. The inhibitory effect on the T large fibre from the contralateral tympanic nerve fibres appeared with the time delay of 4·6 msec. (Suga & Katsuki, 1961). Fig. 4 shows schematically the functional connexions between the tympanic nerves and two T large fibres.
(2) Synaptic transmission
From the fact that the pharmacological agents described above, known to act on the synapses of Mammalia and Crustacea, gave rise to the change in impulse discharges of the T large fibre, it may be concluded that synaptic transmission between the tympanic neurons and the T large fibre and between both neurons and the inhibitory interneurons is carried out through some chemical processes. The concentrations of the agents used in the present work were higher than those reported by other authors as applied to such isolated materials. If the agents were applied after opening the sheath of the prothoracic ganglion, events similar to those described above were caused by the solutions of lower (by almost 10%) concentration in all cases. As it was found that the inhibitory effect on the T large fibre was weakened by peeling off the sheath of the prothoracic ganglion and impulse discharges on the T large fibre increased, no attempt was made to discover the minimum effective concentration of the test solution, because the condition of the material was unstable.
It is well known that picrotoxin and GABA are competitive antagonists at the excitatory synapses of the cat brain (Purpura et al. 1957) and at the inhibitory synapses of crustacean stretch-receptors (Elliott & Florey, 1956; Edwards & Kuffler, 1957) and muscle (Grundfest et al. 1959; Reuben et al. 1959), so it is highly probable that these agents act antagonistically on the synapses in the prothoracic ganglion as on those of Crustacea and that an antagonism might be also found between acetylcholine and D-tubocurarine. Acetylcholine and D-tubocurarine which can Cause certain effects on the cat brain have been revealed to be without effect on lobster neuromuscular synapses, although acetylcholine acted as an excitor on the insect synapses. Both strychnine and metrazol suppress the activity of the T large fibre. However, the response of the T large fibre to a sound stimulus returned after washing the prothoracic ganglion with the physiological saline solution. Therefore it seems that these two drugs act inhibitorily on the insect synapse. These results suggest that synaptic transmission in the prothoracic ganglion may be carried out by a mechanism different from that in mammalian synapse. It should be possible to reveal more details of the mechanism by intracellular potential recording, but this has been unsuccessful as yet.
ACKNOWLEDGEMENTS
This work was supported by grants from the Rockefeller Foundation (GA. MNS. 59115) and also from the Ministry of Education of Japan.
REFERENCES
NaCI, 12·16 g. ; KCl,0·23 g. ; CaCl2(2H2O), 0·27 g. ; Na2HPO4(2H2O), 1·07 g. ; NaH2PO4 (2H2O), 0·05 g. ; H2O, 1000 g.