The effects of drugs on the neurones of the snail Helix aspersa

Comp. Biochem. Physiol., 1961, Vol. 3, pp. 143 to 160. Pergamon Press Ltd., London. Printed in Great Britain

THE EFFECTS OF DRUGS ON THE NEURONES THE SNAIL HELIX ASPERSA G. A. K E R K U T

OF

and R. J. W A L K E R

D e p a r t m e n t of Physiology and Biochemistry, T h e U n i v e r s i t y of S o u t h a m p t o n (Received 9 February 1961)

A b s t r a c t - - ( 1 ) The effect of adding various chemicals to the isolated brain of the snail Helix aspersa is described. The reactions were followed by measuring the effect of the chemicals on the resting potential and the spontaneous action potentials of the nerve cells. (2) All the following chemicals were effective at dilutions between 10 -~ and 10 11 g/ml. The sign indicates their overall acceleratory ( + ) or inhibitory ( - ) effect on the spontaneous activity. Acetylcholine ( + ) Glutamic acid ( + ) Adrenaline ( + ) Histamine ( + ) Cocarboxylase ( + ) Nor-adrenaline ( 4- ) Dimethyl-amino-ethanol ( - ) 5-HT ( + ) Dopamine ( - ) Phenylalanine ( - ) T-Aminobutyric acid ( + ) Thiamine hydrochloride ( + ) (3) D°pamine and phenylalanine caused a hyperp°larizati°n °f the resting p°tential" (4) Phenylalanine, glutamic acid and cocarboxylase could protect the cell against the action of high concentrations of acetylcholine. (5) It is thought that most of the above chemicals may occur naturally in the snail and that acetylcholine and dopamine are the most likely transmitter substances. (6) Most of the drugs had a dual action; inhibiting some cells and accelerating others. It is suggested that there is a marked chemical heterogeneity in the CNS with cells reacting specifically but differently to a given chemical. INTRODUCTION

IN R~CErrTYEA~Sinterest has been aroused in the study of the pharmacology of the invertebrates. Various invertebrate preparations such as the leech muscle (Minz, •932; Feldberg & Krayer, 1933) and the heart of Venus mercenaria (Welsh & Taub, 1948) have proved of especial use in drug assay. The actions of various drugs have been determined for selected invertebrate preparations: i.e. anemone sphincter muscle (Ross, 1960a, b); cockroach nerve cord (Twarog & Roeder, 1957); crayfish nerve cord (Wiersma & Schallek, 1948; Hichar, 1960@ The molluscan nervous system has been to some extent neglected and the present paper will describe a series of experiments on the effect of various naturally occurring substances on the resting potential and the spontaneous activity of neurones from t h e c o m m o n g a r d e n snail H e l i x aspersa. The compounds used were either naturally occurring substances or precursors o f s u c h s u b s t a n c e s , a n d in t h e m a i n t h e y w e r e c h o s e n so as to a l l o w s o m e i n s i g h t i n t o t h e n a t u r e a n d a c t i o n o f t h e c h e m i c a l t r a n s m i t t e r s at t h e s y n a p t i c j u n c t i o n . II

143

144

G.A.

KERKUT AND R. J. WALKER METHODS

T h e experiments were carried out on brains from locally collected snails

Helix aspersa. T h e brains were dissected and placed on a glass slide. T h e connective tissue surrounding the brain was carefully removed and the brain then placed on a black glass slide and then put in a small (10 ml) bath containing Cardot's ringer (1921). 5-30 m e g o h m glass microelectrodes filled with 3M KC1 were inserted into the nerve cells of the parietal and visceral ganglia. T h e electrodes led to a cathode follower and thence to a Tektronix 502 oscilloscope. T h e potentials were monitored on a twin channel tape recorder and simultaneouslv led to an oscilloscope camera. T h e tape records were replayed either onto an Ediswan Pen recorder or the camera.

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FIo. 1. T h e resting potential in snail nerve cells. T h e f r e q u e n c y of obtaining the various potentials is plotted. T h e majority of t h e n e u r o n e s h a d a potential b e t w e e n 20 a n d 40 m V .

T h e test solutions were usually made up and stored in a refrigerator. T h e y were diluted to the required range just prior to the experiment. All concentrations were added in series, the most dilute being used first of all. T h e final volume of the bath led to a tenfold dilution of the added material. All the drug concentrations are expressed in terms of grams per millilitre. T h u s ]0 -6 means that 1 ml of the final solution would contain one millionth of a

T H E EFFECTS OF DRUGS O N T H E N E U R O N E S OF T H E S N A I L

145

gram of material. The only liquid used, DMAE, was made up v/v instead of w/v. Dilutions greater than 10_6 are considered as being physiological whilst those less than 10-6 are considered as being pharmacological levels. Many of the winter snails that were used were inactive and in a hibernating condition. It had previously been found that such animals had nerve cells with a low resting potential and very little spontaneous activity (Kerkut & Walker, 1961). The animals were brought into activity in the following manner. The snails were placed in ajar containing ~ in. of water over crumpled chromatography paper. The lid of the jar was screwed down and the jar then brightly illuminated with two bench lamps for a couple of hours. The results of the humid atmosphere, the light and possibly the increased temperature led the animals to emerge from their shells and crawl round and round the jar. The brains of such animals were indistinguishable from those of normally active animals; they had a high resting potential and many of the cells were spontaneously active. During the summer and autumn, normal active snails were used from the garden. No difference was noted between the reactions of activated snails and normally active snails. 14C

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RESULTS (a) The properties of the resting potential and the action potential The resting potential varied in value between 7 and 50 mV. Fig. 1 shows a histogram of the frequency distribution of 247 resting potentials all of which were

146

G.A. Keu~t-w AND R. J. WA~.i{~u

taken from nerves showing spontaneous activity. Of these values, 210 fell between 20 and 45 mV, with the maximum number of cells having a potential between 30 and 39 inV. The degree of depolarization that occurred preceding each action potential varied slightly from one neurone to the next, but in forty-eight out of sixty-six cells where this phenomenon was carefully noted, the depolarization was between 4 and 6 mV; i.e. the resting potential fell this amount before the action potential was initiated. The complete range itself lay between 2 and 22 mV. ACh

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FIG. 3. T h e action of acetylcholine. T h i s figure is a p e n recording of t h e activity of a single n e u r o n e , t h e t h r e e parts of t h e figure b e i n g c o n t i n u o u s . Acetylcholine at 10 -n g/ml was a d d e d at t h e arrow, a n d t h e n e r v e r e s p o n d e d 4½ sec later by acceleration.

The action potential varied slightly in shape from one neurone to another, though "typical" action potentials with overshoots of 20-30 mV were often found. On other occasions, however, the action potential would fail to overshoot the base line, and the action potential would thus be less than the resting potential. The pattern of potential was constant for any one neurone under observation. On occasions multiple spikes could be observed, the spikes firing off at independent frequencies; on other occasions the post synaptic potentials could be clearly seen. Yet other occasions exhibited a double synchronous spike. The duration of the action potential showed a considerable range, lasting from 2 to 100 msec. Fig. 2 shows the frequency distribution of the duration of the action potential in 374 different cells. The majority of the potentials were of short duration.

THE EFFECTS OF DRUGS ON THE NEURONES OF THE SNAIL

147

(b) The effect of drugs on the spontaneous activity of snail neurones

1. Acetylcholine (ACh). Acetylcholine at physiological levels was found to affect the spontaneous activity of nerve cells. Fig. 3 shows a pen recording of an experiment in which the preparation responded to the addition of 10 -G g/ml ACh. T h e r e was a delay of approximately 4 sec and the activity of the unit then markedly increased. This increased activity was maintained for over 20 sec. T h e most sensitive preparations responded to 10 -8 g/ml, the response being an increase in the spontaneous activity. ACh ~dded

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T h e action of acetylcholine.

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T h e effects o f a d d i n g v a r i o u s c o n c e n t r a t i o n s of

acetylcholine to a single preparation are shown in this graph. 10-7 g/ml caused an acceleration; 10-s caused a slight inhibition. The inhibition was more marked at the concentrations 10-5, 10 -4. Fig. 4 shows the effect of different concentrations of ACh on the activity of a single nerve cell. 10 -7 caused a slight increase in the activity; 10 -6 caused a decrease in activity that lasted for 1 min, after which the activity increased. Higher concentrations of ACh caused an increase in the duration of the inhibition. This experiment demonstrates that ACh has a dual effect on the spontaneous activity; it can cause an increase in activity and it can also cause a decrease in activity--the difference sometimes depending on the concentration of the ACh. Other cells have been observed which would only show inhibition following acetylcholine addition. T h e maximum dilution that has been observed to bring about inhibition is 10 -~ g/ml. Higher concentrations of ACh (10 -4) caused a

(;. A. KERKUTAND R. J. WALKER

148

brief increase in activity and then a marked inhibition. This was usually accompanied by a lasting depolarization of the resting potential almost to zero. Levels such as 10 -~, 10 -G, 10 -7 rarely had such a marked effect on the resting potential. Certain chemicals tended to protect the membrane against the action of 10 -4 ACh; they either prevented the complete depolarization or tended to make the depolarization last for a shorter time. This effect was most marked after application of fi-phenylalanine > glutamic acid > cocarboxylase, the drugs being arranged in order of decreasing protection against ACh. y-Aminobutyric acid and noradrenaline did not appear to provide any such protection to the membrane. 60 o---

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FIc. 5. The action of acetylcholine. The resting potential (Q) and the frequency of the action potentials (©) are shown in the figure. Acetylcholine caused a sudden decrease in the resting potential and an increase in the frequency of the action potentials. As the resting potential returned towards the initial level, the frequency of action potential decreased,

Fig. 5 illustrates the relationship between the resting potential and the frequency of the action potentials. In this particular case the membrane had been pretreated with phenylalanine. ACh at a concentration of 10 .4 was then added. It will be seen that the resting potential fell from 60 to 36 mV whilst the action potentials increased in frequency from 1 per 15 sec to 30 per 15 sec. As the resting potential slowly recovered, so the frequency of the spikes decreased. Had the nerve been treated to 10 -~ without any pre-treatment to phenylalanine, the resting potential would have fallen to zero and remained there. The action potentials would, after an initial increase in activity, have been completely inhibited. This experiment is also of interest in that it indicates that the drugs are acting on the actual cell under observation. The electrode would measure the resting

149

THE EFFECTS OF DRUGS ON THE NEURONES OF THE SNAIL

potential only of the impaled cell and the activity of this cell closely followed the resting potential values. This fact is of importance in considering the action of drugs on the cells in the brain since the cells remain in contact with one another and it is often difficult to decide whether the effect that one sees is a direct effect of the drug on that cell or whether the drug has acted on other cells that are in synaptic connexion with the observed cell.

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FIG, 6. T h e action of dimethyl-amino-ethanol ( D M A E ) . D M A E caused an increase in the resting potential ( 0 ) and a decrease in the frequency of the action potentials ( Q ) .

Soaking the preparation in 10 -5 g/ml of physostigmine potentiated the action to ACh so that the preparations became approximately 100 times more sensitive. On the other hand, addition of 2 x 10 -7 g/ml atropine had no effect either on the spontaneous activity or on the sensitivity to ACh. ACh, besides causing a change in the frequency of spontaneously firing cells, could sometimes also initiate activity in resting cells. On the other hand, a certain number of neurones were found that were spontaneously active but which were unaffected by the addition of ACh at physiological levels. 2. Dimethyl-amino-ethanol (DM~4E). Dimethyl-amino-ethanol has been suggested as a possible natural precursor of choline (Pepev et al., 1960). We did not try the action of choline on the preparation because we thought that choline would be produced after the esterase had acted on the acetylcholine. We found that in general, at physiological concentrations, D M A E tended to inhibit the spontaneous activity of the nerve cells. Fig. 6 shows the effect of adding 10 8 g/ml of D M A E to a spontaneously active nerve cell. The frequency of activity fell

150

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(-;. A. KERKUT AND R.

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from 40 per 15 sec to zero. At the same time the resting potential showed a hyperpolarization from 45 to 70 mV. As the resting potential recovered back to its initial level, so the frequency of action potentials increased. Higher concentrations GABA

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FIG. 7. T h e action o f y - a m i n o b u t y r i c acid (GABA). T h e above pen recording shows ~ continuous recording from a single unit. G A B A at a concentration of 10 -u g/ml was added to the nerve cell and caused an increase in the frequency of the action potentials.

of D M A E (10 4, 10-3, 10 2) tended to depolarize completely the membrane and maintain the resting potential at zero. This was the only drug, apart from acetylcholine and adrenaline, that caused complete depolarization. A few nerve cells showed slight acceleration following addition of DMAE, the threshold concentration being 10 s. Other nerve cells were quite unaffected by D M A E at physiological levels.

THE EFFECTS OF DRUGS ON THE NEURONES OF THE SNAIL

151

3. Glutamic acid. Glutamic acid appeared to act on some neurones by accelerating them and on others by inhibition. The threshold for acceleration was 10 -7 g/ml whilst that for inhibition was 10 --2°. The inhibitory reaction occurred

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within 5-10 sec after adding the glutamic acid, but the acceleratory effect was only noticeable about 1 min after application. Glutamic acid did not elicit activity from a resting cell.

152

G. A. KERKUT AND R.

J.

WALKER

4. 7-Aminobutyric acid (GABA). T h e overall effect of GABA was acceleration though it did cause inhibition to some neurones. T h e threshold for both acceleration and inhibition was 10 -1° g/ml, though it was more usual for the effect to come in at 10 -8. Fig. 7 shows the effect of adding 10 9 GABA on a spontaneous neurone; the activity showed a marked increase 5 sec after the drug had been added. GABA did not bring the resting cell into activity. 5. 5-Hydroxytryptamine (5-HT). 5 - H T caused both inhibition and acceleration of the spontaneous activity, the effect depending upon the cell under observation and the concentration of 5 - H T used. In general the overall effect of 5 - H T was acceleration. T h e reaction occurred within 5-10 sec of adding the 5 - H T . T h e threshold for acceleration was 10 9 g/ml. T h e threshold for inhibition was 1 0 8 g/ml. 5 - H T did not bring the resting neurone into activity. 6. Histamine hydrochloride. Histamine can cause acceleration or inhibition of the spontaneous activity depending on the type of neurone and the concentration of the drug. In general the type of neurone was more important. T h e reaction took place 5-10 sec after adding the histamine. T h e threshold for acceleration was 10 -9 g/ml whilst that for inhibition was 10 -8. An accelerator cell would also be stimulated by higher concentrations of histamine. Fig. 8 shows the effect of adding 10 7 to an accelerator cell. This cell was also stimulated by 10 6 and 10 -5. Silent cells were often stimulated into activity by the addition of 10 ~ histamine. Inhibitor cells could be affected by quite dilute solutions, but it is of interest to note that some cells were inhibited by 10 -8 but stimulated by 10 6. T h e inhibitory effect of histamine diminished with repeated application, thus successive applications of histamine at 10 -~ to an inhibitory neurone caused inhibition of activity for 42 rain the first time, 23 min the second time and 15 sec the third time. Histamine has been detected in the snail brain. 7. Nor-adrenaline. T h e overall effect of nor-adrenaline was an inhibitory one, though some neurones reacted by accelerating. T h e threshold for acceleration and inhibition was 10 9 g/ml. T h e r e was a general tendency for the response to nor-adrenaline to be a delayed one, the effect occurring a minute or so after addition of the drug. Nor-adrenaline did not affect the resting cell. 8. Adrenaline. Adrenaline affected the nerve cells in the main by causing an acceleration of the activity. On the other hand, a few" cells did react by being inhibited. T h e threshold for both acceleration and inhibition was 10 v g/ml. T h e more concentrated solutions of adrenaline tended to cause a proportional increase in the activity. This is illustrated in Fig. 9 where the maximum rate of action potentials is plotted against the added concentration of adrenaline. T h e r e was a linear relationship. T h o u g h adrenaline did cause an acceleration and a depolarization of the potential, it did not reduce the resting potential to zero even at high concentrations. 9. Dopamine (3-hydroxytyramhw). T h e overall physiological effect of dopamine was inhibition. It did cause acceleration to some cells, the threshold for acceleration was 10 -t° g/ml and that for inhibition 10 -11, though the usual threshold was 10 : for acceleration and 10 :~ for inhibition. Dopamine affected the resting

THE EFFECTS OF DRUGS ON THE NEURONES

153

OF THE SNAIL

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Fzo. 10. T h e action of dopamine. D o p a m i n e caused a sudden increase in the resting potential ( O ) and a decrease in the frequency of the action potentials ( O ) . As the resting potential returned to the normal level, so the frequency of action potentials returned back to normal.

154

( ; . A. KERKCT AND R . J. \V.~,L~ER

potential for the cells as is illustrated in Fig. 10. The addition of dopamine 10 '~ caused a decrease in the frequency of the action potentials from 50 per 15 sec to zero. At the same time it hyperpolarized the membrane from 28 to 50 inV. As the

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T h e action of cocarboxylase. Addition of cocarboxylase caused an increase in the frequency of the action potentials.

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resting potential slowly decreased to its original value, so the action potentials increased in frequency back to their original level. 10. DL-phenylalanine. The addition of phenylalanine to spontaneously active neurones tended to decrease their activity. The threshold concentration was

THE EFFECTS OF DRUGS ON THE NEURONES OF THE S N A I L

155

10 -s g/ml. The drug tended to cause a hyperpolarization of the resting potential and it could protect the membrane from complete permanent depolarization by 10 -4 ACh. The response occurred within 5-10 sec of adding the drug. A few cells did not respond at all to the addition of phenylalanine. 11. Cocarboxylase. Cocarboxylase (thiamine pyrophosphate) elicited acceleration in some neurones, inhibition in others, and no effect in still others. The threshold for acceleration was 10 -9 g/ml whilst that for inhibition was 10 -s g/ml. Fig. 11 shows the effect of adding 10 -s cocarboxylase to a preparation. The preparation showed an increase in activity much higher than anything recorded for that unit. 12. Thiamine hydrochloride. The general effect of thiamine was that of acceleration though quite a few cells were unaffected. The maximum threshold for acceleration was 10 -s g/ml though the average threshold was 10 -7. Fig. 12 shows the effect of adding 10 -7 thiamine to a unit; the activity increased. No inhibition was noted following the addition of thiamine. DISCUSSION The snail preparation proved to be sensitive to a majority of the chemicals described in the previous section, the reactions occurring at physiological drug levels of 10 -8 g/ml or less. This is perhaps to be expected if the results are to have any natural significance since the drugs were being applied directly to the nerve cell under observation; there was no connective tissue between the chemicals and the nerve to act as a diffusion barrier. Most of the drugs that had an effect on the preparation elicited their effect within 5-10 sec after addition. There were some exceptions to this, thus nor-adrenaline took a minute or so to obtain a reaction, and glutamic acid required over a minute for its acceleratory effect to be seen. The quick response to the addition of a drug, and the relationship between the resting potential and the frequency of the action potentials, indicate that it is most likely that the drug is acting directly on the cell under observation, and not via the nerve cells in the rest of the brain. The drugs described usually had a triple effect on the nerve cells; they accelerated, they inhibited, and they had no effect! This result can be seen more clearly from Table 1 and it raises several problems. Are the results due to chance, experimental variability, concentration effects, or are they due to cellular differences ? From our results it would appear that the last two of these explanations are correct. The most important variable would appear to be the cell under observation. Certain cells, no matter what concentration range was used, would only show inhibition of their activity following addition of the drug. Other cells would give acceleration if the drug was administered at low concentrations, and inhibition if the drug was added at a higher concentration. Other cells, still, were unaffected by the administration of the drug. The results obtained following administration of a given chemical were usually striking and determined, even if they were exactly opposite to those obtained in a previous experiment. These results make it imperative to know the precise histological localization of each cell

156

G . A . KERKUT AND R. J. WALKER

u n d e r observation before one can state with certainty the effect of a chemical on the nerve cell. T h i s conclusion is not altogether unexpected. Bradley & Mollica (1958) w o r k i n g on single units in the m e s e n c e p h a l o n and medulla of the decerebrate cat f o u n d that adrenaline accelerated some cells, inhibited others, and had no effect on still others. Acetylcholine on the other h a n d always had an effect t h o u g h in some cases it was an acceleration and in other cases an inhibition. T h e most reasonable conclusion is that the nervous system shows heterogeneity in its reactions to chemicals; certain cells react in one m a n n e r whilst other cells react in another to the administration of a given chemical. TABLE 1

Drug Acetylcholine Adrenaline Cocarboxylase Dimethyl-aminoethanol Dopamine GABA Glutamic acid Histamine HCI Nor-adrenaline 5-HT Phenylalanine Thiamine HCI

No, of

expts. 39 19 12 11 21 11 14 15 19 20 9 8

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T h e second p r o b l e m c o n c e r n s the evidence that any of the tested chemicals is a natural transmitter in the central nervous system of the snail. L e t us briefly consider the possibility for each of the tested chemicals. Acetylcholine. Acetylcholine acts on the snail nerves at a physiological level (10 -8 g/ml). It has b o t h a stinmlatory and an inhibitory effect. A similar conclusion was drawn by T a u c & G e r s c h e n f e l d (1960) for the nerve cells of Helix pomatia. T h e y f o u n d that 10 -8 stimulated and 10 5 inhibited, and have p u t forward the interesting suggestion that each nerve cell was capable of reacting to A C h , the stimulation or inhibition d e p e n d i n g on the resting potential of the cell. I f the potential was above 50 mV, then A C h stimulated; if it was below 50 m V then A C h inhibited. T h e critical voltage (50 mV) varied f r o m cell to cell. W e have f o u n d no clear relationship between a cell's resting potential and the m a n n e r in w h i c h it reacts to acetylcholine. T h u s , cells with resting potentials varying f r o m - 50 to - 10 m V have had their s p o n t a n e o u s activity inhibited b y the addition of acetylcholine. O t h e r cells with resting potentials varying over the same range have had their s p o n t a n e o u s activity increased b y the addition of acetylcholine. Yet a third g r o u p of cells with the same range of resting potentials have been unaffected by acetylcholine.

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Other workers have found ACh to have an effect on gastropod nerves. Duncan (1961) showed that ACh at 2 x 10 -5 stimulated the isolated pedal nerves of the snail Physa. We have found an acetylcholine-like substance present in Helix brain. When tested on the rat jejunum it gave a concentration equivalent to 5-10 /xg/g of brain tissue. Chromatographic analysis also indicated an ACh-like substance to be present. Choline esterases are known to be present in the blood (Augustinsson, 1946) and we have shown the presence of cholinesterase histochemically in snail nerve cells. Thus, within the snail brain there is an ACh-like substance, the nerve cells are sensitive to ACh at levels lower than those found in the whole brain, and their sensitivity is increased by eserine. T h e evidence is reasonably strong that ACh could be a natural CNS transmitter in the snail. Dimethyl-amino-ethanol. Dimethyl-amino-ethanol in general had an inhibitory effect (10-6). Addition of D M A E did not have an immediate effect on the neurone; there was normally a delay of 60-100 sec before a response occurred. T h e hyperpolarization of the cells and the inhibition of the activity resemble the inhibitory action of acetylcholine and it is possible that D M A E might be converted into ACh though there is, at present, no definite evidence that can support this view. 7-Aminobutyric acid (GABA). It was suggested by Bazemore et al. (1957) that GABA was the material responsible for the physiological action of the Factor I extract from the mammalian brain. More recently Florey & Chapman (1961) have shown that Factor I and GABA are not synonymous since Factor I is a much more powerful inhibitor than GABA. Nevertheless, GABA has a most marked inhibitory action on the crustacean stretch receptor. We found that the overall effect of GABA was a stimulatory one though a few units did respond by decreasing their activity. T h e effect of GABA is, in general, a temporary one, and the accelerated neurone soon returned back to its initial firing rate. Hirchar (1960b) found that GABA at concentrations from 10 -1° to 10 -2 M had no effect on the spontaneous activity of the nerves in the fifth abdominal ganglion of the crayfish Orconectes virilis, which may indicate that the central neurones of the crayfish may react differently from the peripheral nerves in the reaction to GABA. Glutamic acid. Glutamic acid is considered to be the metabolic precursor of GABA. It also plays a role in the metabolism of the nervous system (Strecker, 1957). We found that the nerve cells were sensitive to the addition of glutamic acid (inhibition threshold, 10 10; acceleration threshold, 10 8). T h e r e was, however, no overall effect of this drug, the acceleratory effect was equally common to the inhibitory effect. It was noticeable, however, that the inhibition was in general irreversible--the cells did not recover. With an inhibitor such as dopamine, on the other hand, the overall effect was that inhibition and the results were reversible. We have detected glutamic acid in the brain of the snail. Phenylalanine. We investigated this compound for two reasons ; first, because it has been found *hat Helix hearts kept in saline, enriched with phenylalanine, will beat for 24 days (Jullien et al., 1956). Secondly, phenylalanine is a possible intermediate in the formation of dopamine. T h e administration of C 14 phenylalanine

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to rats resuhed in the recovery of radioactive adrenaline (Gurin & Delluva, 1947). We found that the effect of phenylalanine at 10 -s was an inhibitory one, and that in particular it could repolarize the nerve membrane and protect it against the action of strong concentration (10 -5) of acetylcholine. Dopamine. T h e overall effect of dopamine was to inhibit the activity of the snail cells (maximum threshold, 10 L1). T h e majority of the responses were delayed ones, i.e. they took over a minute to have an effect and thus it is possible that cells other than the recorded ones might be affected by dopamine. Dopamine hyperpolarized the nerve membrane and in doing so it was the most effective of all the drugs tested. Ostlund (1954) was unable to find dopamine in the molluscs though he did find an unidentified catechol amine which he called "catechol 4". Price (unpublished) has isolated a material from the brain of gastropods which gives a spot at the same R s as dopamine, and which, like dopamine, inhibited the isolated frog heart. Thus, there is a fair possibility that dopamine exists in the snail brain. It has a possible role as an inhibitory compound in the snail brain. Adrenaline and nor-adrenaline. T h e preparations were more sensitive to noradrenaline than adrenaline. T h e effects of nor-adrenaline were mixed ; it stimulated some neurones and inhibited others. Adrenaline, on the other hand, had mainly an excitatory effect. We have not been able to detect adrenaline or nor-adrenaline in chromatograms of snail material and think it unlikely that these substances occur naturally in the animal. 5-Hydroxytryptamine. 5 - H T has been suggested as a cardio-regulator in molluscs by Bacq et al. (1952) and Welsh (1953). Its distribution in the invertebrates has been investigated by Welsh & Moorhead (1960), and they have found quantities in the gastropods ranging from 2.6 /xg/g brain tissue in Polynices keros to 11.0/xg/g brain tissue in Melongena. We have, so far, been unable to find any valid concentrations of 5 - H T in our chromatograms of the snail-brain extract (Kerkut & Laverack, 1960). Our recent investigations have indicated that if 5 - H T is present, it will be in concentration less than 0.5 #g/g. On the other hand, we have found that the overall effect of 5 - H T is to accelerate the neurones, the drug being effective at a concentration of 10 -9 g/ml. Tkiamine kydrockloride and cocarboxylase. Von Muralt (1945, 1958) suggested that thiamine compounds played a double role in the nervous system. Cocarboxylase (thiamine pyrophosphate) was important in metabolic systems whilst thiamine was more important in the transmission of the nerve impulse. We found that cocarboxylase could both excite and inhibit and which ever it did was effective in reasonably high dilutions (10 -s and 10 -9 g/ml). Thiamine, on the other hand, was an excitant (10-8). This tends to agree with yon Muralt's finding that more thiamine was present in excited nerves than non-excited nerves. Similarly, Petropulos (1960) found that antithiamine compounds caused a hyperpolarization of the nerves of Rana esculenta. Augustinsson (1946, 1948) has described an enzyme in Helix dart sac and blood which will hydrolyse acetylthiamine. Histamine. ttistamine could cause both excitement and inhibition depending on the cell under investigation. T h e threshold for acceleration was 1 0 :~ and for

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inhibition 10 -8. T h e stimulatory effect soon faded, but the inhibitory response was more lasting and the neurone in general failed to return to its initial rate of discharge. Preliminary investigations indicate that histamine may be present in the snail ganglia. Thus, of the chemicals tested, acetylcholine, dopamine, glutamic acid and histamine have been found in the snail brain; 5-hydroxytryptamine, thiamine, cocarboxylase, dimethyl-amino-ethanol, y-aminobutyric acid and phenylalanine are possibly present. We doubt whether adrenaline and nor-adrenaline are present. It is difficult to conclude whether any of the above chemicals are natural excitants and inhibitors of the nervous system because the results so far merely indicate that excitation and inhibition can occur. T h e fact that all these chemicals have an effect at physiological concentrations is of interest, and the further fact that some of them are known to be present in the snail strengthens their claim as natural neuro-chemical transmitters. T h e strongest case, so far, is that for acetylcholine which also has an esterase present. Dopamine is a possible inhibitory substance. However, it is clear that further work on specific nerve cells whose anatomical relationships are well known and precisely defined will be necessary before one can conclude the extent to which any given chemical acts as a neurotransmitter and also the extent to which chemical heterogeneity exists in the central nervous system.

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