Effects of postulated neurochumoral transmitters on the isolated radula protractor of Busycon canaliculatum

Comp. Biochem. PhysioL, 1970, Vol. 33, pp. 249 to 258. Pergamon Press. Printed in Great Britain

EFFECTS OF POSTULATED NEUROHUMORAL TRANSMITTERS ON THE ISOLATED RADULA PROTRACTOR OF B U S Y C O N C A N A L I C U L A T U M R. B. H I L L Department of Zoology, University of Rhode Island, Kingston, Rhode Island 02881 (Received 4 ffuly 1969) A b s t r a c t - - 1 . Acetylcholine potentiates twitches in low concentrations and

induces contracture in high concentrations. Acetylcholine contracture is temperature dependent. 2. Tryptamine is more effective than 5-hydroxytryptamine in potentiating twitches and in inducing rhythmicity. 3. High concentrations of noradrenaline, tyramine and GABA potentiate twitches. 4. High concentrations of adrenaline, dopamine and glutamic acid depress twitches. 5. At comparable concentrations, tryptamine is more effective than 5-hydroxytryptamine in relaxing acetyJcholine contracture. INTRODUCTION FRESHLY isolated muscles of the radula apparatus of Busycon canaliculatum (Hill, 1956, 1958) or Buccinura undatum (F/inge & Mattisson, 1958a, b) are not spontaneously active but may be provoked to rhythmical activity as, for example, by the simultaneous presence in the bathing fluid of acetylcholine and 5-hydroxytryptamine. F~inge & Mattisson (1958a, b) used the entire bundle of buccal muscles and reported rhythmicity (superimposed on contracture) when acetylcholine alone had been added, but the rhythmicity was increased by small doses of tryptamine. I used isolated individual muscles (radula retractor, radula protractor, odontophore retractor, odontophore protractor) and found in each (Hill, 1956, 1958) that acetylcholine alone never induced rhythmicity, unless the muscle had previously been exposed to tryptamine or 5-hydroxytryptamine. F/inge & Mattisson pointed out the resemblance to cardiac pulsations of the rhythmical contractions which occur in the combined presence of certain concentractions of hcetylcholine and 5-hydroxytryptamine. I (Hill, 1958) remarked that, in the presence of acetylcholine and tryptamine, "rhythmicity may be as regular in rate and amplitude as a heartbeat". Thus the radula muscle preparation is of interest as a possible model of cardiac rhythmicity. It has also been used for bioassay by F/inge (1963), Feder & Arvidsson (1967) and Feder & Lasker (1968). Since interest in the radula protractor preparation, used as a model of cardiac rhythmicity or for bioassay, centers around its responses to acetylcholine and to 249

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R . B . HILL

a n a l o g u e s of 5 - h y d r o x y t r y p t a m i n e , it s e e m e d w o r t h w h i l e to e x a m i n e its p h a r m a cology m o r e closely. A n e c e s s a r y first s t e p was to e x a m i n e t h e effect on t h e i s o l a t e d r a d u l a p r o t r a c t o r of a n u m b e r o f s u b s t a n c e s , b e l i e v e d on g o o d e v i d e n c e to be c h e m i c a l n e u r o t r a n s mitters. MATERIALS AND METHODS Procedures T h e radula protractor of Busycon (Busycotypus) canaliculatum L. 1758 has been used for pharmacological investigations by three procedures. T h e first is simply to study the effect of drugs in inducing responses of the quiescent isolated muscle (Hill, 1958). T h e second is to use pharmacological agents to modulate the response of the radula protractor to direct stimulation with electrical shocks (Hill, 1963). T h e third method is to record response in the muscle to stimulation of the nerve, while bathing the muscle in a solution containing the agent under investigation (Hill, 1962). While the third method has the apparent added complication of requiring consideration of the effects of drugs on axonal transmission and on neuromuscular transmission, it may be that neither factor can be eliminated from consideration when studying the isolated muscle. Mattisson & Arvidsson (1966) found that after 5-12 hr isolated radula muscles began spontaneous activity. I have never observed spontaneous activity, but it does seem that after 16 hr in isolation induced rhythmicity of the radula protractor becomes much better co-ordinated. Such observations could be taken as hints that functional denervation sets in after some hours in isolation, but in any case it seems very probable that in the first few hours after dissection there are still nerve fibers capable of response to the electrical shocks used to drive an isolated radula protractor. Activity of central origin is, of course, eliminated from the isolated radula protractor but it may also be eliminated from the nerve-muscle preparation by cutting all nerve trunks coursing from the buccal and cerebral ganglia into the proboscis (Herrick, 1906; Hill, 1962). Thus the isolated radula protractor preparation may be preferred to a nerve-muscle preparation for the sake of greater facility in mechanical recording, stimulation and application of drugs, but it must be borne in mind that the elimination of nerve involvement may require use of pharmacological agents blocking axonal conduction or neuromuscular transmission. Experiments reported in this series of papers followed either the first method (effects of drugs on quiescent muscle) or the second method (effects of drugs on responses to electrical stimulation). Wherever feasible, the second method was used as it has proved capable of revealing lower thresholds. Kerkut & Leake (1966) used regular stimulation of the garden snail brain-nerve-pharyngeal retractor preparation and reported that it had the merit of rendering the effects of added drugs more consistent because spontaneous activity of the pharyngeal retractor interferes with the interpretation of drug effects. However, since the denervated radula protractor is not spontaneously active there is not quite so much advantage to the use of electrical stimulation and it was omitted where it appeared that the use of stimulation would have made the experimental design too complex. Twitches were evoked by 10-msec shocks of a nominal 80 V, applied across bath electrodes as previously reported (Hill et al., 1968). Muscles were left to equilibrate to bath temperature under tension, in natural sea water, for ~--2 hr before use. Pharmacological agents were applied by injection into the bath of appropriate quantities of stock solutions to produce the desired final bath concentrations. The following substances were used: acetylcholine iodide (ACh), tryptamine HCI, 5-hydroxytryptamine creatinine sulfate (SHT), tyramine HC1, 3-hydroxytyramine HC1 (dopamine), adrenaline bitartrate, noradrenaline bitartrate, glutamic acid, gamma-aminobutyric acid (GABA), pentylamine and hexylamine.

EFFECT OF NEUROHUMORS ON RADULA PROTRACTOR

251

C o n t r a c t i o n s were r e c o r d e d o n a s m o k e d d r u m u s i n g a n isotonic lever s y s t e m w i t h a load of 0"037 g. Solutions were n o t buffered, since t h e r a d u l a p r o t r a c t o r is n o t sensitive to p H c h a n g e w i t h i n t h e r a n g e involved. S e a - w a t e r p H was consistently 8"4.

RESULTS

1. Acetylcholine As has been reported before (Hill, 1958), 10 -5 M aeetylcholine induces contracture of the radula protractor, but 10 -7 or 10 -s M acetylcholine (Hill, 1962) increases amplitude of twitches by one-third. Fiinge & Mattisson (1958b) reported temperature dependence of the acetyeholine response curve of the entire buccal muscle mass isolated from Buccinum undatum. The effect of temperature on acetylcholine contracture of the radula protractor of B. canaliculatum is very similar. Acetylcholine contracture is maintained longer and attains a greater degree of shortening at 8°C than at higher temperatures. For this series of experiments, a compromise temperature was chosen between low temperatures at which contracture might not relax rapidly and higher temperatures at which the speed of contraction is more rapid. In some preliminary experiments that compromise temperature was set at 12.5°C, but in most of the experiments 15°C was used. The chosen temperature was maintained by circulating coolant through the outer jackets of the aerated baths of natural sea water in which the muscles were mounted. The effect on twitches of added KC1 or of post-tetanic potentiation (Hill, 1963) is much like that of low concentrations of ACh, except that below the threshold for ACh contracture (in the region of 5 x 10 -8 M) aeetylcholine has no potentiating effect on twitches. In order to provide a basis for differentiating cholinergic effects, we have tested a series of known ACh antagonists against ACh contracture. Each antagonist was tested on two muscles, at bath concentrations of 10 -6 M, 10 -5 M and 10 -4 M, for effectiveness in blocking ACh (10 -6 M) contracture. Results are reported in Table 1, as average percentage reduction of contracture. D-Tubocurarine chloride, sodium pentobarbital and dimethisoquin HC1 were then selected for more detailed study. Concentration-action curves for ACh from 10 -s M to 10 -3 M, with washing between steps, were run against each antagonist from 10 -6 M to 10 -3 M in eight replications for each experiment. The results, as presented in Fig. 1, suggest competitive inhibition (Goldstein et al., 1968) for all three. 2. Effects of other postulated neurohumors on electrically driven twitches Substances which have been postulated to be chemical neurotransmitters (see Discussion) were assessed on optimal twitches of the radula protractor, that is: twitches elicited by stimuli evoking large contractions but little increase in tonus (some muscles responded with increased tonus to any stimulus which would elicit twitches). After ten control twitches a substance in solution would be injected

252

R. B. HILL TABLE 1--EFFECTIVENESS OF ACETYLCHOLINE ANTAGONISTS

Concentrations

Antagonist

10 -* M (%)

10 -5 M (~o)

10 -6 M ( °o )

Cocaine HCI Dimethisoquin HC1 9-Tubocurarine Mytolon ® Crude curare Dimethyltubocurare Sodium pentobarbital Atropine sulfate Hexamethonium TEA Procaine Pentamethonium Gallamine Decamethonium Hemicholinium Methylpentynol Mytelase ® Methylene blue

- 100 - 100 - 100 - 95 - 98 - 90 - 83 - 79 - 68 - 68 - 68 - 46 - 46 - 32 - 22 - 18 - 14 - 12

- 75 -67 - 43 - 46 - 32 - 39 - 21 - 27 - 24 - 20 - 9 - 20 - 14 - 20 - 13 - 10 0 + 12

- 27 - 11 - 15 0 - 21 - 2 - 20 - 8 - 14 0 - 5 - 4 0 0 - 8 - 22 0 +5

i n t o t h e b a t h , b r i n g i n g t h e final b a t h c o n c e n t r a t i o n to t h e d e s i r e d level of p o s t u l a t e d n e u r o h u m o r . A f t e r t e n m o r e t w i t c h e s , t h e m u s c l e w o u l d b e a l l o w e d to rest for 5 m i n w i t h t h e k y m o g r a p h d r u m s t o p p e d a n d t h e n t e n m o r e t w i t c h e s w o u l d b e r e c o r d e d b e f o r e t h e b a t h was w a s h e d out. E x a m p l e s of t h e k i n d o f r e c o r d o b t a i n e d are s h o w n in Fig. 2 w h e r e t h e effects of 10 -5 M , 10 -4 M a n d 10 -3 M 5 - h y d r o x y t r y p t a m i n e are c o m p a r e d to t h o s e o f t h e s a m e c o n c e n t r a t i o n s of t r y p t a m i n e . T h e effects o b t a i n e d i n c l u d e d increase in t w i t c h a m p l i t u d e w i t h o u t increase in t o n u s (10-5 M 5 H T ) , i n c r e a s e in t w i t c h a m p l i t u d e w i t h increase in t o n u s (10 -4 5 H T ) , o n s e t of r h y t h m i c i t y (10 -3 M t r y p t a m i n e ) a n d i n c r e a s e in t w i t c h a m p l i t u d e after w a s h i n g o u t a h i g h c o n c e n t r a t i o n (10 -3 M 5 H T ) . T h e m o s t c o n s i s t e n t effect for assessing n e u r o h u m o r s a p p e a r e d to b e i n c r e a s e in t w i t c h a m p l i t u d e . T r y p t a m i n e was b y far t h e m o s t effective of t h e p o s t u l a t e d n e u r o h u m o r s in potentiating twitches. When compared with 5-hydroxytryptamine, the striking difference (Fig. 3) is t h a t a l t h o u g h t h e t h r e s h o l d for b o t h is a b o u t 10 -7 M , t h e r i s i n g c u r v e of effectiveness for 5 - h y d r o x y t r y p t a m i n e levels off at 10 -5 M , w h e r e a s t h a t for t r y p t a m i n e c o n t i n u e s to rise u n t i l at 10 -3 M t h e effect o f t r y p t a m i n e o n t w i t c h e s is o b s c u r e d b y i n d u c e d r h y t h m i c i t y (Fig. 2). N o r a d r e n a l i n e p o t e n t i a t e d t w i t c h e s s l i g h t l y at c o n c e n t r a t i o n s f r o m 5 × 10 -6 M to 10 -3 M b u t a d r e n a l i n e ( F i g . 4) h a d a d e p r e s s a n t effect t h a t i n c r e a s e d w i t h c o n c e n t r a t i o n f r o m 5 × 10 -5 M to 10 -8 M . H i g h c o n c e n t r a t i o n s o f t y r a m i n e will p o t e n t i a t e t w i t c h e s (Fig. 5) w i t h m a x i m a l effect at 10 -* M , b u t t h e s a m e c o n c e n t r a t i o n s o f 3 - h y d r o x y t y r a m i n e

EFFECT OF N E U R O H U M O R S

253

ON RADULA PROTRACTOR

(dopamine) will not. Glutamic acid in concentrations from 3 x 10 -6 M to 3 x 10 -s M had no significant immediate effect, but did have a strongly depressing effect after 5 min. GABA had no significant immediate effect, but after 5 rain concentrations from 10 -6 to 10 -~ M slightly potentiated twitches. . f o ~ l

I00 Dimelhisoquin

5C

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FIG. 1. Concentration-action curves for ACh in the presence of three antagonists.

3. Effectiveness of postulated neurohumors in relaxing acetylcholine contracture A set procedure was used for studying the effectiveness of neurohumors in relaxing contracture. After 3 rain of contracture, induced by 2.5 x 10 -5 M ACh, the bath was made up to the desired concentration of tryptamine or 5-hydroxytryptamine, still in the presence of the original acetylcholine concentration. After 12 min the bath was rinsed out for 3 rain. Tryptamine was tested on eight preparations from B. canaliculatum, in concentrations from 10 -6 M to 6 x 10-3M. The lowest effective concentrations relaxed the acetylcholine contracture, with rhythmicity superimposed. Relaxation

254

R.B. HILL

and the amplitude of rhythmicity increased with increasing concentration. Rhythmicity tapered off toward the end of the 12 min at higher concentrations (Fig. 6a). 5-Hydroxytryptamine in a range of concentrations from 10 -6 M to 6 × 10 -7 M was tested on four preparations from Busycon contrarium. The lowest.effective concentrations simply relaxed the acetylcholine contracture. Increasing concentrations also tended to produce rhythmicity toward the end of the 12 min. Note short-term regularity of rhythmicity as shown by high drum speed inset for 6× 10 -3 M (Fig. 6b). At comparable concentrations, tryptamine was more effective than 5-hydroxytryptamine in relaxing acetylcholine contracture. 400

ere/e/r

• Tryptamine

300

%

200 e~....e

5HT

I00

I0-'

I 0 -~

I0 - s

I0 -~

FIG. 3. Ordinate: percentage increase in twitch amplitude after 5 min. Abscissa: molar concentration of tryptamine and 5-hydroxytryptamine. r indicates rhythmicity obscuring the amplitude of twitches.

5°r

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%

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M

FIG. 4. Ordinate: percentage change in twitch amplitude after 5 min. Abscissa: molar concentration of adrenaline or noradrenaline.

Four consecutive applications of 6 x 10 -3 M 5HT would produce the same reproducible "relaxation with rhythmicity" of the standard ACh contracture,

5HT

[0 "m M

IO-SM

I 0 "4 M

I0 -4 M

10-3M

I0 -= M

FIG. 2. Kymograph records showing potentiation of twitches by 5-hydroxytryptamine and by tryptamine. Time signal = 10sec. Bar indicates drum stopped 5 rain.

Fro. 6. a. Effect of t r y p t a m i n e in the indicated molar c o n c e n t r a t i o n s in relaxing contracture i n d u c e d by 2'5 x 10 5 M A C h (B. canaliculatum, 0.02 g load), b. Effect of 5 - h y d r o x y t r y p t a m i n e in the indicated molar concentrations in relaxing contracture i n d u c e d by 2'5 x 10 5 M A C h (B. contrarium, s t a n d a r d load). Regular time signal = 10 sec. Inset time signal = 10 see and 1 sec.

3 x l O -5

3xlO -6

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t

Acetylcholine

1

t Tryptamine

1

t Hexylomine

I

5xlO -~

3 x l O -3

8'5 x I0 "4

t

Pentylarnine

1

FI~. 7. Kymograph records of the effects of the indicated molar concentrations of pharmacological agents in inducing contractures or spontaneous activity

Fro. 8.

S x IO "4 Tryp ta mine

1 6 xlO -S

l",~'-"

t

....

Effects of the indicated molar concentrations of 5-hydroxytryptamine and tryptamine in inducing contracture and rhythmic contractions.

I

i

5+~Hyd f o x y t ryp t am; ne

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EF~Cr OF N~UaOrIUMORSON aADULAPROraACrOa

255

in two preparations from B. contrarium. A similar reproducibility was obtained using 10 -3 M tryptamine on two preparations.

Jso

L -501 -r

/ ~ e

3-HTyramine 10-6

10-s

10-4

10-3

M FIG. 5. Ordinate: percentage change in amplitude after 5 min. Abscissa: molar concentration of tyramine or 3-hydroxytyramine.

4. Effectiveness of pharmacological agents in inducing contractures or spontaneous activity The set procedure was to make the bath up to the desired concentration of an agent, record for 12 min and wash for 3 min. Acetylcholine alone (Fig. 7) produced graded contractures in concentrations of 8.5 x 10 -~ M, 3 x 10 -6 M and 3 x 10 -s M, but never induced any rhythmicity of the isolated radula protractor. Tryptamine alone (Fig. 7) proved capable of inducing rhythmic bursts of contractions in the absence of acetylcholine. A concentration of 9 x 10 -5 M was below theshold, 9 × 10 -4 M induced a pattern of slow contractions with superimposed rapid contractions, and at 3 x 10 -8 M the rapid contractions were markedly improved in amplitude and regularity. The higher concentrations of tryptamine, e.g. 6 x 10 -3 M, induced a gradual rise in base-line (Fig. 8). In the absence of acetylcholine, 5-hydroxytryptamine (Fig. 8), pentylamine and hexylamine (Fig. 7) all proved capable of inducing sporadic contractions but all were inferior to tryptamine with regard to amplitude and regularity of induced rhythmicity. DISCUSSION The radula protractor preparation originally seemed of particular interest because it could be provoked to rhythmicity in the simultaneous presence of two naturally occurring neurohumors of opposing action, thus offering a possible model for the induction of pacemaker activity in myogenic cardiac tissue. As knowledge of the radula protractor preparation accumulates, it appears less of a simple straightforward model but the basic features of interest remain. The

256

R.B. HILL

most regular rhythmicity is still that obtained by tryptamine or 5-hydroxytryptamine acting on an acetylcholine contracture. A disturbing feature (as a model) of the rhythmicity induced by naturally occurring neurohumors is the high concentration of those substances required. The threshold for acetylcholine contracture is ordinarily at the level of 5 x 10 .7 M although acetylcholine will potentiate twitches at lower levels. (Acetylcholine alone (Fig. 7) never induces spontaneous phasic activity). The threshold for induction of spontaneous phasic activity by tryptamine is below 10 .5 M and that for 5-hydroxytryptamine is at the level of 5 x 10 .4 M (Fig. 8). Tryptamine is most effective in inducing a rhythmic relaxation of an acetylcholine contracture at the level of 10 -a M to 2.5 x 10 -a M (Fig. 6a) and 5-hydroxytryptamine is most effective thus at the level of 5 x 10 .3 M to 7.5 x 10 -7 M (Fig. 6B). However, the threshold for both in potentiating twitches is at the level of 10 .7 M (Fig. 3) which corresponds more closely to the thresholds for acetylcholine. Other biogenic amines, in addition to tryptamine and 5-hydroxytryptamine, are postulated chemical neurotransmitters for the Mollusca (Hill & Welsh, 1966) and some of those were studied. Noradrenaline increases amplitude of twitches relatively slightly (Fig. 4) over the range of concentrations from 10 -5 M to 10 .3 M, but adrenaline has a depressing effect on twitch amplitude, increasing over the same range of concentrations. The difference in action of adrenaline and noradrenaline was not predicted, since both are excitatory for gastropod hearts (Hill, 1958; S.-Rdsza & Zs.-Nagy, 1967). Tyramine has the effect of increasing twitch amplitude (Fig. 5) with a gradually increasing effectiveness over the concentration range 10 -~ M to 10 .4 M at which latter level it is more than three times as effective as noradrenaline. 3-Hydroxytyramine (dopamine) has a weakly inhibitory effect on twitches, gradually increasing over the same range of concentrations. Here again the result was not to be predicted from experiments on hearts since both tyramine and dopamine are excitatory on cephalopod hearts (Fgnge & Ostlund, 1954) and dopamine is excitatory on gastropod hearts (S.-Rdsza & Zs.-Nagy, 1967). However, there is a case for dopamine as the chemical transmitter of long-lasting inhibition by hyperpolarization of gastropod neurons (Kerkut & Horn, 1968), and it is thus conceivable that dopamine may hyperpolarize radula muscle. It is of interest that for two pairs of substances, both of which generally excite molluscan hearts, the smooth muscle of the radula protractor is excited by one and depressed by the other: a situation roughly parallel to vertebrate heart vs. smooth muscle. It may be of some significance that both noradrenaline (which potentiates twitches) and dopamine (which depresses twitches) have been identified in molluscan nervous tissue (Cottrell, 1967), and that acetylcholine and dopamine have been said to be likely transmitter substances for the snail brain (Kerkut & Walker, 1961). There is some evidence that gamma-amino-butyric acid (GABA) and glutamate (Kerkut, 1967; Usherwood & Machili, 1968) act as chemical transmitters in Crustacea, insects and vertebrates. The radula protractor could perhaps be used in differential bioassay, since twitches are depressed by glutamic

EFFECT OF NEUROHUMORS ON RADULA PROTRACTOR

257

acid and potentiated by GABA. However, tryptamine remains the most interesting of the postulated chemical neurotransmitters in its ability to induce rhythmicity. SUMMARY Of the postulated chemical transmitters tested, tryptamine and 5-hydroxytryptamine are the most effective in inducing rhythmicity and potentiating twitches of the isolated radula protractor.

Achnowledgements--I wish to acknowledge financial assistance from an N.S.F. research grant (GB-1001) and the technical assistance of Mrs. Linden Vargish. REFERENCES

COTTRELL G. (1967) Occurrence of dopamine and noradrenaline in the nervous tissue of some invertebrate species. Br. J. Pharmacol. 29, 63-69. FANGE R. (1963) Toxic factors in starfishes. Sarsia 10, 19-21. FANGE R. & MATTISSONA. (1958a) Rhythmical properties of a smooth muscle, rich in haematin compounds. Ark. Zool. 11, 112. FANGE R. & MATTISSONA. (1958b) Studies on the physiology of the radula-muscle of Buccinum undatum. Acta zool. 39, 53-64. FANGE R. & OSTLUNDE. (1954) The effects of adrenaline, noradrenaline, tyramine and other drugs on the isolated heart from marine vertebrates and a cephalopod (Eledone cirrosa). Acta zool. 35, 289-305. FEDER H. M. & ARVlDSSONJ. (1967) Studies on a sea-star (Marthasterias glacialis) extract responsible for avoidance reactions in a gastropod (Buccinum undatum). Ark. zool. 19, 369-379. FEDER H. M. & LASKERR. (1968) A radula muscle preparation from the gastropod, Kelletia keUetii, for biochemical assays. Veliger 10, 283-285. GOLDSTEINA., ARONOWL. & KALMANS. M. (1968) Principles of Drug Action. Harper & Row, New York. HERRICKJ. C. (1906) Mechanism of the odontophoral apparatus in Sycotypus canaliculatus. Am. Nat. 40, 707-737. HILL R. B. (1956) Regulation of rhythmic activity in two types of red muscle in Busycon canaliculatum. Anat. Rec. 125, 613. HILL R. B. (1958) The effects of certain neurohumors and of other drugs on the ventricle and radula protractor of Busycon canaliculatum and on the ventricle of Strombus gigas. Biol. Bull. 155, 471-482. HILL R. B. (1962) Pharmacology of the radula protractor of Busycon canaliculatum. Biol. Bull. 127, 499. HILL R. B. (1963) Physiology of the radula protractor of Busycon canaliculatum. Fedn Proc. 22, 461. HILL R. B., MARANTZE., BEATTIEB. & LOCKHARTJ. (1968) Mechanical properties of the radula protractor of Busycon canaliculatum. Experientia 24, 91-92. HILL R. B. & WELSH J. H. (1966) Heart, circulation, and blood cells. In Physiology of Mollusca (Edited by WILBURK. & YONGEC. M.), Vol. II, pp. 125-174. Academic Press, New York. KERKUT G. A. (1967) Biochemical aspects of invertebrate nerve cells. In Invertebrate Nervous Systems. Their Significance for Mammalian Neurophysiology (Edited by WIERSMAC.), pp. 5-37. University of Chicago Press, Chicago. KERKUT G. A. & HORN N. (1968) Dopamine and long lasting inhibition of snail neurons. Life Sci. 7, 567-569.

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KERKUT G. A. & LEAKE L. D. (1966) T h e effect of drugs on the snail pharyngeal retractor muscle. Comp. Biochem. Physiol. 17, 623-633. I~RKUT G. A. & WALKER R. J. (1961) T h e effect of drugs on the neurons of the snail Helix aspersa. Comp. Biochem. Physiol. 3, 143-160. MATTISSON A. G. M. & ARVIDSSON J. A. (1966) Some effects of electrical stimulation and exogenous metabolites on the contractile activity and the ultrastructure of the radulamuscle of Buceinum undatum. Z. Zellforsch. 73, 37-55. S.-R6szA K. & Zs.-NAGY I. (1967) Physiological and histochemical evidence for neuroendocrine regulation of heart activity in the snail Lymnaea stagnalis. Comp. Biochem. Physiol. 23, 373-382. USHERWOOD P. N. R. & MACrlILI P. (1968) Pharmacological properties of excitatory neuromuscular synapses in the locust, ft. exp. Biol. 49, 341-361.

Key Word IndexmMuscle contractants; radula protractor; Busycon canaliculatum; acetylcholine; tryptarnines; 5-hydroxytryptamine; noradrenaline; GABA; adrenaline; dopamine; glutamic acid.