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The assumed participation of 5-hydroxytryptamine in regulation of the circadian rhythm of locomotor activity in Acheta Domesticus L.
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Comp. gen. Pharmac.
THE ASSUMED PARTICIPATION OF 5 - H Y D R O X Y T R Y P T A M I N E IN R E G U L A T I O N OF THE CIRCADIAN R H Y T H M OF L O C O M O T O R ACTIVITY IN ACHETA DOMESTICUS L. BRONISLAW CYMBOROWSKI Department of Animal Physiology, Zoological Institute, University of Warsaw, Poland (Received 31 March, 197o)
ABSTRACT 1. Locomotor activity of Acheta domesticus L. from alternating light and dark conditions was studied after injecting reserpine, LSD, and 5-hydroxytryptamine. 2. Great disturbances to locomotor activity, consisting of reduction of the level of locomotor activity and abolition of its rhythm, were caused by injecting reserpine and LSD. 3. These changes, as well as the effect of 5-HT, suggest that serotonin participates in regulation of circadian rhythm of locomotor activity of these insects.
House crickets, Acheta domesticus L., kept from the time of hatching to attainment of sexual maturity under LD I2:12 (light 12 hours: dark i~ hours) conditions exhibit very distinct locomotor activity rhythm (Cymborowski, 1968 , i969a , I97oa ). The characteristic rhythm is also maintained for a certain time after these insects are transferred to constant light conditions (LL). We are thus faced with the problem of the endogenic regulation of this process. A large number of experiments have been carried out on the mechanism regulating locomotor activity in insects which point to the fact that the neurohormonal system takes part in this process (Harker, I955, 1956 , I96oa, b; Roberts, I966; Brady, 1967a, b, 1969; Nishiitsutsuji-Uwo, Petropulos, and Pittendrigh, I967 ; Cymborowski, I968). It has been shown in studies on crickets taken from LD i2:12 conditions that there is circadian rhythm of R N A synthesis in the neurosecretory cells of pars intercerebralis and the suboesophageal ganglion (Cymborowski and Dutkowski, x968, t 969; Cymborowski, I97ob), and of protein synthesis in these centres (Cymborowski and Dutkowski, I97O ).
Simultaneously rhythm is observed to exist in accumulation and liberation of neurosecretion in the neurosecretory cells of these insects (Cymborowski, i97ob). The histochemical changes presented above and in particular the variations in accumulation and liberation of neurosecretion have been linked to the locomotor activity rhythm in the insects examined. This connexion would appear to be justified in view of the results of analogous studies made using crickets which did not exhibit locomotor activity rhythm and had been kept under LL conditions for the whole period. N o rhythmically occurring histochemical changes in the C.N.S. centres examined were observed in this group of insects. Data in literature show that the neurosecretion is a protein substance (Arvy and Gabe, I962; Ichikawa and Ishizaki, 1963). It can, however, be only a carrying substance in relation to the biologically active compound (Scharrer and Scharrer, 1954; Acher and Fromageot, x957). In the present study pharmacological investigations have been undertaken for the purpose of defining the hypothetical nature
197o , x, 3 1 6 - 3 2 2
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o f the n e u r o s e c r e t o r y s u b s t a n c e t a k i n g p a r t in r e g u l a t i o n o f the l o c o m o t o r a c t i v i t y r h y t h m in insects. A t t e n t i o n has b e e n p a i d to 5 - H T , the p r e s e n c e o f w h i c h in the n e r v o u s s y s t e m o f insect w o u l d a p p e a r to be indisputable (Welsh and Moorhead, t96o ; Colh o u n , i 9 6 o , i964). K e r k u t (1967) , after r e v i e w i n g the o c c u r r e n c e o f 5 - H T in i n v e r t e b r a t e s , was i n c l i n e d
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Locomotor activity in these insects was determined in an actograph of the author's own construction (Cymborowski, 1969b ). The conditions in the actograph were similar to the culture conditions. Only those insects exhibiting very distinct locomotor activity rhythm were used for the pharmacological examinations. Observations were made of the effect of reserpine (serpasil), diethylamide of lysergic acid (LSD-25), and serotonin (5-HT) on the loco-
Fie;. I.--Activity records of A. domesticus taken from L D 12:12 conditions. After finding that there were no changes in activity rhythm following injection of distilled H20, 5 lag. of reserpine per g. bodyweight of insect were injected into its haemolymph. The same amount of reserpine was given the next day. Each horizontal line represents the 24-hour activity of the cricket. The vertical white lines indicate the movements made by the insect. A time scale is added at the bottom. The beginning and end of the darkness period are marked.
Fie,. 2.--Activity records of A. domesticus, from LD 12 : x2 conditions, which had 7 lag. of reserpine per g. body-weight injected into its haemolymph on the fifth day after transfer to the actograph. Disturbances were observed in locomotor activity rhythm for two whole days (5, 6). Further explanations as tbr/'~g. I. to a s s u m e t h a t it is a n e u r o t r a n s m i t t e r s u b s t a n c e , a l t h o u g h it does n o t m e e t all the c r i t e r i a u s u a l l y a p p l i e d to such c o m p o u n d s . MATERIAL AND METHOI)S Experiments were made using male house crickets (Acheta domesticus L.) at the age of about i week after ecdvsis. The insects were kept in a large glass container at a temperature of about 29:" C. under conditions of alternating light and darkness LD 12:x2 (that is, ~2 hours light and 12 hours darkness during the 24-hour period). In this particular culture darkness began at 18.oo. Light was supplied by a 4 o W. electric bulb.
motor activity rhythm of the crickets. These substances were injected in a sterilized H~O solution in the following doses: reserpine 5 - i o lag. per g. of the insects' body-weight; LSD 2 -Io lag. per g. of body-weight; and 5 - H T i - 5 lag. per g. of body-weight. The injections were made into the haemolymph using a microsyringe. The volume of the injected fluid was 5/al. In the case of 5 - H T injections were also made into the protocerebrum using a microcapillary syringe, approximately 5o la in diameter. The volume of the fluid injected was 0- 5 lal. Injections were made without using anaesthetics, but the insects were subjected to a short period of hypothermy. The control experiments
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CYMBOROWSKI
showed that this operation does not affect the crickets' locomotor activity rhythm. Control operations were also made by injecting a given amount of distilled water in which the substances investigated were next dissolved. Observation of the insects' locomotor activity was begun immediately after iil.jection. Each experiment was repeated at least 5 times. RESULTS Reserpine i n t r o d u c e d into the h a e m o l y m p h in a m o u n t s o f 5 lag- per g. of the
Comp. gen. Pharmac.
a n d the first h a l f of the day. After this p e r i o d the r h y t h m takes on a c h a r a c t e r similar to initial r h y t h m a n d d i s t u r b a n c e does not occur again. A p p l i c a t i o n of a single dose of reserpine in a m o u n t s of 7 Pg. per g. of the insect's b o d y weight causes abolition of the insect's locom o t o r activity r h y t h m for 2 days (Fig. 2). I m m e d i a t e l y after giving reserpine considerable inhibition of l o c o m o t o r activity is observed lasting for the whole of the 24-hour
FIo. 3.--Activity records ofA. domesticus from LD 12 : x2 conditions, which had xo lag. of reserpine per g. body-weight injected into its haemolymph on the sixth day after transfer to the actograph. Considerable inhibition of locomotor activity was observed for two whole days. Further explanations as tbr Fig. x.
Fro. 4.--Activity records of A. domesticus from LD 12 : x2 conditions, which had 5 lag. of LSD-25 per g. body-weight injected into its haemolymph at different hours. Disturbances in locomotor activity rhythm were observed on the sixth day. Further explanations as for Fig. I. cricket's b o d y - w e i g h t does n o t cause disturbances in the l o c o m o t o r activity r h y t h m of this insect (Fig. I, d a y 4)- W h e n the same a m o u n t o f reserpine is a d m i n i s t e r e d again on the next d a y it causes a shift in p e a k locom o t o r activity from the hours of I 8 - I 9 to 23-24 (Pig. I, d a y 5). After this injection ( d u r i n g the following day) d i s t u r b a n c e o f activity is still observed, consisting p r i m a r i l y in its increase, which is p a r t i c u l a r l y easy to observe d u r i n g the second h a l f of the n i g h t
period. O n the second d a y after injection a g r a d u a l increase is observed in the insect's activity until h y p e r a c t i v i t y occurs (Fig. 2, d a y 6). W h e n reserpine is a d m i n i s t e r e d in a m o u n t s of i o lag. p e r g. of b o d y - w e i g h t it causes a b o l i t i o n of locomotor activity r h y t h m for 2 days also, but d u r i n g this p e r i o d the cricket's activity is greatly r e d u c e d in comparison with the previous dose (Fig. 3). O n the third d a y after giving reserpine a p e a k of locomotor
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R E G U L A T I O N OF LOCOMOTOR A C T I V I T Y
activity appears at the normal time of the day, but even so it is far smaller than the normal. Continued increase in doses of reserpine usually causes the insect's death. T h e short outline given here of experiments using reserpine illustrates the typical course they take. Certain modifications were obtained in some repeats of the experiments, consisting mainly in changes in the level of the locomotor activity of the insects concerned.
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injection (Fig. 4, day 6). It is remarkable here that such disturbances occur more often when using the same doses of LSD, but given at different times during the 24-hour period (F/g. 4). It is not until a dose is given twice as great as the preceding one (i.e., io lag. per g. bodyweight) that it causes abolition of locomotor activity r h y t h m a n d reduction in its level, but only over the course of 2 4 hours (Fig. 5). T h e normal peak of locomotor activity usually
Fro. 5.--Activity records ofA. domesticus from LD 12 : 12 conditions, which had xo lag. of LSD per g. body-weight injected into its haemolymph on the fifth day after transfer to the aclograph. Inhibition of locomotor activity was observed for one whole day (5). Further explanations as for Fig. I.
Fro. 6.--Activity records of A. domesticus from LD x2 : x2 conditions, which had I lag. of 5-HT per g. body-weight injected into haemolymph. There were no changes in locomotor activity. Further explanations as for Fig. I. Injection of LSD into the h a e m o l y m p h of rhythmic crickets produces, in respect of locomotor activity, results differing greatly from results obtained when using reserpine. Single doses of 5 lag- per g. of the insect's body-weight do not cause changes in locom o t o r activity rhythm. Similarly if the same dose is given again on the second day it does not cause any changes. In certain cases slight disturbances were observed in locomotor activity r h y t h m occurring during the second day after
occurred on the next day. Hyperactivity was sometimes observed after this period. Injection ofserotonin into the h a e m o l y m p h of crickets, using amounts from I to 5 tag. per g. of the insect's body-weight, causes practically no significant changes in their activity r h y t h m (Figs. 6, 7). In some cases, after using 5 - H T in doses of 5 lag • per g. body-weight, slight changes occur in activity consisting in increase in activity immediately after injection, and shortening of the activity peak (Fig. 7).
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However, when 5 - H T is injected in doses of i - 5 lag. per g. body-weight into the protocerebrum of r h y t h m i c crickets it causes considerable changes in their locomotor activity. Activity is observed to increase, however, without any distinct changes in its rhythm. After giving 5 lag. ofserotonin per g. of body-weight a very great increase takes place in the cricket's activity (Fig. 8). T h e crickets usually exhibit no great changes during the first day tbllowing this dose, but
per the the
Comp. gen. Pharmac.
g.
body-weight they usually exhibit typical reaction obtained in previous experiments (Fig. 8, days
9-12). DISCUSSION T h e data presented show that reserpine and LSD affect the cricket's locomotor activity. Reserpine exerts a particularly distinct action inhibiting locomotor activity. This would suggest that disturbances are
I'm. 7.--Activity records of A. domesticus from LD I2 : I2 conditions, which had 5 lag. of 5-HT per g. body-weight injected into its haemolymph on the fourth day after transfer to the aetograph. Slight disturbances in locomotor rhythm were observed atier injection. Further explanations as tbr Fig. I.
Fro. 8.--Activity records ofA. domesticus from LD 3~>: x2 conditions, which had 5 lag. of 5-HT per g. body-weight injected into the protocerebrum on the third day after transfer to the actograph. A gradual increase in locomotor activity was observed. On the ninth day of observations 7 lag. of reserpine per g. body-weight were injected into the haemolymph. Inhibition of locomotor activity is observed (9, 3o). Further explanations as for Pig. I. intensified activity takes place on the second day. T h e particularly great increase in activity after darkness falls is remarkable. T h e insects usually die within I-3" 5 weeks after such an injection. I f the insects which had received 5 - H T and exhibit considerable activity are injected with reserpine in amounts from 7 to IO lag.
created in the integrating centres of C.N.S. in these insects. These results are confirmed by data obtained in studies carried out on ants (Formica rufa L.) by Kostowski, Beck, and Meszaros (1966). W h e n these insects are given o ' t - o " 5 lag. of reserpine per mg. bodyweight with their food it causes marked inhibition of spontaneous locomotor activity,
I970 , l
REGULATION
OF LOCOMOTOR
and also changes in the bioelectric activity of the protocerebrum. Variations in the action of reserpine as the result of previous application of niamides lead these authors to the conclusion that the effect of reserpine depends on the concentration and distribution of biogenic amines, in the same way as in vertebrates. It is aLso known that reserpine reduces the serotonin level in the brain of certain invertebrates (see review by Gottrell and Laverack, x968). LSD also limits the action by serotonin, although in this case the mechanism is different from that connected with the effect of reserpine. The results of the action of LSD in the cricket's locomotor activity are not unequivocal and are very difficult to interpret. However, when doses of xo Ixg. per g. of the insect's body-weight are given they cause abolition of rhythm and inhibition of locomotor activity for a period of 24 hours. In none of the experiments did it prove possible to obtain abolition of rhythm for the duration of 2 days, as had been achieved by using reserpine. In the experiments on ants the administration of large doses of LSD (25-xoo lag. per g. of the body-weight) did not bring about any distinct changes in the locomotor activity of these insects (Kostowski and others, x966). It must be emphasized that in this case LSD was administered per os, which might account for the differences which occurred. These authors showed that despite the failure of LSD to affect locomotor activity, it causes considerable changes in E E G of ants. T h e data presented suggest that the effect on locomotor activity of the psychotropic drugs used in the experiments is exerted through the medium of the C.N.S., primarily as the result of disturbances in the action of 5-HT, and consequently ofbioelectric activity. The results of experiments using 5 - H T confirm the above findings. Injection of 5 - H T into the cricket's protocerebrum causes a general increase in the insect's locomotor activity, but without disturbing its rhythm. The lack of distinct changes in locomotor activity after injecting different doses of 5 - H T into the cricket's
ACTIVITY
32x
haemolymph may be caused by the existence of the haemolymph-brain barrier. T h e results of the action of 5 - H T would appear to be understandable in the light of the studies made by Kerkut and Walker (x962), who established the responses of neurons in molluscs after injecting this substance into the interior of the nerve ganglion, and aiso those made by Dudel (x965a , b) who showed that 5-HT exerts a facilitatory action on the neuromuscular junctions in crayfish. It would therefore seem that 5 - H T may participate in regulating locomotor activity rhythm in crickets, the action probably consisting in circadian variations in the concentration of this neurohormone in C.N.S. This suggestion is confirmed by the results of experiments by Fowler and Goodnight (x966a , b), who showed that 5-HT is produced in a cyclic manner in the brain and intestine of Arachnidae (Leibunum logipes) and attains a maximum at o2.oo hours. These authors suggest that there is a correlation between concentration of 5 - H T in the tissues examined and the increase in the locomotor activity of these animals. Similar experiments are being carried out on crickets at the present time. ACKNOWLEDGEMENT
I thank Docent Dr. J. Gill for his critical reading of the manuscript. REFERENCES
ACHBR, R., and FRObtAGBOT, C. (x957), ' T h e relationship of oxytocin and vasopre~sin to the active proteins of posterior pituitary origin. Studies concerning the existence or nonexistence of a single neurohypophysial hormone ', in The aVeuroh.ypophysis (ed. H~LLm% H.), pp. 395o. London: Butterworths. ARV% L., and G~E, M. (I962), ' Histochemistry of the neurosecretory product of the pars intercerebralis of Pterygote insects ', in Areurosecretion (ed. HELLZR, H., and CLARK, R. B.), pp. 33x-344. New York: Academic Press. BRADY, J. (i967a), 'Control of the circadian rhythm of activity in the cockroach. I. The role of the corpora cardiaca, brain and stress ', .7. exp. Biol., 47, x53-x63. - - - - (i967b), ' Control of the circadian rhythm of activity in the cockroach. II. The role of the sub-ocsophageal ganglion and ventral nerve cord ', Ibid., 47, 165-I78-
322
C Y M B O R O W S K I
Bg~vY,J. 0969), 'How areimect circadianrhythms controlled? ', Nature, Lond., aa'3b 78x-784. ~OLHOUN, E. H. (196o), 'Acetylocholine in Periplaneta americana L. IV. The significance of esterase inhibition in intoxication, acetyleholine levels, and nervous conduction ', Can. 07. Biochem. Physiol., 38, 1363-1376. - - - - ( I 9 6 4 ) , Comparative aVeurocherm'stry, p. 333Oxford: Richter. Coa-rP.~ta~, G. A., and I.~VE~a~CK, M. S. (1968), ' Invertebrate pharmacology ', A. Rev. Pharma¢.,
8, -,+~3-0,~.
CYm3oaowsm, B. (1968), ' Badania nad neurohormonaln~ regulacj~ dobowego cyklu aktywno[ci ruchowej .~wierszcza domowego (Achaa domes~us L.)', Doctoral Thesis, University of Warsaw. - - 0969a), ' T h e effect of light on the circadian rhythm of locomotor activity of the house-cricket (Acheta domesticus L . ) ' , 2~oologica Pol., xg, 85-95. - - (t969b), ' Aparaty do rejestracji cykliczno~ci ruchowej owad6w ', Ekol. pol. B, x.~ x29t38. ----097oa), 'Investigations on the neurohormonal factors controlling circadian rhythm of locomotor activity in the house cricket (Acheta d0mesticus L.). I. The role of the brain and suboesophageal ganglion ', ,~oologica Pol. 2o, io3-i25. ----(i97ob), 'Investigations on the neurohormonal factors controlling circadian rhythm of locomotor activity in the house cricket (Acheta domesticus L.). II. Daily histochemical changes in the neurosecretory cells of the pars intercerebralis and suboesophageal ganglion ', Ibid., ao~ I27-I49. - - and Dtrr~ows~a, A. (t968), ' R N A synthesis in the neurosecretory cells of the brain and in the suboesophageal ganglion of the house cricket (Acheta domesticus L.), Orthoptera: Gryllidae', Bull. Acad. pol. Sci., s6~ 497-499. (t969), ' C i r c a d i a n changes in R N A synthesis in the neurosecretory cells of the brain and suboesophageal ganglion of the house cricket ', 07. Insect Physiol., xS, t 187-x ~97. (t97o), ' Circadian changes in protein synthesis in the neurosecretory ceils of the central nervous system of Acheta dom~sticus', Ibid., x6, 34~-348. DVDEL, J. (x965a),' Presynaptic and postsynaptic effects of inhibitory drugs on the crayfish neuromuscular junction ', Pfl,~gers Arch. ges. Physiol., ~ ' ~ ~o4-~ x8. - - - - 0965b), ' The mechanism of presynaptic inhibition at the crayfish neuromuscular junction ', Ibid., a~.b 66-8o.
FOWX~R, D. J., and GoonmonT, C. J. 0966a), ' The cyclic production of 5-hydroxytryptamine in the opilionid ', Am. Z0ol., 6, tf7-x93. (I966b), ' D a i l y rhythms in the arachnid, Leionunum longipes ', Ibid., 6, 529 . ~n,J. E. (t955), ' Control of diurnal rhythms of activity in Periplanaa americana L.', .Nature, Lond., a75, 773. - - - - ( x 9 5 6 ) , 'Factors controlling the diurnal rhythm of activity of Periplaneta americana L.', 3. exp. Biol., 3 3 ~ 224-234. - - - (x96oa), ' Endocrine and nervous factors in insect circadian rhythms ', Cold Spring Hurb. Syrup. quant. Biol., 25~ 279-287. - - (x96ob), ' Internal factors controlling the suboesophageal ganglion neurosecretory cycle in Periplaneta americana L.', 07. exp. Biol., 37, x64-17o. Icmga~wA, M., and I s t a m ~ , H. (t963), ' Protein nature of the brain hormone of ~ t s ', Nature, Lond., x98, 3o8-3o 9. K~m~rr, G. A. (t967) , 'Biochemical aspects of invertebrate nerve ceils ', in The Invertebrate .NervousSystem (ed. W m ~ , C. A. G.), pp. 5-37. Chicago: Chlcago University Press. - - - - and WALKER,R. J. (t962), ' The specific chemical sensitivity of Helix nerve cells', Comp. Biochem. Physiol., 7, a77-288. KOSTOWS~, W., BZCK, J., and M~zAgos, J. (t966), ' Studies on the effect of certain neurohormones and psychotropic drugs on bioelectric activity of the central nervous system and behaviour in ants (Formica tufa L.)', Acta physiol. pol., x7, 98-I xo. Ntsnn-rstrr~jbUwo, J., PETROPULOS, S. F., and Pri-r~m~mos, C. S. (1967), ' C e n t r a l nervous system control of circadian rhythmicity in the cockroach. I. The role of the pars intercerebralis ', Biol. Bull. mar. biol. Lab., Woods Hole, t33, 679-696. RonEg~, S. K. DE F. 0966), ' Circadian activity rhythm in cockroaches. I I I . The role of endocrine and neural factors ', 07. cell. comp. Physiol., 67, 473-486. SCHAm~R, E., and S C H W a , B. (x954), ' Hormones produced by neurosecretory cells ', Rec. Prog. Horm. Res., xo~ I83-232. WEmH, J. H., and MoonH~a~v, M. (x96o), ' The quantitative distribution of 5-hydroxytryptamine in the invertebrates, especially in their nervom system ', 07. Neurochem., 6, t46-x69.
Key Word Index: Circadian rhythm, locomotor activity, LSD, reserpine, serotonin, 5-hydroxytryptamine, house-crlcket, Acheta domesticus, l~ychotropic drugs.
Comp. gen. Pharmac.
THE ASSUMED PARTICIPATION OF 5 - H Y D R O X Y T R Y P T A M I N E IN R E G U L A T I O N OF THE CIRCADIAN R H Y T H M OF L O C O M O T O R ACTIVITY IN ACHETA DOMESTICUS L. BRONISLAW CYMBOROWSKI Department of Animal Physiology, Zoological Institute, University of Warsaw, Poland (Received 31 March, 197o)
ABSTRACT 1. Locomotor activity of Acheta domesticus L. from alternating light and dark conditions was studied after injecting reserpine, LSD, and 5-hydroxytryptamine. 2. Great disturbances to locomotor activity, consisting of reduction of the level of locomotor activity and abolition of its rhythm, were caused by injecting reserpine and LSD. 3. These changes, as well as the effect of 5-HT, suggest that serotonin participates in regulation of circadian rhythm of locomotor activity of these insects.
House crickets, Acheta domesticus L., kept from the time of hatching to attainment of sexual maturity under LD I2:12 (light 12 hours: dark i~ hours) conditions exhibit very distinct locomotor activity rhythm (Cymborowski, 1968 , i969a , I97oa ). The characteristic rhythm is also maintained for a certain time after these insects are transferred to constant light conditions (LL). We are thus faced with the problem of the endogenic regulation of this process. A large number of experiments have been carried out on the mechanism regulating locomotor activity in insects which point to the fact that the neurohormonal system takes part in this process (Harker, I955, 1956 , I96oa, b; Roberts, I966; Brady, 1967a, b, 1969; Nishiitsutsuji-Uwo, Petropulos, and Pittendrigh, I967 ; Cymborowski, I968). It has been shown in studies on crickets taken from LD i2:12 conditions that there is circadian rhythm of R N A synthesis in the neurosecretory cells of pars intercerebralis and the suboesophageal ganglion (Cymborowski and Dutkowski, x968, t 969; Cymborowski, I97ob), and of protein synthesis in these centres (Cymborowski and Dutkowski, I97O ).
Simultaneously rhythm is observed to exist in accumulation and liberation of neurosecretion in the neurosecretory cells of these insects (Cymborowski, i97ob). The histochemical changes presented above and in particular the variations in accumulation and liberation of neurosecretion have been linked to the locomotor activity rhythm in the insects examined. This connexion would appear to be justified in view of the results of analogous studies made using crickets which did not exhibit locomotor activity rhythm and had been kept under LL conditions for the whole period. N o rhythmically occurring histochemical changes in the C.N.S. centres examined were observed in this group of insects. Data in literature show that the neurosecretion is a protein substance (Arvy and Gabe, I962; Ichikawa and Ishizaki, 1963). It can, however, be only a carrying substance in relation to the biologically active compound (Scharrer and Scharrer, 1954; Acher and Fromageot, x957). In the present study pharmacological investigations have been undertaken for the purpose of defining the hypothetical nature
197o , x, 3 1 6 - 3 2 2
R E G U L A T I O N OF L O C O M O T O R A C T I V I T Y
o f the n e u r o s e c r e t o r y s u b s t a n c e t a k i n g p a r t in r e g u l a t i o n o f the l o c o m o t o r a c t i v i t y r h y t h m in insects. A t t e n t i o n has b e e n p a i d to 5 - H T , the p r e s e n c e o f w h i c h in the n e r v o u s s y s t e m o f insect w o u l d a p p e a r to be indisputable (Welsh and Moorhead, t96o ; Colh o u n , i 9 6 o , i964). K e r k u t (1967) , after r e v i e w i n g the o c c u r r e n c e o f 5 - H T in i n v e r t e b r a t e s , was i n c l i n e d
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Locomotor activity in these insects was determined in an actograph of the author's own construction (Cymborowski, 1969b ). The conditions in the actograph were similar to the culture conditions. Only those insects exhibiting very distinct locomotor activity rhythm were used for the pharmacological examinations. Observations were made of the effect of reserpine (serpasil), diethylamide of lysergic acid (LSD-25), and serotonin (5-HT) on the loco-
Fie;. I.--Activity records of A. domesticus taken from L D 12:12 conditions. After finding that there were no changes in activity rhythm following injection of distilled H20, 5 lag. of reserpine per g. bodyweight of insect were injected into its haemolymph. The same amount of reserpine was given the next day. Each horizontal line represents the 24-hour activity of the cricket. The vertical white lines indicate the movements made by the insect. A time scale is added at the bottom. The beginning and end of the darkness period are marked.
Fie,. 2.--Activity records of A. domesticus, from LD 12 : x2 conditions, which had 7 lag. of reserpine per g. body-weight injected into its haemolymph on the fifth day after transfer to the actograph. Disturbances were observed in locomotor activity rhythm for two whole days (5, 6). Further explanations as tbr/'~g. I. to a s s u m e t h a t it is a n e u r o t r a n s m i t t e r s u b s t a n c e , a l t h o u g h it does n o t m e e t all the c r i t e r i a u s u a l l y a p p l i e d to such c o m p o u n d s . MATERIAL AND METHOI)S Experiments were made using male house crickets (Acheta domesticus L.) at the age of about i week after ecdvsis. The insects were kept in a large glass container at a temperature of about 29:" C. under conditions of alternating light and darkness LD 12:x2 (that is, ~2 hours light and 12 hours darkness during the 24-hour period). In this particular culture darkness began at 18.oo. Light was supplied by a 4 o W. electric bulb.
motor activity rhythm of the crickets. These substances were injected in a sterilized H~O solution in the following doses: reserpine 5 - i o lag. per g. of the insects' body-weight; LSD 2 -Io lag. per g. of body-weight; and 5 - H T i - 5 lag. per g. of body-weight. The injections were made into the haemolymph using a microsyringe. The volume of the injected fluid was 5/al. In the case of 5 - H T injections were also made into the protocerebrum using a microcapillary syringe, approximately 5o la in diameter. The volume of the fluid injected was 0- 5 lal. Injections were made without using anaesthetics, but the insects were subjected to a short period of hypothermy. The control experiments
3x8
CYMBOROWSKI
showed that this operation does not affect the crickets' locomotor activity rhythm. Control operations were also made by injecting a given amount of distilled water in which the substances investigated were next dissolved. Observation of the insects' locomotor activity was begun immediately after iil.jection. Each experiment was repeated at least 5 times. RESULTS Reserpine i n t r o d u c e d into the h a e m o l y m p h in a m o u n t s o f 5 lag- per g. of the
Comp. gen. Pharmac.
a n d the first h a l f of the day. After this p e r i o d the r h y t h m takes on a c h a r a c t e r similar to initial r h y t h m a n d d i s t u r b a n c e does not occur again. A p p l i c a t i o n of a single dose of reserpine in a m o u n t s of 7 Pg. per g. of the insect's b o d y weight causes abolition of the insect's locom o t o r activity r h y t h m for 2 days (Fig. 2). I m m e d i a t e l y after giving reserpine considerable inhibition of l o c o m o t o r activity is observed lasting for the whole of the 24-hour
FIo. 3.--Activity records ofA. domesticus from LD 12 : x2 conditions, which had xo lag. of reserpine per g. body-weight injected into its haemolymph on the sixth day after transfer to the actograph. Considerable inhibition of locomotor activity was observed for two whole days. Further explanations as tbr Fig. x.
Fro. 4.--Activity records of A. domesticus from LD 12 : x2 conditions, which had 5 lag. of LSD-25 per g. body-weight injected into its haemolymph at different hours. Disturbances in locomotor activity rhythm were observed on the sixth day. Further explanations as for Fig. I. cricket's b o d y - w e i g h t does n o t cause disturbances in the l o c o m o t o r activity r h y t h m of this insect (Fig. I, d a y 4)- W h e n the same a m o u n t o f reserpine is a d m i n i s t e r e d again on the next d a y it causes a shift in p e a k locom o t o r activity from the hours of I 8 - I 9 to 23-24 (Pig. I, d a y 5). After this injection ( d u r i n g the following day) d i s t u r b a n c e o f activity is still observed, consisting p r i m a r i l y in its increase, which is p a r t i c u l a r l y easy to observe d u r i n g the second h a l f of the n i g h t
period. O n the second d a y after injection a g r a d u a l increase is observed in the insect's activity until h y p e r a c t i v i t y occurs (Fig. 2, d a y 6). W h e n reserpine is a d m i n i s t e r e d in a m o u n t s of i o lag. p e r g. of b o d y - w e i g h t it causes a b o l i t i o n of locomotor activity r h y t h m for 2 days also, but d u r i n g this p e r i o d the cricket's activity is greatly r e d u c e d in comparison with the previous dose (Fig. 3). O n the third d a y after giving reserpine a p e a k of locomotor
I97o , I
R E G U L A T I O N OF LOCOMOTOR A C T I V I T Y
activity appears at the normal time of the day, but even so it is far smaller than the normal. Continued increase in doses of reserpine usually causes the insect's death. T h e short outline given here of experiments using reserpine illustrates the typical course they take. Certain modifications were obtained in some repeats of the experiments, consisting mainly in changes in the level of the locomotor activity of the insects concerned.
319
injection (Fig. 4, day 6). It is remarkable here that such disturbances occur more often when using the same doses of LSD, but given at different times during the 24-hour period (F/g. 4). It is not until a dose is given twice as great as the preceding one (i.e., io lag. per g. bodyweight) that it causes abolition of locomotor activity r h y t h m a n d reduction in its level, but only over the course of 2 4 hours (Fig. 5). T h e normal peak of locomotor activity usually
Fro. 5.--Activity records ofA. domesticus from LD 12 : 12 conditions, which had xo lag. of LSD per g. body-weight injected into its haemolymph on the fifth day after transfer to the aclograph. Inhibition of locomotor activity was observed for one whole day (5). Further explanations as for Fig. I.
Fro. 6.--Activity records of A. domesticus from LD x2 : x2 conditions, which had I lag. of 5-HT per g. body-weight injected into haemolymph. There were no changes in locomotor activity. Further explanations as for Fig. I. Injection of LSD into the h a e m o l y m p h of rhythmic crickets produces, in respect of locomotor activity, results differing greatly from results obtained when using reserpine. Single doses of 5 lag- per g. of the insect's body-weight do not cause changes in locom o t o r activity rhythm. Similarly if the same dose is given again on the second day it does not cause any changes. In certain cases slight disturbances were observed in locomotor activity r h y t h m occurring during the second day after
occurred on the next day. Hyperactivity was sometimes observed after this period. Injection ofserotonin into the h a e m o l y m p h of crickets, using amounts from I to 5 tag. per g. of the insect's body-weight, causes practically no significant changes in their activity r h y t h m (Figs. 6, 7). In some cases, after using 5 - H T in doses of 5 lag • per g. body-weight, slight changes occur in activity consisting in increase in activity immediately after injection, and shortening of the activity peak (Fig. 7).
32o
CYMBOROWSKI
However, when 5 - H T is injected in doses of i - 5 lag. per g. body-weight into the protocerebrum of r h y t h m i c crickets it causes considerable changes in their locomotor activity. Activity is observed to increase, however, without any distinct changes in its rhythm. After giving 5 lag. ofserotonin per g. of body-weight a very great increase takes place in the cricket's activity (Fig. 8). T h e crickets usually exhibit no great changes during the first day tbllowing this dose, but
per the the
Comp. gen. Pharmac.
g.
body-weight they usually exhibit typical reaction obtained in previous experiments (Fig. 8, days
9-12). DISCUSSION T h e data presented show that reserpine and LSD affect the cricket's locomotor activity. Reserpine exerts a particularly distinct action inhibiting locomotor activity. This would suggest that disturbances are
I'm. 7.--Activity records of A. domesticus from LD I2 : I2 conditions, which had 5 lag. of 5-HT per g. body-weight injected into its haemolymph on the fourth day after transfer to the aetograph. Slight disturbances in locomotor rhythm were observed atier injection. Further explanations as tbr Fig. I.
Fro. 8.--Activity records ofA. domesticus from LD 3~>: x2 conditions, which had 5 lag. of 5-HT per g. body-weight injected into the protocerebrum on the third day after transfer to the actograph. A gradual increase in locomotor activity was observed. On the ninth day of observations 7 lag. of reserpine per g. body-weight were injected into the haemolymph. Inhibition of locomotor activity is observed (9, 3o). Further explanations as for Pig. I. intensified activity takes place on the second day. T h e particularly great increase in activity after darkness falls is remarkable. T h e insects usually die within I-3" 5 weeks after such an injection. I f the insects which had received 5 - H T and exhibit considerable activity are injected with reserpine in amounts from 7 to IO lag.
created in the integrating centres of C.N.S. in these insects. These results are confirmed by data obtained in studies carried out on ants (Formica rufa L.) by Kostowski, Beck, and Meszaros (1966). W h e n these insects are given o ' t - o " 5 lag. of reserpine per mg. bodyweight with their food it causes marked inhibition of spontaneous locomotor activity,
I970 , l
REGULATION
OF LOCOMOTOR
and also changes in the bioelectric activity of the protocerebrum. Variations in the action of reserpine as the result of previous application of niamides lead these authors to the conclusion that the effect of reserpine depends on the concentration and distribution of biogenic amines, in the same way as in vertebrates. It is aLso known that reserpine reduces the serotonin level in the brain of certain invertebrates (see review by Gottrell and Laverack, x968). LSD also limits the action by serotonin, although in this case the mechanism is different from that connected with the effect of reserpine. The results of the action of LSD in the cricket's locomotor activity are not unequivocal and are very difficult to interpret. However, when doses of xo Ixg. per g. of the insect's body-weight are given they cause abolition of rhythm and inhibition of locomotor activity for a period of 24 hours. In none of the experiments did it prove possible to obtain abolition of rhythm for the duration of 2 days, as had been achieved by using reserpine. In the experiments on ants the administration of large doses of LSD (25-xoo lag. per g. of the body-weight) did not bring about any distinct changes in the locomotor activity of these insects (Kostowski and others, x966). It must be emphasized that in this case LSD was administered per os, which might account for the differences which occurred. These authors showed that despite the failure of LSD to affect locomotor activity, it causes considerable changes in E E G of ants. T h e data presented suggest that the effect on locomotor activity of the psychotropic drugs used in the experiments is exerted through the medium of the C.N.S., primarily as the result of disturbances in the action of 5-HT, and consequently ofbioelectric activity. The results of experiments using 5 - H T confirm the above findings. Injection of 5 - H T into the cricket's protocerebrum causes a general increase in the insect's locomotor activity, but without disturbing its rhythm. The lack of distinct changes in locomotor activity after injecting different doses of 5 - H T into the cricket's
ACTIVITY
32x
haemolymph may be caused by the existence of the haemolymph-brain barrier. T h e results of the action of 5 - H T would appear to be understandable in the light of the studies made by Kerkut and Walker (x962), who established the responses of neurons in molluscs after injecting this substance into the interior of the nerve ganglion, and aiso those made by Dudel (x965a , b) who showed that 5-HT exerts a facilitatory action on the neuromuscular junctions in crayfish. It would therefore seem that 5 - H T may participate in regulating locomotor activity rhythm in crickets, the action probably consisting in circadian variations in the concentration of this neurohormone in C.N.S. This suggestion is confirmed by the results of experiments by Fowler and Goodnight (x966a , b), who showed that 5-HT is produced in a cyclic manner in the brain and intestine of Arachnidae (Leibunum logipes) and attains a maximum at o2.oo hours. These authors suggest that there is a correlation between concentration of 5 - H T in the tissues examined and the increase in the locomotor activity of these animals. Similar experiments are being carried out on crickets at the present time. ACKNOWLEDGEMENT
I thank Docent Dr. J. Gill for his critical reading of the manuscript. REFERENCES
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Key Word Index: Circadian rhythm, locomotor activity, LSD, reserpine, serotonin, 5-hydroxytryptamine, house-crlcket, Acheta domesticus, l~ychotropic drugs.