- Email: [email protected]
The role of serotonin in regulation of the circadian rhythms of locomotor activity in the cricket (Acheta domesticus L.) II. Distribution of serotonin and variations in different brain structure
('omp. BIochem Physiol. 1978~ Vol 59C. pp 17 to 20 Per¢lamon Pres.~ Prmted m Great Britain
THE ROLE OF S E R O T O N I N IN R E G U L A T I O N OF THE CIRCADIAN RHYTHMS OF L O C O M O T O R ACTIVITY IN THE CRICKET (ACHETA DOMESTICUS L.) II. D I S T R I B U T I O N OF S E R O T O N I N A N D VARIATIONS IN D I F F E R E N T BRAIN S T R U C T U R E M. MUSZY.~SKA-PYTELand B. CYMBOROWSKI Department of Animal Physiology, University of Warsaw. Zwirki i Wigury 93. 024)89 Warszawa. Poland
(Received 21 April 1977) Abstract--l. Investigations were made on the distribution of 5-hydroxytryptamine (5-HT) level during 24 hr in the different structures of brain of crickets from LD 12:12 conditions. 2. It was found that there was a distinct correlation between 5-HT level in the central body and intensity of locomotor activity during the course of 24 hr. 3. The hypothesis was made on the role of 5-HT and neurosccretion in regulation of circadian rhythms of locomotor activity in the insects investigated.
INTRODUCTION
Serotonin is one of the biogenic amines occurring generally in invertebrates. It has been found to be present inter alia in the insect brain, where its level is high, being for instance in Perigplaneta americana 0.08/lmol/g of tissue weight (Osborn & Neuhoff. 1974), in Locusta migratoria 2.34 + 0.60~ug/g (Hiripi & S-R6zsa, 1973), in Schistocerca gregaria from 0.79 to 1.47,ug/g (Klemm & Axelsson, 1973) and in Acheta domesticus, depending on the time of day, from 1.43 to 2.66ng/mg (Muszyfiska-Pytel & Cymborowski, Part 1 of this study). In addition to the brain the presence of 5-HT has also been found in the frontal ganglion and in corpora cardiaca in Periplaneta americana (Hentschel, 1974) and in corpora cardiaca of Acheta domesticus (Muszyfiska-Pytel, in preparation). Serotonin probably plays a dual role in insects: as neurotransmitter and as hormone. It is not as yet possible exactly to define the processes it controls, but it would seem that one of them is regulation of behaviour, in particular of the circadian rhythm of the locomotor activity of insects. This is shown by the results obtained in experiments made by Kostowski et al. (1966) on the effect of reserpine a serotonin depletor on the behaviour of the ant, Formica tufa L. These authors found considerable inhibition of spontaneous locomotor activity and differences in behaviour consisting of reciprocal attacks upon one another and lack of attention to their surroundings after administering this psychotropic compound to these insects. Hinks (1967) found that administration of 5-HT to the moth, Noctua pronuba caused prolongation of the night phase of flight. Disturbance to this phase of flight was also caused by destruction of the neurosecretory cells of pars intercerebralis. Hinks (1967) found serotonin in type A cells. The level of this amine was correlated with the rhythm of flight activity. He therefore suggested that activation of the night flight phase takes place by means of secretion of serotonin from type A serotoninergic cells. In the 17 CaP
59/Ic- B
house cricket also increase in serotonin level causes hyperactivity, and disturbance in circadian rhythms of locomotor activity (Cymborowski, 1970a; Cymborowski & Muszyfiska, 1974). The centre regulating the circadian rhythm of locomotor activity in the cricket is pars intercerebralis (Cymborowski, 1970b. 1973). It was assumed that, as in the case of Noctua pronuba, some of the neurosecretory cells of pars intercerebralis will be serotoninergic cells and that they will form the serotoninergic centre taking part in regulation of locomotor activity rhythm in crickets. The existence of this centre has been suggested in Part 1 of this paper. It was also anticipated that serotonin concentration in this centre would be closely correlated with intensity of locomotor activity in the crickets. It was therefore decided to examine the distribution of 5-HT in the brain, and in turn its variations in intensity in the centre or centres in which it occurs at different times of the day in insects taken from cultures synchronized under LD 12:12 conditions. MATERIAL AND METHODS
The studies were carried out on insects cultured under LD 12:12 conditions 2hr after start of the light period, in the middle of the light phase and 2 hr after the onset of the period of darkness, this corresponded to the hours of 08.00, 12.00, 20.00 of the photoperiod applied. After excising the brains they were fixed for 4 hr in 10% formaldehyde solution, then rinsed for 3 hr in running water, washed in distilled water, dehydrated and embedded in paraffin, sectioned to 81~m and stained by the DMAB method after Adams (1957). RESULTS
1. Localization of serotonin in the brain of crickets Serotonin was not discovered in the neurosecretory cells of pars intercerebralis in the examinations made, but 5-HT was found to occur in the following structures of the cricket's brain: protocerebral bridge (Fig.
18
M. MUSZYNSKA-P','TEI.and B. CVMFI(:,ROWSKI
Fig. 1. Cross-section through protoccrcbral bridge (Mp) of the brain of a cricket. Brain taken for examination 2 hr after the start of the light period from an insect culturcd under LD 12:12 conditions. Preparation stained by the DMAB method for presence of serotonm. Fig. 2. ('ross-section through central bod~ (Cc) of the brain of a cricket. Brain taken for examination 2 hr after the start of the light period from an insect cultured under LD 12:12 conditions. Preparation stained by the DMAB method for presence of serotonin. Fig. 3. Cross-section through calyx of mushroom bod,~ (KI of the brain of a cricket. Brain taken for examination 2 hr after the start of the light period from an insect cultured under LD 12:12 conditions. Preparation stained by the method for presence of serotonin. Fig. 4. ('ross-section through the brain of a cricket taken for examination 2 hr after the start of the light period from insect cultured under LD 12:12 conditions. Preparation stained by the method for presence of serotonin. 1), central body (Figs 2 and 4). calyces of m u s h r o o m bodies (Fig. 3), pedicles, :z- and fl-lobes of m u s h r o o m bodies (Fig. 4) and in the antennal lobes of the deutocerebrum. Serotonin level was not identical in these structures. In the central b~x:ly, calyces of m u s h r o o m bodies and protocerebral bridge 5-HT concentration was higher than in :~- and fl-lobes and the pedicles of m u s h r o o m bodies and in the antennal lobes. It was also found that serotonin level varied in these structures during the course of 24 hr in crickets cultured under LD 12:12 conditions. The results of these studies are given below. 2. Variations in serotonin let'el in di(ferent brain stru. tures at diflt, rent timex ol the day The results of histochemical studies on variations in serotonin level in different brain structures dr, ring periods of low locomotor activity (hours: 08.(R) and 12.00) and intensive locomotor activity (20.00) carried out on rhythmic crickets obtained from L D 12:12 conditions are shown in diagram form in Fig. 5. Serotonin concentration was defined on the basis of degree of intensity of staining of the given structure.
The degree of the five-point scale used to define staining correspond to scrotonin levels. Two hours after the start of the light period serotonin level was found to be very high in protocerebral bridge, high m the central body and calyces, medium m :~- and fl-lobes of m u s h r o o m bodies and Iov, in the pedicles. In the middle of the day 5-HT level m the central body rose to a very high level, remained unchanged in calyces, was low in protocerebral bridge and :~-Iobes of mushroom bodies, very low m fl-lobes and pedicles of m u s h r o o m bodies. Two hours after the onset of darkness scrotonin concentration markedly decreased in some of the structures (to low level in the central body. in cal',ces to medium and in x-lobes to very low level), and in other parts remained unchanged (in //-lobes and pedicles of mt,shroom bodies on a very low level and on a low level in protocerebral bridgel. I ) I S ( ' I 'SSI()N
Adams' method (1957) used in this study is not specific for serotonin. It reveals scrotonm and also
19
Serotonin regulation of circadian rhythms other indole compounds: tryptophan, 5-hydroxytryptophan and 5-hydroxyindole acetic acid. This technique was used to examine localization of serotonin and to define variations in its level after having established that the chief indole compound of the central nervous system of these insects is serotonin (Muszyfiska-Pytel, in preparation).
/,
Neurosecretory
I"
cells
o f pars intercerebrallS r8 16
-*
[4
12
24
6
5-H T C e n t r a l body
LD800 Locomotor
activity
Fig. 6. ilypothetical diagram of locomotor activity regulation in the house cricket.
LD ; 2 0 0
~
)
~
~
~
~
~
LD2000
Fig. 5. Variations in scrotonin level at different times of the day in different brain structures of crickets cultured under LD conditions. Pi pars interccrebralis: Mp protocerebral bridge: Cc ..-central bod): K cal)x of mushroom bod); a .:c-lobe of mushroom body: b ..//-lobe of mushroom body. Degrees of scale used to define the serotonin levels.
:'.".?i:.:,5".." very low
low'
medium
high
II ,,cry high
Studies made so far on distribution of serotonin in insect brains relied chiefly on the fluorescent method. Regions of the brain in which 5-HT has been found to be present are: protoccrebral bridge, corpus ventrale, deuterocerebrum (Klemm, 1974), calyces of mushroom bodies (Schi.irmann & Klemm, 1973) and optic lobes (Elofsson & Klemm, 1972; Klemm & Axelsson, 1975). Use of the D M A B method revealed additional areas in which serotonin occurred in crickets, namely ~- and fl-lobes, pedicles of mushroom bodies and the central body, but serotonin was not discovered in the neurosecretory cells of pars intercerebralis. It was however found, as expected, that serotonin level varied in different brain structures during the day. Analysis of results obtained, made from the aspect of locomotor activity, led to the conclusion that in crickets the serotonincrgic centre taking part in regulation of their circadian rhythm of activity is the central body. This conclusion was reached on the grounds of the fact that there is a distinct correlation between 5-HT Icvel in the central body and intensity of locomotor activity. The central body was the only structure with a different distribution of 5-HT intensity during the 24-hr period. In all the others the greatest amount of scrotonin occurred at the start of the light period, which was followed by a gradual decrease in the contents of this amine or, in the case of calyces, a high level was maintained throughout the whole light period. In the central body, however, the highest 5-HT level was in the middle of the light period, while liberation of the amine probably began towards the end of the day period, since serotonin concentration was low during the first hours of night. During the same period, as that in which serotonin disappeared from the area of the central body, the level of locomotor activity rose abruptly, as did the amount of neurosecretion in the cells of pars intercerehralis (Cymborowski, 1970c). On the other hand throughout the whole period of high 5-HT levels in the region of the central body the light period-both the degree of accumulation of neurosecretion on
20
M. MUSZY.~SKA-PY/EL and B. CYMBOROWSKI
the cells of pars intercerebralis, and also the level of activity, were low. Variations in serotonin level in the area of the central body, variations in level of neurosecretion in the neurosecretory cells of pars intercerebralis and intensity of locomotor activity of crickets during the 24-hr period are shown in diagram form in Fig. 6. Neurosecretion in the neurosecretory cells of pars intercerebralis inhibits the locomotor activity of insects (Cymborowski. 1970b). Blocking of its liberation should therefore cause intensification of activity. It would seem that it is serotonin occurring in the region of the central body which performs this function and this is probably the mechanism of the part played by this structure in regulation of locomotor activity rhythm in the cricket. The mechanism of this regulation (Fig. 6) would therefore be as follows: during the day. when serotonin accumulates in the area of the central body. it does not affect the liberation of ncurosccretion from the cells of pars intercerebralis. The liberated ncurosecretion from the cells of pars intercerebralis inhibits locomotor activity, hence the level of the letter is low. At night, on the other hand, liberation of serotonin takes place from the area of the central body, which as a result causes intensification of locomotor activity. In the light of the hypothesis presented here the result, consisting in obtaining considerable inhibition of locomotor activity in insects after administration of serotonin depletors, become easy to understand. Reduction in 5-HT level caused increased liberation of neurosecretion from the cells of pars intercerebralis and in consequence inhibition of activity, whereas increase in serotonin level in the brain caused inhibition of neurosecretion, resulting in intensification of activity. REFERENCES ADAMS C. W. M. (1957) A p-dimethylaminobenzaldehyde nitrite method for the histochcmical demonstration of tryptophan and related compounds. J. clin. Path. 10, 56-62. CVMnOROWSKI B. (1970a) The assumed participation of 5-hydroxytryptamine in regulation of the circadian rhythm of locomotor activity in Aeheta domesticus L. Comp. Gen. Pharmac. 1, 316-.322. CVMBOROWSK| B. (1970b) Investigations on the neuro-
hormonal factors controlling circadian rhythm of locomotor activity in the house cricket (Acheta domesticus L.). I. The role of the brain and subesophageal ganglion. Zooloyiea Pol. 20, 103-125. CVMBOROWSKt B. (1970c) Investigation on the neurohormonal factors controlling circadian rhythm of locomotor activity in the house cricket (Acheta domesticus L.I. II. Daily histochemicaL changes in the neurosecretory cells of pars intercerebralis and subesophageal ganglion. Zooloyica Pol. 20, 127-147. CYMBOROWSKI B. (1973) Control of the circadian rhythm of locomotor activity in the house cricket. J. Insect Physiol. 16, 341 348. CVMnOROWSKI B. & Mt:SZVrqSKA M. (1974) The effect of some psychotropic drugs on the circadian rhythm of locomotor activity of Acheta domesticus L J. Interdisciplinary Cycle Res. 5, 3 4. 362 370. ELOVSSO~ R. & KLEMM N. (1972) Monoamine-containing neurones in the optic ganglia of Crustaceans and Insects. Z. ZelIJorsch. mikrosk. Anat. 133, 475-499. HI-NTSf'HEL E. (1974) Experimental investigations on the influence of ovulation in Periplaneta americana L. by aminergic substances. Gen. Comp. Endocr. 22, 411. HtNKS C F. (1967) Relationship between serotonin and the circadian rhythm in some nocturnal moths. Nature. Lond 214, 386-387. HmlPl L & S-RozsA K. (1973) Fluorimetric determination of 5-hydroxytryptamine and catecholamines in the central nervous system and heart of Loeusta migratoria Migratorioides. J. Insect Physiol. 19, 1481-1485. KLEMM N. (1974) Vergleichend-histochemische Unlersuchungen fiber die verteilung monoamin-haltiger Structuren im Oberschlundganglion yon Angehorigen verschiedener Insecten-Ordnungen. Entomologica Germ. I, 21 -49. KL[MM N. d~. AXELSSON S. (19731 Detection of dopamine. noradrenaline and 5-hydroxytryptamine in the cerebral ganglion of the desert locust Schistocerca 9reqaria Forsk. (Insecta, Orthoptera). Brain Res. 57. 289 298. Kos'rowsKl W., BECK J. & MESZAROSJ. (1966) Studies on the effect of certain neurohormones and psychotropic drugs of bioelectrical activity of the central nervous system and behaviour in ants. Formica rufa L. Acta physiol. Pol. XVII (1), 98-110. MUSZYS~SKA-PYTEL & CYMBOROWSK1 B. (in press). OSBORN N. & NEUHOFF V. 0974) Amino acid and serotonin content in the nervous system, muscle and blood of the cockroach Periplaneta. Brain Res. 80, 251-264. SCIIURMANN F. & KLEMM N. (1973) Zur Monoaminverteilung in den Corpora pedunculata des Gehirns yon Acheta domesticus L. (Orthoptera, Insecta). Z Zellforsch. mikrosk. Anat. 136(3). 393-414.
THE ROLE OF S E R O T O N I N IN R E G U L A T I O N OF THE CIRCADIAN RHYTHMS OF L O C O M O T O R ACTIVITY IN THE CRICKET (ACHETA DOMESTICUS L.) II. D I S T R I B U T I O N OF S E R O T O N I N A N D VARIATIONS IN D I F F E R E N T BRAIN S T R U C T U R E M. MUSZY.~SKA-PYTELand B. CYMBOROWSKI Department of Animal Physiology, University of Warsaw. Zwirki i Wigury 93. 024)89 Warszawa. Poland
(Received 21 April 1977) Abstract--l. Investigations were made on the distribution of 5-hydroxytryptamine (5-HT) level during 24 hr in the different structures of brain of crickets from LD 12:12 conditions. 2. It was found that there was a distinct correlation between 5-HT level in the central body and intensity of locomotor activity during the course of 24 hr. 3. The hypothesis was made on the role of 5-HT and neurosccretion in regulation of circadian rhythms of locomotor activity in the insects investigated.
INTRODUCTION
Serotonin is one of the biogenic amines occurring generally in invertebrates. It has been found to be present inter alia in the insect brain, where its level is high, being for instance in Perigplaneta americana 0.08/lmol/g of tissue weight (Osborn & Neuhoff. 1974), in Locusta migratoria 2.34 + 0.60~ug/g (Hiripi & S-R6zsa, 1973), in Schistocerca gregaria from 0.79 to 1.47,ug/g (Klemm & Axelsson, 1973) and in Acheta domesticus, depending on the time of day, from 1.43 to 2.66ng/mg (Muszyfiska-Pytel & Cymborowski, Part 1 of this study). In addition to the brain the presence of 5-HT has also been found in the frontal ganglion and in corpora cardiaca in Periplaneta americana (Hentschel, 1974) and in corpora cardiaca of Acheta domesticus (Muszyfiska-Pytel, in preparation). Serotonin probably plays a dual role in insects: as neurotransmitter and as hormone. It is not as yet possible exactly to define the processes it controls, but it would seem that one of them is regulation of behaviour, in particular of the circadian rhythm of the locomotor activity of insects. This is shown by the results obtained in experiments made by Kostowski et al. (1966) on the effect of reserpine a serotonin depletor on the behaviour of the ant, Formica tufa L. These authors found considerable inhibition of spontaneous locomotor activity and differences in behaviour consisting of reciprocal attacks upon one another and lack of attention to their surroundings after administering this psychotropic compound to these insects. Hinks (1967) found that administration of 5-HT to the moth, Noctua pronuba caused prolongation of the night phase of flight. Disturbance to this phase of flight was also caused by destruction of the neurosecretory cells of pars intercerebralis. Hinks (1967) found serotonin in type A cells. The level of this amine was correlated with the rhythm of flight activity. He therefore suggested that activation of the night flight phase takes place by means of secretion of serotonin from type A serotoninergic cells. In the 17 CaP
59/Ic- B
house cricket also increase in serotonin level causes hyperactivity, and disturbance in circadian rhythms of locomotor activity (Cymborowski, 1970a; Cymborowski & Muszyfiska, 1974). The centre regulating the circadian rhythm of locomotor activity in the cricket is pars intercerebralis (Cymborowski, 1970b. 1973). It was assumed that, as in the case of Noctua pronuba, some of the neurosecretory cells of pars intercerebralis will be serotoninergic cells and that they will form the serotoninergic centre taking part in regulation of locomotor activity rhythm in crickets. The existence of this centre has been suggested in Part 1 of this paper. It was also anticipated that serotonin concentration in this centre would be closely correlated with intensity of locomotor activity in the crickets. It was therefore decided to examine the distribution of 5-HT in the brain, and in turn its variations in intensity in the centre or centres in which it occurs at different times of the day in insects taken from cultures synchronized under LD 12:12 conditions. MATERIAL AND METHODS
The studies were carried out on insects cultured under LD 12:12 conditions 2hr after start of the light period, in the middle of the light phase and 2 hr after the onset of the period of darkness, this corresponded to the hours of 08.00, 12.00, 20.00 of the photoperiod applied. After excising the brains they were fixed for 4 hr in 10% formaldehyde solution, then rinsed for 3 hr in running water, washed in distilled water, dehydrated and embedded in paraffin, sectioned to 81~m and stained by the DMAB method after Adams (1957). RESULTS
1. Localization of serotonin in the brain of crickets Serotonin was not discovered in the neurosecretory cells of pars intercerebralis in the examinations made, but 5-HT was found to occur in the following structures of the cricket's brain: protocerebral bridge (Fig.
18
M. MUSZYNSKA-P','TEI.and B. CVMFI(:,ROWSKI
Fig. 1. Cross-section through protoccrcbral bridge (Mp) of the brain of a cricket. Brain taken for examination 2 hr after the start of the light period from an insect culturcd under LD 12:12 conditions. Preparation stained by the DMAB method for presence of serotonm. Fig. 2. ('ross-section through central bod~ (Cc) of the brain of a cricket. Brain taken for examination 2 hr after the start of the light period from an insect cultured under LD 12:12 conditions. Preparation stained by the DMAB method for presence of serotonin. Fig. 3. Cross-section through calyx of mushroom bod,~ (KI of the brain of a cricket. Brain taken for examination 2 hr after the start of the light period from an insect cultured under LD 12:12 conditions. Preparation stained by the method for presence of serotonin. Fig. 4. ('ross-section through the brain of a cricket taken for examination 2 hr after the start of the light period from insect cultured under LD 12:12 conditions. Preparation stained by the method for presence of serotonin. 1), central body (Figs 2 and 4). calyces of m u s h r o o m bodies (Fig. 3), pedicles, :z- and fl-lobes of m u s h r o o m bodies (Fig. 4) and in the antennal lobes of the deutocerebrum. Serotonin level was not identical in these structures. In the central b~x:ly, calyces of m u s h r o o m bodies and protocerebral bridge 5-HT concentration was higher than in :~- and fl-lobes and the pedicles of m u s h r o o m bodies and in the antennal lobes. It was also found that serotonin level varied in these structures during the course of 24 hr in crickets cultured under LD 12:12 conditions. The results of these studies are given below. 2. Variations in serotonin let'el in di(ferent brain stru. tures at diflt, rent timex ol the day The results of histochemical studies on variations in serotonin level in different brain structures dr, ring periods of low locomotor activity (hours: 08.(R) and 12.00) and intensive locomotor activity (20.00) carried out on rhythmic crickets obtained from L D 12:12 conditions are shown in diagram form in Fig. 5. Serotonin concentration was defined on the basis of degree of intensity of staining of the given structure.
The degree of the five-point scale used to define staining correspond to scrotonin levels. Two hours after the start of the light period serotonin level was found to be very high in protocerebral bridge, high m the central body and calyces, medium m :~- and fl-lobes of m u s h r o o m bodies and Iov, in the pedicles. In the middle of the day 5-HT level m the central body rose to a very high level, remained unchanged in calyces, was low in protocerebral bridge and :~-Iobes of mushroom bodies, very low m fl-lobes and pedicles of m u s h r o o m bodies. Two hours after the onset of darkness scrotonin concentration markedly decreased in some of the structures (to low level in the central body. in cal',ces to medium and in x-lobes to very low level), and in other parts remained unchanged (in //-lobes and pedicles of mt,shroom bodies on a very low level and on a low level in protocerebral bridgel. I ) I S ( ' I 'SSI()N
Adams' method (1957) used in this study is not specific for serotonin. It reveals scrotonm and also
19
Serotonin regulation of circadian rhythms other indole compounds: tryptophan, 5-hydroxytryptophan and 5-hydroxyindole acetic acid. This technique was used to examine localization of serotonin and to define variations in its level after having established that the chief indole compound of the central nervous system of these insects is serotonin (Muszyfiska-Pytel, in preparation).
/,
Neurosecretory
I"
cells
o f pars intercerebrallS r8 16
-*
[4
12
24
6
5-H T C e n t r a l body
LD800 Locomotor
activity
Fig. 6. ilypothetical diagram of locomotor activity regulation in the house cricket.
LD ; 2 0 0
~
)
~
~
~
~
~
LD2000
Fig. 5. Variations in scrotonin level at different times of the day in different brain structures of crickets cultured under LD conditions. Pi pars interccrebralis: Mp protocerebral bridge: Cc ..-central bod): K cal)x of mushroom bod); a .:c-lobe of mushroom body: b ..//-lobe of mushroom body. Degrees of scale used to define the serotonin levels.
:'.".?i:.:,5".." very low
low'
medium
high
II ,,cry high
Studies made so far on distribution of serotonin in insect brains relied chiefly on the fluorescent method. Regions of the brain in which 5-HT has been found to be present are: protoccrebral bridge, corpus ventrale, deuterocerebrum (Klemm, 1974), calyces of mushroom bodies (Schi.irmann & Klemm, 1973) and optic lobes (Elofsson & Klemm, 1972; Klemm & Axelsson, 1975). Use of the D M A B method revealed additional areas in which serotonin occurred in crickets, namely ~- and fl-lobes, pedicles of mushroom bodies and the central body, but serotonin was not discovered in the neurosecretory cells of pars intercerebralis. It was however found, as expected, that serotonin level varied in different brain structures during the day. Analysis of results obtained, made from the aspect of locomotor activity, led to the conclusion that in crickets the serotonincrgic centre taking part in regulation of their circadian rhythm of activity is the central body. This conclusion was reached on the grounds of the fact that there is a distinct correlation between 5-HT Icvel in the central body and intensity of locomotor activity. The central body was the only structure with a different distribution of 5-HT intensity during the 24-hr period. In all the others the greatest amount of scrotonin occurred at the start of the light period, which was followed by a gradual decrease in the contents of this amine or, in the case of calyces, a high level was maintained throughout the whole light period. In the central body, however, the highest 5-HT level was in the middle of the light period, while liberation of the amine probably began towards the end of the day period, since serotonin concentration was low during the first hours of night. During the same period, as that in which serotonin disappeared from the area of the central body, the level of locomotor activity rose abruptly, as did the amount of neurosecretion in the cells of pars intercerehralis (Cymborowski, 1970c). On the other hand throughout the whole period of high 5-HT levels in the region of the central body the light period-both the degree of accumulation of neurosecretion on
20
M. MUSZY.~SKA-PY/EL and B. CYMBOROWSKI
the cells of pars intercerebralis, and also the level of activity, were low. Variations in serotonin level in the area of the central body, variations in level of neurosecretion in the neurosecretory cells of pars intercerebralis and intensity of locomotor activity of crickets during the 24-hr period are shown in diagram form in Fig. 6. Neurosecretion in the neurosecretory cells of pars intercerebralis inhibits the locomotor activity of insects (Cymborowski. 1970b). Blocking of its liberation should therefore cause intensification of activity. It would seem that it is serotonin occurring in the region of the central body which performs this function and this is probably the mechanism of the part played by this structure in regulation of locomotor activity rhythm in the cricket. The mechanism of this regulation (Fig. 6) would therefore be as follows: during the day. when serotonin accumulates in the area of the central body. it does not affect the liberation of ncurosccretion from the cells of pars intercerebralis. The liberated ncurosecretion from the cells of pars intercerebralis inhibits locomotor activity, hence the level of the letter is low. At night, on the other hand, liberation of serotonin takes place from the area of the central body, which as a result causes intensification of locomotor activity. In the light of the hypothesis presented here the result, consisting in obtaining considerable inhibition of locomotor activity in insects after administration of serotonin depletors, become easy to understand. Reduction in 5-HT level caused increased liberation of neurosecretion from the cells of pars intercerebralis and in consequence inhibition of activity, whereas increase in serotonin level in the brain caused inhibition of neurosecretion, resulting in intensification of activity. REFERENCES ADAMS C. W. M. (1957) A p-dimethylaminobenzaldehyde nitrite method for the histochcmical demonstration of tryptophan and related compounds. J. clin. Path. 10, 56-62. CVMnOROWSKI B. (1970a) The assumed participation of 5-hydroxytryptamine in regulation of the circadian rhythm of locomotor activity in Aeheta domesticus L. Comp. Gen. Pharmac. 1, 316-.322. CVMBOROWSK| B. (1970b) Investigations on the neuro-
hormonal factors controlling circadian rhythm of locomotor activity in the house cricket (Acheta domesticus L.). I. The role of the brain and subesophageal ganglion. Zooloyiea Pol. 20, 103-125. CVMBOROWSKt B. (1970c) Investigation on the neurohormonal factors controlling circadian rhythm of locomotor activity in the house cricket (Acheta domesticus L.I. II. Daily histochemicaL changes in the neurosecretory cells of pars intercerebralis and subesophageal ganglion. Zooloyica Pol. 20, 127-147. CYMBOROWSKI B. (1973) Control of the circadian rhythm of locomotor activity in the house cricket. J. Insect Physiol. 16, 341 348. CVMnOROWSKI B. & Mt:SZVrqSKA M. (1974) The effect of some psychotropic drugs on the circadian rhythm of locomotor activity of Acheta domesticus L J. Interdisciplinary Cycle Res. 5, 3 4. 362 370. ELOVSSO~ R. & KLEMM N. (1972) Monoamine-containing neurones in the optic ganglia of Crustaceans and Insects. Z. ZelIJorsch. mikrosk. Anat. 133, 475-499. HI-NTSf'HEL E. (1974) Experimental investigations on the influence of ovulation in Periplaneta americana L. by aminergic substances. Gen. Comp. Endocr. 22, 411. HtNKS C F. (1967) Relationship between serotonin and the circadian rhythm in some nocturnal moths. Nature. Lond 214, 386-387. HmlPl L & S-RozsA K. (1973) Fluorimetric determination of 5-hydroxytryptamine and catecholamines in the central nervous system and heart of Loeusta migratoria Migratorioides. J. Insect Physiol. 19, 1481-1485. KLEMM N. (1974) Vergleichend-histochemische Unlersuchungen fiber die verteilung monoamin-haltiger Structuren im Oberschlundganglion yon Angehorigen verschiedener Insecten-Ordnungen. Entomologica Germ. I, 21 -49. KL[MM N. d~. AXELSSON S. (19731 Detection of dopamine. noradrenaline and 5-hydroxytryptamine in the cerebral ganglion of the desert locust Schistocerca 9reqaria Forsk. (Insecta, Orthoptera). Brain Res. 57. 289 298. Kos'rowsKl W., BECK J. & MESZAROSJ. (1966) Studies on the effect of certain neurohormones and psychotropic drugs of bioelectrical activity of the central nervous system and behaviour in ants. Formica rufa L. Acta physiol. Pol. XVII (1), 98-110. MUSZYS~SKA-PYTEL & CYMBOROWSK1 B. (in press). OSBORN N. & NEUHOFF V. 0974) Amino acid and serotonin content in the nervous system, muscle and blood of the cockroach Periplaneta. Brain Res. 80, 251-264. SCIIURMANN F. & KLEMM N. (1973) Zur Monoaminverteilung in den Corpora pedunculata des Gehirns yon Acheta domesticus L. (Orthoptera, Insecta). Z Zellforsch. mikrosk. Anat. 136(3). 393-414.