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Circadian changes in protein synthesis in the neurosecretory cells of the central nervous system of Acheta domesticus
J. Insect Physiol., 1970, Vol. 16,~~. 341 to 348. Pergamon Press. Printed in Great Britain
CIRCADIAN CHANGES IN PROTEIN SYNTHESIS IN THE NEUROSECRETORY CELLS OF THE CENTRAL NERVOUS SYSTEM OF ACHETA DOMESTICUS BRONISLAW
and ANDRZEJ
CYMBOROWSKI
DUTKOWSKI
Department of Comparative Animal Physiology, Zoological Institute, University of Warsaw, and Department of Biochemistry, M. Nencki Institute of Experimental Biology, Polish Academy of Sciences (Received 29 April 1969; revised SJuly
1969)
Abstract-Synthesis of proteins and accumulation of neurosecretion in the neurosecretory cells of the pars intercerebralis and of the suboesophagea1 ganglion of Acheta domesticus were studied in the diurnal cycle by the autoradiographic method and by staining the neurosecretion. The insects were cultured under LD 12 : 12 conditions. The light period started at 6.00. It was demonstrated that both processes occur in a cyclic manner. The maximum of protein synthesis in the neurosecretory cells of the pars intercerebralis takes place in the light period at 14.00. In the neurosecretory cells of the suboesophageal ganglion this peak occurs during the period of darkness at about 24.00. After a period of intensified protein synthesis, an increase was observed in the degree to which neurosecretion accumulated in the cells studied. The changes observed were associated with the changes in the locomotor activity of the insects studied.
INTRODUCTION
DURING the development of some insects changes were- observed in the amount of neurosecretion in the neurosecretory cells of the brain and the suboesophageal ganglion (FUKUDA, 1951a-c; HASEGAWA, 1952; HIGHNAM, 1961, 1962). This suggested that these central nervous system cells participate in various physiological processes. Recently the participation of these cells in the regulation of the diurnal cycle of locomotor activity has been postulated increasingly often (ROBERTS, 1966; NISHIITSUTSUJI-UWOet al., 1967; CYMBOROWSKI,1970). As demonstrated in a previous paper, in the diurnal cycle of locomotor activity in Acheta domesticus a cyclic sequence of RNA synthesis occurs in the neurosecretory cells of the pars intercerebralis and of the suboesophageal ganglion (CYMBOROWSKI and DUTKOWSKI, 1969). The neurosecretion produced in the territory of those cells seems to be of a proteinic nature (SLOPER,1957; PIPA, 1961; ARVY and GABE, 1962; ICHIKAWA and ISHIZAKI, 1963). It appeared therefore that a study of protein synthesis in the neurosecretory cells of the brain and suboesophageal ganglion of A. domesticus in a circadian rhythm of their locomotor activity might yield interesting results. 343
342
BRONISLAW CYMBOROWSKI ANDANIWZEJDUTKOWSKI MATERIAL
AND METHODS
The studies were carried out on male individuals of the house cricket about a week after ecdysis. These insects were cultured from the time they hatched from eggs to the attainment of sexual maturity under alternating conditions of light and darkness (LD 12 : 12) and showed a distinct rhythm of diurnal locomotor activity. Under the experimental conditions the period of darkness began at 18.00 and of light at 6.00. The details of the culture conditions have been described in another paper (CYMBOROWSKIand DUTKOWSKI,1969). Protein synthesis was studied by means of the autoradiographic method. Brains and suboesophageal ganglia were isolated without narcosis at different The sampled tissues were incubated at intervals during the course of 24 hr. about 30°C in normal saline solution for insects (YEAGER and HAGER, 1934) containing a mixture of the following labelled amino acids: 3H L-arginine (217.75 mcjm-mole), 3H n,L-leucine (14,740 me/m-mole), 3H I+-phenylalanine These amino acids (328.6 me/m-mole), and 14C n,L-serine (5.25 me/m-mole). were dissolved in amounts of 2 &ml of incubation fluid. After 1 hr _t 5 min of incubation, the tissues were washed in the water solution of the same non-labelled amino acids as used in the incubation fluid and at approximately the same concentration. Material was fixed with absolute ethanol-glacial acetic acid mixture (19 : 1 v/v) at 4°C. After fixation, tissues were dehydrated and embedded in Sections 5 p thick were hydrated pa&in following the standard procedure. and rinsed again for 15 min in a solution of unlabelled amino acids and then in distilled water. The autoradiograms were made by the stripping film method of Pelt (cj. PEARSE,1960), using Kodak AR10 plates. Exposure time for the autoradiograms was 3 weeks. Control autoradiograms were made after digestion of sections with trypsin and subsequently with pepsin after PEARSE(1960). After exposure, the autoradiograms were given the normal photographic procedure. Prior to assessment, the autoradiograms were stained with methyl green pyronine mixture. The quantitative estimation of autoradiograms was made by calculating the number of reduced silver grains/unit of area. In order to determine the degree of accumulation of neurosecretion in the neurosecretory cells of the pars intercerebralis and of the suboesophageal ganglion these organs were isolated at 3-hourly intervals over the course of 24 hr. Tissues were fixed with Bouin fluid and embedded in paraffin. Sections 5 p thick were stained with chrome-haematoxylin and floxine (GOMORI, 1941, 1950) or with aldehyde fuchsin after HALMI (1952) as modified by CAMERONand STEELE(1959). The degree of accumulation of neurosecretion was determined by the percentage of cells filled with secretion in relation to the general number of neurosecretory cells. RESULTS The changes in the intensity of protein synthesis in the neurosecretory cells of the pars intercerebralis and suboesophageal ganglion of crickets are presented in Fig. 1. These data show that there is a distinct cyclicity in protein synthesis in the neurosecretory cells of the pars intercerebralis. One hr after the onset of
CIRCADIAN
CHANGES
IN
PROTEIN
SYNTHESIS
IN
ACXETA
DUMESTKWS
343
the light period, an increase of protein synthesis is observed in the cells studied. Its maximum appears about 14.00. In the dark period the level of protein synthesis is low, whereas the cyclicity of protein synthesis is less distinct in the neurosecretory cells of the suboesophageal ganglion. A gradual rise of incorporation of labelled amino acids also begins in those cells after the onset of the light period, but its maximum occurs during the darkness period at about 24.00.
13
6.00
10.00
14.00
18.00
22.00
26.00
FIG. 1. Changes in the level of protein synthesis in neurosecretory cells of pars intercerebralis and the suboesophageal ganglion of rhythmic house crickets from LD 12 : 12 conditions. -O--O-, Intensity of protein synthesis in neurosecretory cells of pars intercerebralis; - A--A-, intensity of protein synthesis in neurosecretory cells of suboesophageal ganglion. S.E.M. are given.
It should be stressed that the intensity of protein synthesis in the neurosecretory cells of the suboesophageal ganglion fails to reach such a high level as that observed in the neurosecretory cells of the pars intercerebralis. In the neurosecretory cells of both centres of the central nervous system, the incorporation of labelled amino acids was found mainly in the cytoplasm of cells and not in their axons (Fig. 2a, b). The changes in the accumulation of neurosecretion in the neurosecretory cells of the investigated centres of the central nervous system at different periods of the day are presented in Fig. 3. In the pars intercerebralis, the maximal number of neurosecretory cells with accumulated neurosecretion are found immediately after the insects’ transition from light to darkness and amounts to about 40 per cent (Figs. 2c, 3). A sirnilar number of cells with accumulated neurosecretion is
344
BRONIS~AW CYMBOROWSKI ANDANDRZEJDUTKOWSKI
found during the next 3 hr period. Subsequently, the number of cells with a high content of neurosecretion decreases considerably and remains at a constant level for the remaining period of observation. It should be mentioned that a complete absence of neurosecretory cells filled with neurosecretion was not observed in the course of the diurnal cycle (Fig. Zd).
hours
FIG. 3. Changes in the degree of accumulation of neurosecretion in the neurosecretory cells in the pars intercerebralis and suboesophageal ganglion of rhythmic crickets from LD 12 : 12 conditions. -O--O-, Pars intercerebralis; -AA-, suboesophageal ganglion. Each point is the average result of six determinations. In the suboesophageal ganglion, some diurnal changes in the number of neurosecretory cells filled with secretion were observed. These changes were not, however, so distinct as those observed in the pars intercerebralis (Fig. 3). DISCUSSION
The results of the previous paper (CYMBOROWSKIand DUTKOWSKI, 1969) suggest that RNA synthesis in the neurosecretory cells of the pars intercerebralis of A&eta donzesticusoriginating from the LD 12 : 12 conditions is induced by the stimulus of the transition of the insects from darkness to light. Maximal RNA synthesis was observed approximately 30 min after the onset of the light period. As found in the previous study, protein synthesis in the neurosecretory cells of this part of the brain occurs in a cyclic manner, its maximum being delayed for about 7 hr in relation to the maximum of RNA synthesis. It may therefore be assumed that the changed light conditions induce the mRNA synthesis in the cells studied. The cyclicity of protein synthesis would thus be the consequence of cyclic mRNA production.
FIG. 2. Autoradiograms of the neurosecretory cells of (a) the pars intercerebralis and (b) the suboesophageal ganglion. The high degree of incorporation of labelled amino acids into the cytoplasm of these cells at the periods of maximal synthesis of proteins is remarkable. (Exposure time of autoradiograms-3 weeks.) Accumulation of the secretion in the neurosecretory cells in the pars intercerebralis from rhythmic crickets at the period of their maximal locomotor activity (c) and at the period of low locomotor activity (d). At the period of maximal locomotor activity a great number of neurosecretory cells with secretion is observed, contrary to the period of low activity. (Bouin fixative, aldehyde-fuchsine staining.)
CIRCADIAN CHANGES IN PROTEIN SYNTHESIS IN ACHETA
DOMESTICUS
347
The accumulation of neurosecretion, as evaluated by the percentage of cells filled with it in relation to the general number of cells, also occurs in a cyclic manner, as has been demonstrated. This might indicate that the changes in protein synthesis observed in those cells are the changes in synthesis of the neurosecretory substance. The fact is striking that the highest number of cells filled with neurosecretion is observed at the period of the maximal locomotor activity of crickets, i.e. immediately after the onset of the period of darkness. As stated, protein synthesis is very insignificant at this period in the neurosecretory cells of the pars intercerebralis, which agrees with the observations of HIGJXNAM(1962). Consequently, at the period of maximal locomotor activity of the insects, the neurosecretion seems to be transitionally accumulated in the cells of the pars intercerebralis. The fact that only a small number of neurosecretory cells filled with secretion is observed at the period of the decrease in the locomotor activity of crickets allows the assumption that the released neurosecretion inhibits locomotor activity, which agrees with the previously proposed hypothesis (CYMBOROWSKI,1968). The r61e of neurosecretion as a factor inhibiting the locomotor activity of the insects examined and also the experimental data concerning this are discussed in another paper (CYMBOROWSKI,1970). A correlation between RNA and protein synthesis-similar to that presented above-has also been observed in the neurosecretory cells of the suboesophageal ganglion. Both processes are, however, retarded in relation to the same processes occurring in neurosecretory cells of the pars intercerebralis. It should be stressed that while RNA synthesis in the neurosecretory cells of the suboesophageal ganglion is characterized by two periods of increase only one peak is observed in protein synthesis.
REFERENCES ARTY L. and GABEM. (1962) Histochemistry of the neurosecretory product of the pars intercerebralis of Pterygote insects. In Neurosemetion (Ed by HELLERH. and CLARK R. B.), pp. 331-344.
Academic Press, New York.
CAMERON M. L. and STEELE J. E. (1959) Simplified aldehyde fuchsin staining of neurosecretory celIs. Stain Technol. 34, 265-266. CYMBOROWSKI B. (1968) Badania nad neurohormonalna regulacja dobowego cyklu aktywno&i ruchowej gwierszcza domowego (Acheta domesticus L.). Doctoral Thesis, University of Warsaw.
CYMBOROWSKI B. (1970) Investigations on the neurohormonal factors controlling circadian rhythm of locomotor activity in the house cricket (Acheta domesticus L.)-I. The role of the brain and suboesophageal ganglion. 2001. Poloniae.
In press.
CYMBOROWSKI B. and DUTKOWSKI A. (1969) Circadian changes in RNA synthesis in the neurosecretory cells of the brain and suboesophageal ganglion of the house cricket. J. Insect Physiol. 15, 1187-1197. FUKUDAS. (195la) Alternation of voltinism in the silkworm by decapitating the pupa. Zool. Mag.Jap. 60, 119-121. FUKUDAS. (195lb) Factors determining the production of non-diapause eggs in the silkworm. Proc.Jap. Acad. 27, 582-586.
348
BRONISLAWCYMBOROWSKIAND ANDRZEJDUTKOWSKI
FUKUDAS. (1951c) The production of the diapause eggs by transplanting the suboesophageal ganglion in the silkworm. Proc.Jap. Acad. 27, 672-677. GOMORI G. (1941) Observations with differential stains on human islets of Langerhans. Am.J. clin. Path. 17, 395-406. GOMORI G. (1950), Aldehyde-fuchsin: A new stain for elastic tissue. Am. J. clin. Path. 20, 665-666. HALMI N. S. (1952) Differentiation of two types of basophils in the adenohypophysis of the rat and mouse. Stain Technol. 27, 61-64. HASEGAWAIX. (1952) Studies on the voltinism in the silkworm Bombyx mori. Nature, Lond. 179, 1300-1301. HIGHNAM C. K. (1961) Induced changes in the amounts of material in the neurosecretory system of the desert locust. Nature, Lond. 191, 199-200. HIGHNAMC. K. (1962) Neurosecretory control of ovarian development in the desert locust. In Neurosecretion (Ed. by HELLERH. and CLARKR. B.), pp. 379-390. Academic Press, New York. ICHIKAWAM. and ISHIZAKIH. (1963) Protein nature of the brain hormone of insects. Nature, Lond. 198, 308-309. NISHIITSUTSUJI-UWO J., PETROPULOS S. F. and PITTENDRIGHC. S. (1967) Central nervous system control of circadian rhythmicity in the cockroach-I. Role of the pars intercerebralis. Biol. I&&, Woods Hole 133, 679-696, PEARSEA. G. E. (1960) Histochemistry, Theoretical and Applied. Churchill, London. PIPA R. L. (1961) Studies on the hexapod nervous system-IV. A cytological study of neurons and their inclusions in the brain of a cockroach, Periplaneta americana L. Biol. Bull., Woods Hole 121, 521-534. ROBERTSS. K. (1966) Circadian activity rhythms in the cockroach-III. The role of endocrine and neural factors. y. cell. camp. Physiol. 67, 473-486. SLOPERJ. C. (1957) Presence of a substance rich in cystine in the neurosecretory system of an insect. Nature, Lond. 179, 148-149. YEAGERI. F. and HAGERA. (1934) On the rates of contraction of the isolated heart and Malpighian tube of the insect Periplaneta orientalis. Iowa State Coil. J. Sci. 8, 391-395.
CIRCADIAN CHANGES IN PROTEIN SYNTHESIS IN THE NEUROSECRETORY CELLS OF THE CENTRAL NERVOUS SYSTEM OF ACHETA DOMESTICUS BRONISLAW
and ANDRZEJ
CYMBOROWSKI
DUTKOWSKI
Department of Comparative Animal Physiology, Zoological Institute, University of Warsaw, and Department of Biochemistry, M. Nencki Institute of Experimental Biology, Polish Academy of Sciences (Received 29 April 1969; revised SJuly
1969)
Abstract-Synthesis of proteins and accumulation of neurosecretion in the neurosecretory cells of the pars intercerebralis and of the suboesophagea1 ganglion of Acheta domesticus were studied in the diurnal cycle by the autoradiographic method and by staining the neurosecretion. The insects were cultured under LD 12 : 12 conditions. The light period started at 6.00. It was demonstrated that both processes occur in a cyclic manner. The maximum of protein synthesis in the neurosecretory cells of the pars intercerebralis takes place in the light period at 14.00. In the neurosecretory cells of the suboesophageal ganglion this peak occurs during the period of darkness at about 24.00. After a period of intensified protein synthesis, an increase was observed in the degree to which neurosecretion accumulated in the cells studied. The changes observed were associated with the changes in the locomotor activity of the insects studied.
INTRODUCTION
DURING the development of some insects changes were- observed in the amount of neurosecretion in the neurosecretory cells of the brain and the suboesophageal ganglion (FUKUDA, 1951a-c; HASEGAWA, 1952; HIGHNAM, 1961, 1962). This suggested that these central nervous system cells participate in various physiological processes. Recently the participation of these cells in the regulation of the diurnal cycle of locomotor activity has been postulated increasingly often (ROBERTS, 1966; NISHIITSUTSUJI-UWOet al., 1967; CYMBOROWSKI,1970). As demonstrated in a previous paper, in the diurnal cycle of locomotor activity in Acheta domesticus a cyclic sequence of RNA synthesis occurs in the neurosecretory cells of the pars intercerebralis and of the suboesophageal ganglion (CYMBOROWSKI and DUTKOWSKI, 1969). The neurosecretion produced in the territory of those cells seems to be of a proteinic nature (SLOPER,1957; PIPA, 1961; ARVY and GABE, 1962; ICHIKAWA and ISHIZAKI, 1963). It appeared therefore that a study of protein synthesis in the neurosecretory cells of the brain and suboesophageal ganglion of A. domesticus in a circadian rhythm of their locomotor activity might yield interesting results. 343
342
BRONISLAW CYMBOROWSKI ANDANIWZEJDUTKOWSKI MATERIAL
AND METHODS
The studies were carried out on male individuals of the house cricket about a week after ecdysis. These insects were cultured from the time they hatched from eggs to the attainment of sexual maturity under alternating conditions of light and darkness (LD 12 : 12) and showed a distinct rhythm of diurnal locomotor activity. Under the experimental conditions the period of darkness began at 18.00 and of light at 6.00. The details of the culture conditions have been described in another paper (CYMBOROWSKIand DUTKOWSKI,1969). Protein synthesis was studied by means of the autoradiographic method. Brains and suboesophageal ganglia were isolated without narcosis at different The sampled tissues were incubated at intervals during the course of 24 hr. about 30°C in normal saline solution for insects (YEAGER and HAGER, 1934) containing a mixture of the following labelled amino acids: 3H L-arginine (217.75 mcjm-mole), 3H n,L-leucine (14,740 me/m-mole), 3H I+-phenylalanine These amino acids (328.6 me/m-mole), and 14C n,L-serine (5.25 me/m-mole). were dissolved in amounts of 2 &ml of incubation fluid. After 1 hr _t 5 min of incubation, the tissues were washed in the water solution of the same non-labelled amino acids as used in the incubation fluid and at approximately the same concentration. Material was fixed with absolute ethanol-glacial acetic acid mixture (19 : 1 v/v) at 4°C. After fixation, tissues were dehydrated and embedded in Sections 5 p thick were hydrated pa&in following the standard procedure. and rinsed again for 15 min in a solution of unlabelled amino acids and then in distilled water. The autoradiograms were made by the stripping film method of Pelt (cj. PEARSE,1960), using Kodak AR10 plates. Exposure time for the autoradiograms was 3 weeks. Control autoradiograms were made after digestion of sections with trypsin and subsequently with pepsin after PEARSE(1960). After exposure, the autoradiograms were given the normal photographic procedure. Prior to assessment, the autoradiograms were stained with methyl green pyronine mixture. The quantitative estimation of autoradiograms was made by calculating the number of reduced silver grains/unit of area. In order to determine the degree of accumulation of neurosecretion in the neurosecretory cells of the pars intercerebralis and of the suboesophageal ganglion these organs were isolated at 3-hourly intervals over the course of 24 hr. Tissues were fixed with Bouin fluid and embedded in paraffin. Sections 5 p thick were stained with chrome-haematoxylin and floxine (GOMORI, 1941, 1950) or with aldehyde fuchsin after HALMI (1952) as modified by CAMERONand STEELE(1959). The degree of accumulation of neurosecretion was determined by the percentage of cells filled with secretion in relation to the general number of neurosecretory cells. RESULTS The changes in the intensity of protein synthesis in the neurosecretory cells of the pars intercerebralis and suboesophageal ganglion of crickets are presented in Fig. 1. These data show that there is a distinct cyclicity in protein synthesis in the neurosecretory cells of the pars intercerebralis. One hr after the onset of
CIRCADIAN
CHANGES
IN
PROTEIN
SYNTHESIS
IN
ACXETA
DUMESTKWS
343
the light period, an increase of protein synthesis is observed in the cells studied. Its maximum appears about 14.00. In the dark period the level of protein synthesis is low, whereas the cyclicity of protein synthesis is less distinct in the neurosecretory cells of the suboesophageal ganglion. A gradual rise of incorporation of labelled amino acids also begins in those cells after the onset of the light period, but its maximum occurs during the darkness period at about 24.00.
13
6.00
10.00
14.00
18.00
22.00
26.00
FIG. 1. Changes in the level of protein synthesis in neurosecretory cells of pars intercerebralis and the suboesophageal ganglion of rhythmic house crickets from LD 12 : 12 conditions. -O--O-, Intensity of protein synthesis in neurosecretory cells of pars intercerebralis; - A--A-, intensity of protein synthesis in neurosecretory cells of suboesophageal ganglion. S.E.M. are given.
It should be stressed that the intensity of protein synthesis in the neurosecretory cells of the suboesophageal ganglion fails to reach such a high level as that observed in the neurosecretory cells of the pars intercerebralis. In the neurosecretory cells of both centres of the central nervous system, the incorporation of labelled amino acids was found mainly in the cytoplasm of cells and not in their axons (Fig. 2a, b). The changes in the accumulation of neurosecretion in the neurosecretory cells of the investigated centres of the central nervous system at different periods of the day are presented in Fig. 3. In the pars intercerebralis, the maximal number of neurosecretory cells with accumulated neurosecretion are found immediately after the insects’ transition from light to darkness and amounts to about 40 per cent (Figs. 2c, 3). A sirnilar number of cells with accumulated neurosecretion is
344
BRONIS~AW CYMBOROWSKI ANDANDRZEJDUTKOWSKI
found during the next 3 hr period. Subsequently, the number of cells with a high content of neurosecretion decreases considerably and remains at a constant level for the remaining period of observation. It should be mentioned that a complete absence of neurosecretory cells filled with neurosecretion was not observed in the course of the diurnal cycle (Fig. Zd).
hours
FIG. 3. Changes in the degree of accumulation of neurosecretion in the neurosecretory cells in the pars intercerebralis and suboesophageal ganglion of rhythmic crickets from LD 12 : 12 conditions. -O--O-, Pars intercerebralis; -AA-, suboesophageal ganglion. Each point is the average result of six determinations. In the suboesophageal ganglion, some diurnal changes in the number of neurosecretory cells filled with secretion were observed. These changes were not, however, so distinct as those observed in the pars intercerebralis (Fig. 3). DISCUSSION
The results of the previous paper (CYMBOROWSKIand DUTKOWSKI, 1969) suggest that RNA synthesis in the neurosecretory cells of the pars intercerebralis of A&eta donzesticusoriginating from the LD 12 : 12 conditions is induced by the stimulus of the transition of the insects from darkness to light. Maximal RNA synthesis was observed approximately 30 min after the onset of the light period. As found in the previous study, protein synthesis in the neurosecretory cells of this part of the brain occurs in a cyclic manner, its maximum being delayed for about 7 hr in relation to the maximum of RNA synthesis. It may therefore be assumed that the changed light conditions induce the mRNA synthesis in the cells studied. The cyclicity of protein synthesis would thus be the consequence of cyclic mRNA production.
FIG. 2. Autoradiograms of the neurosecretory cells of (a) the pars intercerebralis and (b) the suboesophageal ganglion. The high degree of incorporation of labelled amino acids into the cytoplasm of these cells at the periods of maximal synthesis of proteins is remarkable. (Exposure time of autoradiograms-3 weeks.) Accumulation of the secretion in the neurosecretory cells in the pars intercerebralis from rhythmic crickets at the period of their maximal locomotor activity (c) and at the period of low locomotor activity (d). At the period of maximal locomotor activity a great number of neurosecretory cells with secretion is observed, contrary to the period of low activity. (Bouin fixative, aldehyde-fuchsine staining.)
CIRCADIAN CHANGES IN PROTEIN SYNTHESIS IN ACHETA
DOMESTICUS
347
The accumulation of neurosecretion, as evaluated by the percentage of cells filled with it in relation to the general number of cells, also occurs in a cyclic manner, as has been demonstrated. This might indicate that the changes in protein synthesis observed in those cells are the changes in synthesis of the neurosecretory substance. The fact is striking that the highest number of cells filled with neurosecretion is observed at the period of the maximal locomotor activity of crickets, i.e. immediately after the onset of the period of darkness. As stated, protein synthesis is very insignificant at this period in the neurosecretory cells of the pars intercerebralis, which agrees with the observations of HIGJXNAM(1962). Consequently, at the period of maximal locomotor activity of the insects, the neurosecretion seems to be transitionally accumulated in the cells of the pars intercerebralis. The fact that only a small number of neurosecretory cells filled with secretion is observed at the period of the decrease in the locomotor activity of crickets allows the assumption that the released neurosecretion inhibits locomotor activity, which agrees with the previously proposed hypothesis (CYMBOROWSKI,1968). The r61e of neurosecretion as a factor inhibiting the locomotor activity of the insects examined and also the experimental data concerning this are discussed in another paper (CYMBOROWSKI,1970). A correlation between RNA and protein synthesis-similar to that presented above-has also been observed in the neurosecretory cells of the suboesophageal ganglion. Both processes are, however, retarded in relation to the same processes occurring in neurosecretory cells of the pars intercerebralis. It should be stressed that while RNA synthesis in the neurosecretory cells of the suboesophageal ganglion is characterized by two periods of increase only one peak is observed in protein synthesis.
REFERENCES ARTY L. and GABEM. (1962) Histochemistry of the neurosecretory product of the pars intercerebralis of Pterygote insects. In Neurosemetion (Ed by HELLERH. and CLARK R. B.), pp. 331-344.
Academic Press, New York.
CAMERON M. L. and STEELE J. E. (1959) Simplified aldehyde fuchsin staining of neurosecretory celIs. Stain Technol. 34, 265-266. CYMBOROWSKI B. (1968) Badania nad neurohormonalna regulacja dobowego cyklu aktywno&i ruchowej gwierszcza domowego (Acheta domesticus L.). Doctoral Thesis, University of Warsaw.
CYMBOROWSKI B. (1970) Investigations on the neurohormonal factors controlling circadian rhythm of locomotor activity in the house cricket (Acheta domesticus L.)-I. The role of the brain and suboesophageal ganglion. 2001. Poloniae.
In press.
CYMBOROWSKI B. and DUTKOWSKI A. (1969) Circadian changes in RNA synthesis in the neurosecretory cells of the brain and suboesophageal ganglion of the house cricket. J. Insect Physiol. 15, 1187-1197. FUKUDAS. (195la) Alternation of voltinism in the silkworm by decapitating the pupa. Zool. Mag.Jap. 60, 119-121. FUKUDAS. (195lb) Factors determining the production of non-diapause eggs in the silkworm. Proc.Jap. Acad. 27, 582-586.
348
BRONISLAWCYMBOROWSKIAND ANDRZEJDUTKOWSKI
FUKUDAS. (1951c) The production of the diapause eggs by transplanting the suboesophageal ganglion in the silkworm. Proc.Jap. Acad. 27, 672-677. GOMORI G. (1941) Observations with differential stains on human islets of Langerhans. Am.J. clin. Path. 17, 395-406. GOMORI G. (1950), Aldehyde-fuchsin: A new stain for elastic tissue. Am. J. clin. Path. 20, 665-666. HALMI N. S. (1952) Differentiation of two types of basophils in the adenohypophysis of the rat and mouse. Stain Technol. 27, 61-64. HASEGAWAIX. (1952) Studies on the voltinism in the silkworm Bombyx mori. Nature, Lond. 179, 1300-1301. HIGHNAM C. K. (1961) Induced changes in the amounts of material in the neurosecretory system of the desert locust. Nature, Lond. 191, 199-200. HIGHNAMC. K. (1962) Neurosecretory control of ovarian development in the desert locust. In Neurosecretion (Ed. by HELLERH. and CLARKR. B.), pp. 379-390. Academic Press, New York. ICHIKAWAM. and ISHIZAKIH. (1963) Protein nature of the brain hormone of insects. Nature, Lond. 198, 308-309. NISHIITSUTSUJI-UWO J., PETROPULOS S. F. and PITTENDRIGHC. S. (1967) Central nervous system control of circadian rhythmicity in the cockroach-I. Role of the pars intercerebralis. Biol. I&&, Woods Hole 133, 679-696, PEARSEA. G. E. (1960) Histochemistry, Theoretical and Applied. Churchill, London. PIPA R. L. (1961) Studies on the hexapod nervous system-IV. A cytological study of neurons and their inclusions in the brain of a cockroach, Periplaneta americana L. Biol. Bull., Woods Hole 121, 521-534. ROBERTSS. K. (1966) Circadian activity rhythms in the cockroach-III. The role of endocrine and neural factors. y. cell. camp. Physiol. 67, 473-486. SLOPERJ. C. (1957) Presence of a substance rich in cystine in the neurosecretory system of an insect. Nature, Lond. 179, 148-149. YEAGERI. F. and HAGERA. (1934) On the rates of contraction of the isolated heart and Malpighian tube of the insect Periplaneta orientalis. Iowa State Coil. J. Sci. 8, 391-395.