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Brain creatine kinase activity during ontogeny of the cichlid fish oreochromis mossambicus and the clawed toad xenopus laevis, influence of gravity?
Neurochem. Int. Vol. 22, No. 4, pp. 405-411, 1993 Printedin Great Britain
0197-0186/93$6.00+ 0.00 PergamonPress Ltd
BRAIN CREATINE KINASE ACTIVITY D U R I N G O N T O G E N Y OF THE CICHLID FISH O R E O C H R O M I S M O S S A M B I C U S AND THE CLAWED TOAD X E N O P U S LAEVIS, I N F L U E N C E OF GRAVITY? KLAUS SLENZKA, RAMONA APPEL a n d HINRICH RAHMANN* University of Stuttgart-Hohenheim, Institute of Zoology, Garbenstr. 30, D-7000 Stuttgart 70, Germany (Received 20 May 1992; accepted 3 September 1992)
Abstract--The development of creatine kinase (CK) activity was studied in the brain of cichlid fish and clawed toads. The activity of CK in the whole brain of the fish decreases immediatelyafter hatching (stage 6) from values of about 135 nmol substrate cleaved/mg protein/min to a value of about 105 at stage 8 (5 days post hatch at 20°C). With the exception of a significant peak (125 nmol) between stages 9 and 10 (7 and 9 days respectively,post hatch at 20°C) and a small intermediate peak at stages 12 and 13 (about 10 days post hatch at 20°C) a constant level of about 100 nmol cleaved substrate is maintained until maturity. In contrast, CK activity was determined to be 3-fold higher in the whole brain of the clawed toad. With the exception of two significantpeaks at stages 47 and 49 (5 and 12 days respectively,post fertilization at 23°C) a value of about 360 nmol was found during larval development and metamorphosis, as well as in the adult brain. In investigating the possible influenceof gravity on CK activity during early ontogeny of the brain both animal species were exposed to hyper-gravity (3 + 1 g) for 7 days. A significantdecrease of total CK activity of 20% was found in the fish brain and of about 5% in the toad. Changes in total CK activity during brain development, as well as after the influence of altered gravitational forces, suggests that this enzyme is well adapted to critical phases of brain development and reacts very sensitivelyduring this period to changes in environmental conditions.
Creatine kinase (CK, EC 2.7.3.2) is a dimeric enzyme catalyzing the reversible transfer reaction of energyrich phosphate groups from ATP to creatine : ATP + creatine ~ creatine phosphate + ADP (Dawson et al., 1967, 1969 ; Dawson, 1970). This creatine-creatine phosphate "energy shuttle" has been well described by Bessman and co-workers (Bessman and Fanyo, 1966; Bessman et al., 1978; Bessman and Carpenter, 1985) as well as by Walliman and Eppenberger (1985, 1990). Generally it can be found in vertebrate tissues with a high energy metabolism e.g. in cardiac, skeletal muscles, brain and adipose tissues (Berlet et al., 1976), as well as in tumors (Shatton et al., 1979), and in smaller amounts in many other tissues (Jacobs and Lehninger, 1973). In contrast to this, invertebrate species have developed different enzyme systems, for example phosphorylarginine (Storey, 1977). In general, CK exists in different isoforms, one (CKmito) being located at the outer surface of the inner mitochondrial membrane whereas the others (MM,
*To whom correspondence should be addressed.
MB, BB) being primarily cytosolic (Bessman and Carpenter, 1985 ; Eppenberger et al., 1967 ; Walliman and Eppenberger, 1985, 1990; Walliman et al., 1984, 1989). M M - C K was found to be near the location of ATPase activity and is functionally coupled with it, providing ATP for the ATPase reaction (Saks et al., 1976, 1977, 1978, 1991). Mitochondrial CK producing phosphorylcreatine from ATP has been explained by CK coupling to the ATP/ADP translocase, which has been suggested to supply ATP directly to CK (Jacobs and Deffley, 1986; Saks et al., 1975, 1991). Regarding these basic functions of CK, several investigations were performed concerning clinical aspects as well as the possible influence of exogenously applied drugs or physical factors on CK activity. For example, Christenson et al., (1990) and Geoffrey et al., (1990) reported an increase in CK activity during reperfusion after myocardial infarction. Mahler (1979) demonstrated a dramatic decrease of mitochondrial CK activity in muscle dystrophy. Clarkson and Ebbeling (1988) showed significant alterations of CK activity after muscle damage. Concerning brain CK activity, Schwartz et al. (1989) and Peschke et al., 405
406
Kt, Atis SI l NZKA el a[.
(1989) reported that the C K serum level is a good indicator of the size of various brain lesions or ganglionectomy. To prevent these alterations of CK activity in the cardiac or skeletal system as well as in the brain different attempts were performed in applying exogenous or e n d o g e n o u s substances. Binderman et al. (1988), e.g. reported significant stimulations o f the C K activity by exogenously applying vitamin D analogues. C u p t a et al. ( 1991 ) showed that the application of high doses o f c a r b u f u r a n , induced significant alterations in the activity o f C K and its isoenzymes, as well as in energy-rich phosphates, whereas a disordered serum C K level can be stabilized by the application of dantrolene (Quinlan et al., 1990). In the last decade, research interest arose more and more in to w h a t extent e n v i r o n m e n t a l factors influences e n z y m e - d e p e n d e n t metabolic systems, especially during early ontogeny. In regard to this, analyses o f the functionally coupled ATPases revealed that seasonal a d a p t a t i o n , hydrostatic pressure a n d the application o f exogenously applied m e m b r a n e o u s glycosphingolipids ( = gangliosides) can induce d r a m a t i c changes in the activity o f Ca2+-ATPascs ( G i b b s and Somero, 1990 ; Slenzka et al., 1990, 1991 ). Focusing on the c r e a t i n e creatine p h o s p h a t e "energy shuttle", relevant investigations concerning d e v e l o p m e n t - d e p e n d e n t changes have been concentrated up to now, on the muscular C K (Hoerter et al., 1991); with few reports dealing with this aspect for the brain C K (Lapin et al., 1974; M a n o s et al., 1991). A possible influence o f the e n v i r o n m e n t a l factor gravity was d e m o n s t r a t e d in the slight increase of brain C K activity in rats t h a t h a d been exposed for 22 days to near weightlessness conditions a b o a r d a Soviet C O S M O S satellite (Krasnov, 1975, 1977). In the present study, we report on the develo p m e n t a l profile of the whole brain C K activity in the cichlid fish Oreochromis mossambicus a n d the clawed toad Xenopus laevis, a n d the influence of long-term hyper-gravity (7 days; 3 + 1 g) on the n e u r o n a l C K activity of these species d u r i n g their early ontogenetic development.
EXPERIMENTAL PROCEDURES
Materials All chemicals used in this study were purchased from the Sigma Chemical Co. (Deisenhofen, Germany) Maintenance of animals Mouth-breeding cichlid fish (Oreochromis rnossambicus) and clawed toad larvae (Xenopus laevis) were kept within small round miniaquaria (5 cm diameter, t.5 cm height; Bachofer Co., Reutlingen, Germany) at a density of 5 anim-
als/aquaria and an ambient temperature for both species of 20'C. This temperature was chosen regarding the planned STATEX-II experiment (Statolith experiment) aboard the 2nd German Spacelab Mission D-2 where cichlid fish will be reared beside their preferential temperature of 28' (' at 20 (' (Rahmann and Slenzka, 1991). Hilbig et al., (1992) have shown that this temperature had no major influence either on the normal morphological development or on that of compared to the normal behaviour. Thus, it was assumed that the maintenance temperature of 20' C, also has no m~qor influence on the normal development of the CK system. tlilbig el al. (1992) also reported, that a period of increased gravity during the early ontogenetic development of cichlid fish and toad larvae has no inlluence on the viability of the animals and it was shown thal further on a normal development up to the adult occur. Determination q f nhole hrahl CK actit,il v Determination of the protein content follows the method of Lowry et al. ( 1951). CK assays were carried out according to Oliver (1955) and Wallimann et al. (1984), with modifications for the reported analyses. The reaction was observed spectrophotometrically in a final volume of 3 ml containing 66 mM TEA buffer (pH 7.2), 34 mM EDTA, 40 mM glucose, I mM NADP ~, 51 mM MgCI> 6.7 mM ADP, 2 mM AMP, 1.2 U hexokinase, 1.2 U glucose-6-phosphate dehydrogenase, 12 pg oligomycin, and 35 #g total homogenate protein. After 5 rain preincubation at 25°C, 5 mM phosphocreatine was added to start the reaction. Specific activity was calculated as total activity minus basal activity (tesl run without the addition of MgCI2) and expressed as nmol substrate cleaved/mg protein/rain. Hereafter expressed as nmol substrate cleaved or nmol. Each test was performed five times from a pooled preparation of about 7 brains, and the standard error of the means (SEM) was calculated from these trials. Development-dependent im~estigations Three groups of about 300 cichlid fish larvae (stage 7 ; 2 days post hatch at 2WC) were carefully removed from the mother's mouth, and 5 siblings were transfered to the each miniaquaria. At critical developmental stages, starting with stage 7, up to free swimming (stage 15) and from adult fish the brains were quickly removed and prepared for the CK assay. Additionally, three groups of about 300 fertilized clawed toad eggs were also transfered, 5 per miniaquaria. Starting with developmental stage 14 ( = neurula stage, about 16 h post fertilization at 23'C) up to 66 (58 days post fertilization at 23"C) and from adults, the brains were quickly removed and prepared for the CK assay. The determination of the respective developmental stages lbr the cichlid fish was according to B/iuerle and Voss (1992) and Anken et al. (1992), and according to Nieuwkoop and Faber (1967) for the clawed toad. Gratuity-dependent investigations In regard to a possible influence of altered gravitational forces on the neuronal CK activity of the experimental animals 5 groups of hyper-gravity (3-1-1 g) animals of each species were investigated in comparison to normal 1 g earth controls. For each group 120 siblings of fish or toad larvae were reared from day 2 post hatch (stage 7; cichlid fish) or stage 14 (neurula stage; clawed toad) for 7 days in the
407
Brain creatine kinase and influenceof gravity miniaquaria, 5 each. Sixty of them were subjected to an increased acceleration of 3 + 1 g within a non-vibrating centrifuge (IEC HN-S, Damon/IEC Division, USA) at room temperature versus controls which were raised under identical conditions but at normal 1g earth gravity. Immediately after the hyper-g exposure, brains were removed and prepared for the CK assay.
two significantly increased peaks of enzyme activity at developmental stages 47 and 49, with high values of about 450 nmol (Fig. 2).
Influence of 7 days hyper-gravity on the brain CK activity of developing fish and frog larvae On the basis of our data obtained from the developmental analyses as well as from Krasnovs (1975, 1977) work with rats, it had been of general interest to determine whether or not hyper-gravity might have an influence on the CK activity within the developing brain of aquatic vertebrates. In regard to this, cichlid fish and clawed toad larvae were exposed for 7 days during early ontogenetic development from stage 7 or 8 (about 2 days post hatch) for the fish and stage 14 ( = neurula stage) for the frog, to increased gravitational forces (3 + 1 g within a non-vibrating centrifuge. The data obtained revealed significant alterations of whole brain CK activity in hyper-g fish as well as in hyper-g toad larvae after the exposure period. A statistically significant decrease of about 20% from a value of 91.5-73.3 nmol substrate cleaved (P < 0.0001) was registered in the brains of cichlid fish [Fig. 3(A)] and from about 390 to 370 nmol in the brains of the clawed toads [P < 0.0001 ; Fig 3(B)] These results show that an increasing gravity significantly influences brain CK activity.
RESULTS
CK activity during ontogenetic development of cichlid .fish and clawed toads CK activity of the whole brain of the post hatched cichlid fish Oreochromis mossambicus decreased drastically from about 137 nmot substrate cleaved at stage 7 (2 days post hatch at 20°C) to about 105 nmol at stage 8. A rapid increase was observed from stage 8 to 10 to a value of about 125 nmol followed by a fast decrease to values of about 100 nmol. At stages 12 and 13 a slight enhancement was registered followed by the definite level ( -I- 100 nmol) up to the adult stage (Fig. 1). In comparison to the data obtained from the cichlids, whole brain CK activity of the clawed toad was found with values of about 350 to 450 nmol 3fold higher than in the cichlid. Contrary to the high CK activity level found directly after hatching for the cichlid fish, the CK level in the developing toad brain is nearly the same as that in the adult frog (about 370 nmol). Similar to the fish, the clawed toad expressed
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DISCUSSION
Our objective was to investigate whether or not there would be any alterations in total brain CK activity in the cichlid fish and the clawed toad during early ontogeny as well as whether or not hyper-gravity influences the CK system within the brain of these species. Development-related investigations
Lapin et al. (1974) and Manos et al. (1991) repotted that in the developing rat brain CK activity increases postnatally without intermediate alterations. In contrast to these findings, we found in the fish brain, a dramatic decrease after hatching, which correlates with the phase of synaptogenesis and with the first onset of optokinetic reflexes and swimming movements (Anken et al., 1992; B/iuerle and Voss, 1992). Another small peak was detected at stage 13, which can be correlated with the first food-uptake parallel to the resorption of the yolk sac and with the onset of myelination. Very similar results were obtained from the clawed toad ; at stages 47 and 49, extremely high levels of CK activity were found, correlating to the maturation of the Purkinje cells, the differentiation of the optic tectum, and the start of neurohypophysis development (Nieuwkoop and Faber, 1967). Surprisingly, during metamorphosis no further significant changes in thc, CK activity were observed. Generally, the presence of high levels of CK activity in the mammalian brain (in the order of 2-18 #tool) has been attributed to neuronal energy requirements
either for synaptogenesis or for neuronal growth and myelination (Bessman and Carpenter, 1985 ; Lapin et al., 1974; Maker et al., 1973 ; Manos et al., 1991). Our own data confirms this, insofar as in fish and frogs, significant alterations in neuronal CK activity also occur during critical phases of neuronal development. Gravity-related investigations
The sprouting of nerve fibers, formation of synapses and the onset of first reflexes during early ontogenetic development of the nervous system, is to a great extent genetically determined. However, it is well known that dramatic failures can take place in the development of behaviour, brain organization, and brain ultrastructure and biochemistry, when long-term environmental disturbances (e.g. rearing in total darkness or under altered temperatures) occur (Flohr, 1988; Jeserich and Rahmann, 1979, Sester et al,, 1984; Slenzka et al., 1991; Zeutzius and Rahmann, 1980; Zeutzius et al., 1984). If these disturbances take place during critical developmental phases for relatively brief periods only, the organism is able for compensation to a certain degree by activating related structures (Flohr, 1988). Gravity, having been a stable factor on earth for more than 3.5 billions of years, has constantly influenced life. However, during the last decades, space flights enabled scientists to investigate the influence of alterations of this factor in different ways, for example in the development of the brain. In regard to this, it was reported that functional adaptations within the rat
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Fig. 3. Effect of long-term hyper-gravity (7 days ; 3 _ 1 g) on brain creatine kinase activity in cichlid fish Oreochromis mossambicus (A) as well as in clawed toads Xenopus laevis (B) controls. Specific CK activity is expressed as nmol substrate cleaved/mg protein/min. The values represent the mean + SEM of 80 measurements out of 4 pooled preparations. (**** = P < 0.0001)
brain occur during and especially after space flights, as demonstrated by an increase o f c y t o c h r o m e oxidase activity (Murakami et al., 1985). Based on quantitative histochemical determinations of C K activity, Krasnov (1975, 1977) reported that rats, having been exposed for 22 days to near weightlessness conditions aboard a Soviet C O S M O S satellite, revealed a slight increase of C K activity in several gravity related neuronal integration centers. In our paper data are presented concerning the influence of a one week hyper-gravitv period on brain C K activity during early ontogeny offish and frog larvae. Both the cichlid fish as well as the clawed toad expressed a significant decrease in total brain C K activity after a 7 days hyper-gravity exposure (3 _+ 1 g) Our data can only be subjected to interpretation insofar as brain C K activity of aquatic vertebrates, which has shown to be obviously very sensitive to long-term disturbances of gravity, plays a major role in cellular gravity perception within the brain and possibly in regulating of the cellular energy metabolism in regard to changed environmental conditions. Acknowledgements--This work was supported by a grant
from the German Space Agency DARA (FKZ 01 QV 8774). The help of Mr W. Janson (M. Sci.) in polishing the English style of our manuscript is gratefully acknowledged. REFERENCES
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Brain creatine kinase and influence of gravity Wallimann T. and Eppenberger H. M. (1985) Localization and function of M-line bound creatine kinase. M-band modell and creatine phosphate shuttle. In : Cell and Muscle Motility, (Shay J. W., ed.), Vol 6, pp 239-285. Plenum Press Corp., New York. Wallimann T. and Eppenberger H. M. (1990) The subcellular compartmentation of creatine kinase isoenzymes as a precondition for a proposed phosphoryl-creatine circuit. In : Progress in Clinical and Biological Research, Isoenzymes : Structure, Function, and Use in Biology and Medicine (Ogita Z.-1- and Markert C., eds), Vol. 344, pp. 877-899. Wiley-Liss New York. Wallimann T., SchlSsser T. and Eppenberger H. M. (1984) Function of M-line-bound creatine kinase as intramyofibrillar ATP regenerator at the receiving end of the phosphorylcreatine shuttle in muscle. J. biol. Chem., 259, 5238-5246. Wallimann T., Schnyder T., Schlegel J., Wyss M., Wegmann
411
G., Rossi A.-M., Hemmer W., Eppenberger H. M. and Quest A. F. G. (1989) Subcellular compartmentation of creatine kinase isoenzymes, regulation of CK and octameric structure of mitochondrial CK : important aspects of the phosphorylcreatine circuit. In : Proceedings of the 3rd Int. Congress on Muscle Energetics. (Paul R. J. and Elzinger G., eds.), pp. 159 176. Alan Liss Yufuin. Zeutzius I. and Rahmann H. (1980) Quantitative ultrastructural investigations on synaptogenesis in the cerebellum and the optic tectum of light-reared and darkreared rainbow trout (Salmo Gairdneri, Rich.). Differentiation 17, 181-186. Zeutzius I., Probst W. and Rahmann H. (1984) Influence of dark-rearing on the ontogenetic development of Sarotherodon mossambicus (Cichlidae, TeleosteO : I. Effects on body weight, body growth pattern, swimming activity and visual acuity. II. Effects on allometric growth relations and differentiation of the optic rectum. Exp. Bio143, 77 96.
0197-0186/93$6.00+ 0.00 PergamonPress Ltd
BRAIN CREATINE KINASE ACTIVITY D U R I N G O N T O G E N Y OF THE CICHLID FISH O R E O C H R O M I S M O S S A M B I C U S AND THE CLAWED TOAD X E N O P U S LAEVIS, I N F L U E N C E OF GRAVITY? KLAUS SLENZKA, RAMONA APPEL a n d HINRICH RAHMANN* University of Stuttgart-Hohenheim, Institute of Zoology, Garbenstr. 30, D-7000 Stuttgart 70, Germany (Received 20 May 1992; accepted 3 September 1992)
Abstract--The development of creatine kinase (CK) activity was studied in the brain of cichlid fish and clawed toads. The activity of CK in the whole brain of the fish decreases immediatelyafter hatching (stage 6) from values of about 135 nmol substrate cleaved/mg protein/min to a value of about 105 at stage 8 (5 days post hatch at 20°C). With the exception of a significant peak (125 nmol) between stages 9 and 10 (7 and 9 days respectively,post hatch at 20°C) and a small intermediate peak at stages 12 and 13 (about 10 days post hatch at 20°C) a constant level of about 100 nmol cleaved substrate is maintained until maturity. In contrast, CK activity was determined to be 3-fold higher in the whole brain of the clawed toad. With the exception of two significantpeaks at stages 47 and 49 (5 and 12 days respectively,post fertilization at 23°C) a value of about 360 nmol was found during larval development and metamorphosis, as well as in the adult brain. In investigating the possible influenceof gravity on CK activity during early ontogeny of the brain both animal species were exposed to hyper-gravity (3 + 1 g) for 7 days. A significantdecrease of total CK activity of 20% was found in the fish brain and of about 5% in the toad. Changes in total CK activity during brain development, as well as after the influence of altered gravitational forces, suggests that this enzyme is well adapted to critical phases of brain development and reacts very sensitivelyduring this period to changes in environmental conditions.
Creatine kinase (CK, EC 2.7.3.2) is a dimeric enzyme catalyzing the reversible transfer reaction of energyrich phosphate groups from ATP to creatine : ATP + creatine ~ creatine phosphate + ADP (Dawson et al., 1967, 1969 ; Dawson, 1970). This creatine-creatine phosphate "energy shuttle" has been well described by Bessman and co-workers (Bessman and Fanyo, 1966; Bessman et al., 1978; Bessman and Carpenter, 1985) as well as by Walliman and Eppenberger (1985, 1990). Generally it can be found in vertebrate tissues with a high energy metabolism e.g. in cardiac, skeletal muscles, brain and adipose tissues (Berlet et al., 1976), as well as in tumors (Shatton et al., 1979), and in smaller amounts in many other tissues (Jacobs and Lehninger, 1973). In contrast to this, invertebrate species have developed different enzyme systems, for example phosphorylarginine (Storey, 1977). In general, CK exists in different isoforms, one (CKmito) being located at the outer surface of the inner mitochondrial membrane whereas the others (MM,
*To whom correspondence should be addressed.
MB, BB) being primarily cytosolic (Bessman and Carpenter, 1985 ; Eppenberger et al., 1967 ; Walliman and Eppenberger, 1985, 1990; Walliman et al., 1984, 1989). M M - C K was found to be near the location of ATPase activity and is functionally coupled with it, providing ATP for the ATPase reaction (Saks et al., 1976, 1977, 1978, 1991). Mitochondrial CK producing phosphorylcreatine from ATP has been explained by CK coupling to the ATP/ADP translocase, which has been suggested to supply ATP directly to CK (Jacobs and Deffley, 1986; Saks et al., 1975, 1991). Regarding these basic functions of CK, several investigations were performed concerning clinical aspects as well as the possible influence of exogenously applied drugs or physical factors on CK activity. For example, Christenson et al., (1990) and Geoffrey et al., (1990) reported an increase in CK activity during reperfusion after myocardial infarction. Mahler (1979) demonstrated a dramatic decrease of mitochondrial CK activity in muscle dystrophy. Clarkson and Ebbeling (1988) showed significant alterations of CK activity after muscle damage. Concerning brain CK activity, Schwartz et al. (1989) and Peschke et al., 405
406
Kt, Atis SI l NZKA el a[.
(1989) reported that the C K serum level is a good indicator of the size of various brain lesions or ganglionectomy. To prevent these alterations of CK activity in the cardiac or skeletal system as well as in the brain different attempts were performed in applying exogenous or e n d o g e n o u s substances. Binderman et al. (1988), e.g. reported significant stimulations o f the C K activity by exogenously applying vitamin D analogues. C u p t a et al. ( 1991 ) showed that the application of high doses o f c a r b u f u r a n , induced significant alterations in the activity o f C K and its isoenzymes, as well as in energy-rich phosphates, whereas a disordered serum C K level can be stabilized by the application of dantrolene (Quinlan et al., 1990). In the last decade, research interest arose more and more in to w h a t extent e n v i r o n m e n t a l factors influences e n z y m e - d e p e n d e n t metabolic systems, especially during early ontogeny. In regard to this, analyses o f the functionally coupled ATPases revealed that seasonal a d a p t a t i o n , hydrostatic pressure a n d the application o f exogenously applied m e m b r a n e o u s glycosphingolipids ( = gangliosides) can induce d r a m a t i c changes in the activity o f Ca2+-ATPascs ( G i b b s and Somero, 1990 ; Slenzka et al., 1990, 1991 ). Focusing on the c r e a t i n e creatine p h o s p h a t e "energy shuttle", relevant investigations concerning d e v e l o p m e n t - d e p e n d e n t changes have been concentrated up to now, on the muscular C K (Hoerter et al., 1991); with few reports dealing with this aspect for the brain C K (Lapin et al., 1974; M a n o s et al., 1991). A possible influence o f the e n v i r o n m e n t a l factor gravity was d e m o n s t r a t e d in the slight increase of brain C K activity in rats t h a t h a d been exposed for 22 days to near weightlessness conditions a b o a r d a Soviet C O S M O S satellite (Krasnov, 1975, 1977). In the present study, we report on the develo p m e n t a l profile of the whole brain C K activity in the cichlid fish Oreochromis mossambicus a n d the clawed toad Xenopus laevis, a n d the influence of long-term hyper-gravity (7 days; 3 + 1 g) on the n e u r o n a l C K activity of these species d u r i n g their early ontogenetic development.
EXPERIMENTAL PROCEDURES
Materials All chemicals used in this study were purchased from the Sigma Chemical Co. (Deisenhofen, Germany) Maintenance of animals Mouth-breeding cichlid fish (Oreochromis rnossambicus) and clawed toad larvae (Xenopus laevis) were kept within small round miniaquaria (5 cm diameter, t.5 cm height; Bachofer Co., Reutlingen, Germany) at a density of 5 anim-
als/aquaria and an ambient temperature for both species of 20'C. This temperature was chosen regarding the planned STATEX-II experiment (Statolith experiment) aboard the 2nd German Spacelab Mission D-2 where cichlid fish will be reared beside their preferential temperature of 28' (' at 20 (' (Rahmann and Slenzka, 1991). Hilbig et al., (1992) have shown that this temperature had no major influence either on the normal morphological development or on that of compared to the normal behaviour. Thus, it was assumed that the maintenance temperature of 20' C, also has no m~qor influence on the normal development of the CK system. tlilbig el al. (1992) also reported, that a period of increased gravity during the early ontogenetic development of cichlid fish and toad larvae has no inlluence on the viability of the animals and it was shown thal further on a normal development up to the adult occur. Determination q f nhole hrahl CK actit,il v Determination of the protein content follows the method of Lowry et al. ( 1951). CK assays were carried out according to Oliver (1955) and Wallimann et al. (1984), with modifications for the reported analyses. The reaction was observed spectrophotometrically in a final volume of 3 ml containing 66 mM TEA buffer (pH 7.2), 34 mM EDTA, 40 mM glucose, I mM NADP ~, 51 mM MgCI> 6.7 mM ADP, 2 mM AMP, 1.2 U hexokinase, 1.2 U glucose-6-phosphate dehydrogenase, 12 pg oligomycin, and 35 #g total homogenate protein. After 5 rain preincubation at 25°C, 5 mM phosphocreatine was added to start the reaction. Specific activity was calculated as total activity minus basal activity (tesl run without the addition of MgCI2) and expressed as nmol substrate cleaved/mg protein/rain. Hereafter expressed as nmol substrate cleaved or nmol. Each test was performed five times from a pooled preparation of about 7 brains, and the standard error of the means (SEM) was calculated from these trials. Development-dependent im~estigations Three groups of about 300 cichlid fish larvae (stage 7 ; 2 days post hatch at 2WC) were carefully removed from the mother's mouth, and 5 siblings were transfered to the each miniaquaria. At critical developmental stages, starting with stage 7, up to free swimming (stage 15) and from adult fish the brains were quickly removed and prepared for the CK assay. Additionally, three groups of about 300 fertilized clawed toad eggs were also transfered, 5 per miniaquaria. Starting with developmental stage 14 ( = neurula stage, about 16 h post fertilization at 23'C) up to 66 (58 days post fertilization at 23"C) and from adults, the brains were quickly removed and prepared for the CK assay. The determination of the respective developmental stages lbr the cichlid fish was according to B/iuerle and Voss (1992) and Anken et al. (1992), and according to Nieuwkoop and Faber (1967) for the clawed toad. Gratuity-dependent investigations In regard to a possible influence of altered gravitational forces on the neuronal CK activity of the experimental animals 5 groups of hyper-gravity (3-1-1 g) animals of each species were investigated in comparison to normal 1 g earth controls. For each group 120 siblings of fish or toad larvae were reared from day 2 post hatch (stage 7; cichlid fish) or stage 14 (neurula stage; clawed toad) for 7 days in the
407
Brain creatine kinase and influenceof gravity miniaquaria, 5 each. Sixty of them were subjected to an increased acceleration of 3 + 1 g within a non-vibrating centrifuge (IEC HN-S, Damon/IEC Division, USA) at room temperature versus controls which were raised under identical conditions but at normal 1g earth gravity. Immediately after the hyper-g exposure, brains were removed and prepared for the CK assay.
two significantly increased peaks of enzyme activity at developmental stages 47 and 49, with high values of about 450 nmol (Fig. 2).
Influence of 7 days hyper-gravity on the brain CK activity of developing fish and frog larvae On the basis of our data obtained from the developmental analyses as well as from Krasnovs (1975, 1977) work with rats, it had been of general interest to determine whether or not hyper-gravity might have an influence on the CK activity within the developing brain of aquatic vertebrates. In regard to this, cichlid fish and clawed toad larvae were exposed for 7 days during early ontogenetic development from stage 7 or 8 (about 2 days post hatch) for the fish and stage 14 ( = neurula stage) for the frog, to increased gravitational forces (3 + 1 g within a non-vibrating centrifuge. The data obtained revealed significant alterations of whole brain CK activity in hyper-g fish as well as in hyper-g toad larvae after the exposure period. A statistically significant decrease of about 20% from a value of 91.5-73.3 nmol substrate cleaved (P < 0.0001) was registered in the brains of cichlid fish [Fig. 3(A)] and from about 390 to 370 nmol in the brains of the clawed toads [P < 0.0001 ; Fig 3(B)] These results show that an increasing gravity significantly influences brain CK activity.
RESULTS
CK activity during ontogenetic development of cichlid .fish and clawed toads CK activity of the whole brain of the post hatched cichlid fish Oreochromis mossambicus decreased drastically from about 137 nmot substrate cleaved at stage 7 (2 days post hatch at 20°C) to about 105 nmol at stage 8. A rapid increase was observed from stage 8 to 10 to a value of about 125 nmol followed by a fast decrease to values of about 100 nmol. At stages 12 and 13 a slight enhancement was registered followed by the definite level ( -I- 100 nmol) up to the adult stage (Fig. 1). In comparison to the data obtained from the cichlids, whole brain CK activity of the clawed toad was found with values of about 350 to 450 nmol 3fold higher than in the cichlid. Contrary to the high CK activity level found directly after hatching for the cichlid fish, the CK level in the developing toad brain is nearly the same as that in the adult frog (about 370 nmol). Similar to the fish, the clawed toad expressed
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DISCUSSION
Our objective was to investigate whether or not there would be any alterations in total brain CK activity in the cichlid fish and the clawed toad during early ontogeny as well as whether or not hyper-gravity influences the CK system within the brain of these species. Development-related investigations
Lapin et al. (1974) and Manos et al. (1991) repotted that in the developing rat brain CK activity increases postnatally without intermediate alterations. In contrast to these findings, we found in the fish brain, a dramatic decrease after hatching, which correlates with the phase of synaptogenesis and with the first onset of optokinetic reflexes and swimming movements (Anken et al., 1992; B/iuerle and Voss, 1992). Another small peak was detected at stage 13, which can be correlated with the first food-uptake parallel to the resorption of the yolk sac and with the onset of myelination. Very similar results were obtained from the clawed toad ; at stages 47 and 49, extremely high levels of CK activity were found, correlating to the maturation of the Purkinje cells, the differentiation of the optic tectum, and the start of neurohypophysis development (Nieuwkoop and Faber, 1967). Surprisingly, during metamorphosis no further significant changes in thc, CK activity were observed. Generally, the presence of high levels of CK activity in the mammalian brain (in the order of 2-18 #tool) has been attributed to neuronal energy requirements
either for synaptogenesis or for neuronal growth and myelination (Bessman and Carpenter, 1985 ; Lapin et al., 1974; Maker et al., 1973 ; Manos et al., 1991). Our own data confirms this, insofar as in fish and frogs, significant alterations in neuronal CK activity also occur during critical phases of neuronal development. Gravity-related investigations
The sprouting of nerve fibers, formation of synapses and the onset of first reflexes during early ontogenetic development of the nervous system, is to a great extent genetically determined. However, it is well known that dramatic failures can take place in the development of behaviour, brain organization, and brain ultrastructure and biochemistry, when long-term environmental disturbances (e.g. rearing in total darkness or under altered temperatures) occur (Flohr, 1988; Jeserich and Rahmann, 1979, Sester et al,, 1984; Slenzka et al., 1991; Zeutzius and Rahmann, 1980; Zeutzius et al., 1984). If these disturbances take place during critical developmental phases for relatively brief periods only, the organism is able for compensation to a certain degree by activating related structures (Flohr, 1988). Gravity, having been a stable factor on earth for more than 3.5 billions of years, has constantly influenced life. However, during the last decades, space flights enabled scientists to investigate the influence of alterations of this factor in different ways, for example in the development of the brain. In regard to this, it was reported that functional adaptations within the rat
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Fig. 3. Effect of long-term hyper-gravity (7 days ; 3 _ 1 g) on brain creatine kinase activity in cichlid fish Oreochromis mossambicus (A) as well as in clawed toads Xenopus laevis (B) controls. Specific CK activity is expressed as nmol substrate cleaved/mg protein/min. The values represent the mean + SEM of 80 measurements out of 4 pooled preparations. (**** = P < 0.0001)
brain occur during and especially after space flights, as demonstrated by an increase o f c y t o c h r o m e oxidase activity (Murakami et al., 1985). Based on quantitative histochemical determinations of C K activity, Krasnov (1975, 1977) reported that rats, having been exposed for 22 days to near weightlessness conditions aboard a Soviet C O S M O S satellite, revealed a slight increase of C K activity in several gravity related neuronal integration centers. In our paper data are presented concerning the influence of a one week hyper-gravitv period on brain C K activity during early ontogeny offish and frog larvae. Both the cichlid fish as well as the clawed toad expressed a significant decrease in total brain C K activity after a 7 days hyper-gravity exposure (3 _+ 1 g) Our data can only be subjected to interpretation insofar as brain C K activity of aquatic vertebrates, which has shown to be obviously very sensitive to long-term disturbances of gravity, plays a major role in cellular gravity perception within the brain and possibly in regulating of the cellular energy metabolism in regard to changed environmental conditions. Acknowledgements--This work was supported by a grant
from the German Space Agency DARA (FKZ 01 QV 8774). The help of Mr W. Janson (M. Sci.) in polishing the English style of our manuscript is gratefully acknowledged. REFERENCES
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