Development and altered gravity dependent changes in glucose-6-phosphate dehydrogenase activity in the brain of the cichlid fish Oreochromis Mossambicus

~

Pergamon

Neurochem. Int. Vol. 26, No. 6, pp. 579-585, 1995

0197-0186(94)00176-6

Copyright © 1995ElsevierScienceLtd Printed in Great Britain.All rights reserved 0197~)186/95 $9.50+0.00

DEVELOPMENT AND ALTERED GRAVITY DEPENDENT C H A N G E S IN GLUCOSE-6-PHOSPHATE D E H Y D R O G E N A S E ACTIVITY IN THE B R A I N OF THE CICHLID FISH OREOCHROMIS MOSSAMBICUS K L A U S S L E N Z K A , R A M O N A APPEL and H I N R I C H R A H M A N N * University of Stuttgart-Hohenheim, Institute of Zoology, Garbenstrasse 30, D-70593 Stuttgart, Germany (Received 27 October 1994 ; accepted 20 December 1994)

Abstract--Glucose-6-phosphate dehydrogenase activity was studied in the brain of the cichlid fish Oreochromis mossambicus during early ontogenetic development. In general a slight but continuous decrease in enzyme activity was found (9.5 _+0.5 nmol substrate cleaved per mg protein and per min at developmental stage 13 { = 1 day post hatch at 28°C} to a value of 7.9_+0.6 in adult brain). In order to investigate the possible influence of altered gravity during early ontogenetic brain development, fish larvae were exposed to an increased acceleration of three times earth gravity (3 g) or to functional weightlessness in a fastrotating clinostat for 7 days. A significant increase of brain G6PDH activity of approx. 15% was found after exposure to hyper gravity, whereas a significant decrease of the enzyme activity, ~ 10%, was detected following functional weightlessness in respect to the corresponding 1 g controls. Analyses concerning the regain of normal control enzyme activity of the larvae revealed dramatic fluctuations within the first 5 h after exposure to an increased acceleration of 3 g. Thereafter, between day 1 and day 3 after exposure, brain glucose-6-phosphate dehydrogenase decreased slowly. At day 3 after exposure no further differences of the hyper-g larvae compared to the controls were found. Only slight changes in total brain glucose-6-phosphate dehydrogenase activity occur during ontogenetic development of cichlid fish. This suggests that a more or less constant enzyme activity is important during brain development, but is reacting very sensitively to changes in the environmental factor gravity.

transmitter release and maintenance of the excitability of the synaptic membrane. During neuronal activity PPP is stimulated by increased neurotransmitter biosynthesis, whereby m o n o a m i n e oxidase (MAO), along with catecholamines, was found to be the most stimulating system (Baquer et al., 1975). Another important function served by N A D P H generated in PPP is maintaining m e m b r a n e - b o u n d sulfhydryl groups in the reduced form, and thus keeping the membrane potential and mitochondrial permeability constant, guaranteeing the protection of nervous membranes from free radical attack (Tabakoff et al., 1974), as well as diminishing the peroxidation by H202 of membrane lipids and proteins (Hothersall et aL, 1982; Baquer et al., 1988; Miller et al., 1991). This point is especially important in regard to brain ageing, Alzheimer's disease, and some other neurodegenerative diseases (Chain and Stevens, 1989; Doty, 1989; Kesslak et al., 1988; Miller et aL, 1991). * Author to whom all correspondence should be addressed. 579

Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) is the first and rate limiting enzyme of the pentose phosphate pathway (PPP), which produces N A D P H by the oxidation of glucose-6-phosphate to ribose-5-phosphate. N A D P H is used for reductive synthesis such as lipid production, neurotransmitter formation and degradation, hydrogen peroxide detoxification, maintenance of glutathione in the reduced state, whereas ribose-5-phosphate is a substrate for nucleotide synthesis (Baquer et al., 1988). PPP, although quantitatively a minor route for glucose metabolism, appears to provide essential functions in nervous tissue, and is reported to change its activity during development and especially during ageing of mammals (Baquer et al., 1977, 1988; Hothersall et al., 1981, 1982; Leong et al., 1981). Baquer et al. (1988) also reported that PPP is involved in process occurring in the synaptic regions, such as

580

Klaus Slenzka el a/.

These basic functions of PPP, and especially of the rate limiting enzyme G 6 P D H , require investigation, for instance concerning changes of PPP activity during ontogenetic d e v e l o p m e n t o f fish, and c o m p a r i n g it to the data o b t a i n e d so far from rats, as well as analyses concerning possible influences of e n v i r o n m e n t a l changes. A p a r t from temperature, light and radiation. gravity is one of the most i m p o r t a n t physical factors which influences, by its almost c o n s t a n t acceleration, nearly all living beings during phylogenetic development. In lower aquatic vertebrates, for instance, changes of the gravity vector induce significant alterations of the swimming b e h a v i o u r when fertilization took place long before the exposure (von B a u m g a r t e n et al., 1975; Hoffman et al., 1978; Slenzka et al., 1994). However, no alterations were observed when fish or frog eggs were fertilized shortly before or during altered gravity, e.g. during space flight (von B a u m g a r t e n et al., 1975 ; Scheld et al., 1977 ; H o f f m a n et al., 1978 ; Souza et al., 1994). Since additionally it is well k n o w n that e n v i r o n m e n t a l disturbances during critical develo p m e n t a l phases can be c o m p e n s a t e d by the organism t h r o u g h activating related structures in the central nervous system (Flohr, 1988), the question still remains open to what extent gravity might influence the CNS itself during very early ontogenetic development. In regard to this M u r a k a m i et al. (1985) reported an increase in brain c y t o c h r o m e oxidase activity in rats after space flight. An increase in creatine kinase activity in the rat as well as in the fish brain after space flight was reported by K r a s n o v (1975, 1977). In agreement Slenzka et al. (1993, 1994) d o c u m e n t e d a slight increase in brain creatine kinase of fish after exposure to functional weightlessness within a fast-rotating clinostat, a n d conversely a dramatic decrease in enzyme activity of fish larvae and tadpoles after long-term exposure to an increased acceleration of three times earth gravity (hyper gravity) within a centrifuge. Gravity related investigations concerning PPP activity were performed so far only with h u m a n dermal fibroblasts by G a u b i n et al. (1990) showing an increase o f G 6 P D H activity after exposing the cells to hyper gravity. The present study is concerned with changes in the activity of brain G 6 P D H during ontogenetic develo p m e n t of the m o u t h - b r e e d i n g cichlid fish Oreochromis mossambicus, as well as following the influence of 7 days increased acceleration (3 g) within a centrifuge or 7 days functional weightlessness within a fast-rotating clinostat of this species during its early ontogenetic development.

EXPERIMENTAL PROCEDURES

Maleria/s All chemicals used in this study were purchased from the Sigma Chemical Co. (Deisenhofen, Germany). Derelopment dependent #u'estigations Three groups of about 300 cichlid fish larvae, stage 12 (corresponding to about 3.5 days post fertilization at 28C), were carefully removed from the mother's mouth and transferred to special breeding reservoirs, where they were allowed to float freely under a constant stream of water in order to simulate their steady shifting in the mother's mouth. At critical developmental stages, starting with stage 13 (about 1 day post-hatch at 28cC), up to free swimming (stages 25 = young fish) and from mature adults, the brains were quickly removed and prepared for the G6PDH assay. The determination of the corresponding developmental stages was according to Anken et al. (1993). Hyper gravity/centr![uge experiments In regard to a possible influence of increased acceleration on brain G6PDH of developing cichlid fish nine groups of hyper gravity (3 g) exposed fish were investigated in comparison to normal 1 g controls. For each group 120 siblings were reared from stage 15 within small round vessels (5 cm dia, 1.5 cm height ; Bachofer Co. Reutlingen, Germany) at a density of five animals each. A biofoil as bottoms of the vessels guaranteed gas exchange. Due to the resorption of their yolk sack no additional feeding of the larvae was necessary. Sixty larvae of each group (stage 15 7 days post-hatch at 28'~C) were subjected to an increased acceleration of 3 g for 7 days within a non-vibrating centrifuge (IEC HN-S, Damon/IEC Division, U.S.A.) at an ambient temperature of 2 2 C and compared to controls, which were raised under identical conditions, but at normal l g earth gravity. To prevent dorsal-light reflex orientation during the test runs, they were performed in total darkness. Immediately after exposure, brains were removed and prepared for the G6PDH assay. Determination o / the time course Jor the regain o/" G6PDH actiL,ity to normal, control levels after a long-term exposure to increased aeeeleration Directly after the exposure to an increased acceleration of 3/,, the animals out of six test runs were divided into six groups and transferred to a standard aquaria at natural light/dark cycle, allowing feeding ad lib. after total yolk sack resorption. The first group was immediately sacrificed and brain G6PDH activity was determined as compared to controls. Groups 2 and 3 were prepared 3 and 5 h later respectively. 1, 3 and 5 days after exposure the enzyme activity of the other groups was determined in comparison to that of the controls. Clinostat experiments with.[~mctional weightlessness During three test runs 60 cichlid fish larvae (stage 15) were each exposed to functional weightlessness in total darkness at an ambient temperature of 22"C within a fast-rotating clinostat (86 rpm) for 7 days according to Briegleb (1967). The plastic tubes (25 cm long; 3 mm i.d.) containing the larvae rotated vibration-free within standard aquaria. To guarantee diffusion processes to the surrounding water without any water flow or streaming within the tube they were

Brain glucose-6-phosphate dehydrogenase and influence of gravity perforated with holes of 0.5 mm diam. An appropriate number of 1 g control animals was reared under identical conditions but without rotation. The larvaes' heads were positioned directly in the centre of the rotation axis. Immediately after exposure, the brains were removed and prepared for the G6PDH assay. Using the fast-rotating clinostat we supposed that during early ontogenetic brain development the cells still have to perceive gravity information for target orientation. Certainly the gravity vector is a very weak acceleration and exists as only one of several other parameters, nevertheless might be an important component for adaptive brain development. Assuming that the brain and even each cell can be described as a suspension containing particles with a density higher than that of the surrounding liquid, it can be calculated that the random distribution of gravity by continuous rotation will lead to a certain kind of functional weightlessness around a small diameter from the rotation axis by preventing sedimentation processes of dense particles (Briegleb, 1988). Thus it can be presupposed, that the centrifugal force on the brain cells is less than 1 g. However, it has to be mentioned that a small acceleration gradient still exists and the following results only give an approximation to really reduced gravity and have to be finally proven by experiments under near weightlessness conditions during orbital flight.

Determination of whole brain glucose-6-phosphate dehydrogenase activity Determination of the protein content of the brain tissue followed the method of Lowry et al. (1951). G6PDH activity was measured according to the method of Catalano et al. (1975). The reaction was observed spectrophotometrically at 339 nm over a period of 30 min at 37°C in a final volume of 2 ml, containing 50 mM Tris-HCl buffer (pH 7.2), 0.1 mM CDTA, 50 mM MgCI2, 0.38 mM NADP +, 3.3 mM glucose6-phosphate, 5 mM maleinimide and about 35 pg total homogenate protein. Specific activity was calculated as total activity minus basal activity (test run without the addition of MgCI2) and expressed as nmoles substrate cleaved/mg protein × minute. Hereafter expressed as nmoles substrate cleaved or nmoles.

Statistics In regard to the hyper gravity experiments nine test runs (= centrifugal runs), in regard to experiments in functional weightlessness (clinostat) three test runs, and in regard to the investigations concerning regaining normal enzyme activity six test runs were analysed. Each test run consisted of 60 experimental vs 60 control larvae. From pooled preparation of nine brains each the protein content was determined and at least 20 measurements were performed to determine the enzyme activity. RESULTS

Brain G6PDH activity during ontogenetic development of cichlid fish In general, G 6 P D H activity of the whole brain of post-hatched cichlid fish O. mossambieus (development stage 13) decreased continuously without significant alterations during later ontogenetic development to the adult from values of 9.5 + 0.5 to

581

7.9 + 0 . 6 nmol substrate cleaved per mg protein and minute (Fig. 1).

Brain G6PDH activity of cichlid fish larvae after development under increased acceleration (hyper gravity) or functional weightlessness, respectively On the basis of the data obtained from the developmental analyses and of Gaubin et al. (1990) who is working with human dermal fibroblasts, it was of special interest to investigate whether or not increased acceleration (hyper gravity) or "functional weightlessness" within the fast-rotating clinostat, respectively, might have any influence on the G 6 P D H activity within the developing brain of cichlid fish. In regard to this, cichlid fish larvae were exposed to increased acceleration (3 g) within a non-vibrating centrifuge or to functional weightlessness within the clinostat during early ontogenetic development starting with stage 15 for 7 days at 22°C. The data obtained, revealed significant alterations in brain G 6 P D H activity after the exposure to altered gravity conditions. A highly significant increase (P < 0.0001) in enzyme activity of about 15% (8.9 nmols) was measured after hyper gravity exposure, whereas a significant decrease (P < 0.01) of about 10% (6.59 nmol) was found after clinorotation vs the corresponding 1 g controls (7.35 nmol, Fig. 2). These results clearly show that alterations in the gravity environment during critical developmental phases o f brain development significantly influence at least one pathway of the total brain metabolism as indicated by the brain G 6 P D H activity.

Regain of normal G6PDH activity after hyper gravity conditions Brain G 6 P D H activity of developing cichlid fish was shown to be significantly increased after hyper gravity respectively decreased after clinostat exposure. On this basis the time-course of regaining normal/control enzyme activity was analysed for another 5 days after the transfer of the fish larvae from hyper gravity to 1 g conditions. 3 h after this transfer the enzyme activity in the brains of the former hyper gravity animals decreased significantly whereas an increase occurred in the former dark-kept controls reaching values comparable to animals reared under the normal day and night cycle (Fig. 3). 5 h after transfer the enzyme activity in the brains of the former hyper gravity exposed animals increased and was again significantly higher as compared to the controls. These observed differences and fluctuations of enzyme activity shortly after their transfer to control conditions diminished slowly on day 1 post-exposure and

Klaus Slenzka el a/.

582 'E'

F=

12

X

o O.

E

8

m o

E C

myelinogenesis

synaptogenesis

eyes developed

hatch

/ 0

o

12

13

yolk sack totallyresorpt

I

I

I

~

I

I

14

15

16

17

18

19

free

swimming

~ //---~ 20/21

23•24 youngfish

adult

developmental stages Fig. 1. Whole brain G6PDH activity during development of the cichlid fish O. mossambicus. Specific G6PDH activity is expressed as nmoles substrate cleaved/mg protein x minute. The values represent the mean _+SEM from three batches of pooled preparations. no more differences compared with controls were registered after 3 and 5 days respectively (Fig. 3).

DISCUSSION

Development-dependent

investiyations

The aim of the present study was to investigate whether or not there might be any alterations in total G 6 P D H and thus PPP activity of cichlid fish brain during early ontogenetic development as well as after alterations in the gravity environment. On the one

.m

10'

[]

E X

o

l E

clinostat

[]

lg control

•

3g hypergravity

5'

E

.c. :

I

Fig. 2. Effect of long-term increased acceleration or functional weightlessness (7 days ; 3 g hypergravity respectively 7 days clinorotation) on brain G6PDH activity in the cichlid fish O. mossambicus vs I g controls. Specific G6PDH activity is expressed as nmoles substrate cleaved/mg protein× minute. The values represent the mean + SEM of the number of testruns (hyper gravity: n = 9; clinostat: n = 3: **P < 0.01 : ****P < 0.0001).

hand the activity of G 6 P D H was found to decrease to 1/3 in brain during development of the rat embryo to the adult (Bagdasarian and Hulanicka, 1965). On the other hand, in very detailed studies Baquer et al. (1975, 1977, 1988) and Leong et al. (1981) showed that in the developing rat brain, especially in the cerebellum, an increase of G 6 P D H activity occurred until the tenth day after birth. This rise was proposed to be due to a high rate of lipid synthesis incorporating lipid precursors into myelin (Maker and Hauser, 1967 ; Lehrer et al.. 1970). The diminution in the PPP might additionally have been due to limitations ofcofactors (Baquer et al., 1988). During ageing of the rat brain an increase, rather a decrease of G 6 P D H activity occurred in some brain regions between adulthood and ageing, while the activity in other regions remained unchanged (Leong et al., 1981 ; Mizuno and Ohta, 1986; Miller et al., 1991). In contrast to this particular Miller et al. (1991) reported a highly significant decrease of G 6 P D H activity lbr example in the olfactory epithelium during ageing and discussed it to be most probably a reflection of the decline in the regeneration capacity. The general slight drop of G 6 P D H activity shown in this study might be due to a fish specific continuous turn-over process in some parts of the brain as especially outlined for the optic tectum, where a continuous shifting of synapses had been reported during development (Easter and Stuermer, 1984; Stuermer, 1986). Additionally this process might be combined

Brain glucose-6-phosphate dehydrogenase and influence of gravity

"

583

,'T

01

E

8 0

E

.c.

I I lg control (dark reared) I- - -I 3g hyper gravity (dark reared) i • • • I lg control (dark/light reared)

6 0

0 hrs

I

I

I

I

I

3 hrs

5 hrs

1 day

2 days

3 days

I

5 days

time Fig. 3. Time-course of regaining normal, control G6PDH activity in the brain of developing cichlid fish O. rnossambicus after a 7 day exposure to an increased acceleration of 3 g vs I g controls reared under total darkness and thereafter transferred to normal day/night cycles. Specific G6PDH activity, is expressed as nmoles substrate cleaved/mg protein × minute. The values represent the mean + SEM of six test runs. with a continuous protection of the nerve cells against peroxidative stress. However, for another enzyme system (creatine kinase) located at the plasma membrane Slenzka et al. (1993) had shown previously that in the brain of the same fish species significant alterations occurred during development, which could be paralleled to highly consumptive, critical phases, for instance during synaptogenesis. In summary our data clearly show that during development of cichlid fish beside a general decline of G6PDH activity no significant changes in the PPP activity occur. Gravity-related investigations

During early ontogenetic development of the nervous system on the one hand critical phases occur, for instance concerning the sprouting of nerve fibres, formation of synapses or the onset of first reflexes. On the other hand, it is well known that environmental disturbances during these phases can induce drastic changes in behaviour, in morphological brain organization and in brain biochemistry, thus leading to compensatory reactions of the organism by activating related systems (Flohr, 1988; Slenzka et al., 1991, 1993, 1994; Zeutzius et al., 1984). Gravity, having been a constant vector (acceleration rate of 9.81 m/s 2) since about 3.5 billion years, has continuously influenced life on earth. During the last three decades, especially orbital and space flights have enabled scientists to investigate the influence of

gravity/weightlessness on organisms for example on the development of their behaviour and their vestibular and especially neuronal reactivity. In regard to this, it was reported that functional alterations within the rat brain occur during, and particularly after, space flights as demonstrated by an increase of cytochrome oxidase activity in specific brain regions (Murakami et al., 1985). Based on quantitative histochemical methods, Krasnov (1975, 1977) reported that rats and fish, having been exposed to the conditions of near weightlessness during long-term orbital flights, revealed a slight increase in brain creatine kinase activity (BB-CK). These results had been supported by data of Slenzka et al., (1993, 1994) demonstrating an increase of BB-CK in the developing cichlid fish as well as in the clawed toad brain after exposure to functional weightlessness within the fastrotating clinostat. In contrast to this a highly significant decrease of the enzyme activity after exposure to hyper gravity (3 g) was found (Slenzka et al., 1994). Investigations of other enzymes (succinate dehydrogenase, cytochrome oxidase, acetylcholine esterase, NADPH-diaphorase, lactate dehydrogenase, alkaline and acidic phosphatase, monoamine oxidase) during rat brain development under the conditions of near weightlessness remained uninfluenced (Krasnov et al., 1987). In contrast to this, Paulus et al. (1993) and Anken e t al. (1994) showed by means of histochemical and ultrastructural techniques that brain cytochrome oxidase and succinate dehydrogenase reactivity was

584

Klaus Stenzka et al.

influenced after an exposure of developing cichlid fish to increased acceleration or to the conditions of near weightlessness. Gravity related investigations of PPP or G 6 P D H activity respectively were, up to now, only performed with cell cultures. Gaubin et al. (1990) demonstrated that G 6 P D H activity was enhanced in human dermal fibroblasts after exposure to hyper gravity. |n contrast to this, the same group reported a reduction of G 6 P D H activity in leukaemic murine cells after the same experimental conditions (Croute et al., 1994). The present study shows primarily that changed gravity environments during ontogenetic development of cichlid fish and by this gravity itself has a significant influence on brain G 6 P D H activity. Fishes and amphibians which had developed under altered gravity conditions during orbital flight or during increased acceleration expressed normal swimming behaviour especially when fertilized in the changed gravity environment or transferred to this in a very early development stage (Rahmann et al., 1992, 1995: Slenzka et al., 1994: Souza e t a [ . , 1995). In regard to these findings it can be hypothesized that the observed changes in brain enzyme activities for example in G 6 P D H or BB-CK as reported in this and previous papers might be functionally responsible for the adaptation of the brain metabolism during very early development to long-term changed gravity environments. Additionally the observed changes in the plasma membrane composition as reported in Slenzka et al. (1994) together with results that, for instance, the plasma membrane bound high-affinity CaZ+-ATPase remains uninfluenced in the brain of cichlid fish larvae after exposure to increased accelerations, speak in favour for compensatory reactions of metabolic pathways to guarantee a cellular homeostasis in a new gravity environment. However, behavioural alterations of aquatic vertebrates directly after transfer to a new gravity condition were reported by von Baumgarten et al. (1975) and Rahmann et al. (1992, 1995). Against this background, it had been of interest to investigate the timecourse of regaining normal G 6 P D H control activity after exposure of cichlid fish to increased acceleration. Our results obtained so far clearly show dramatic changes during the first hours after the end of exposure, possibly indicating some kind of overshooting of the brain metabolism, which occurs within the first few days of adaptation. Further investigations have to show whether or not this kind of metabolic adaptation also will take place for other brain enzymes. The clarification of this question is particularly important from an analytical point

of view concerning different durations between the end of exposure and tissue preparation of test animals, which especially happens after space flights due to possible changes in the landing sites of the orbiters and due to security reasons. ,,lcknowledqements The authors gratefully acknowledge the help of Mr M. Metzler and Mr W. Jansen (M.Sci.) in improving our style of English. This work was supported by a grant from the German Space Agency DARA (FKZ 01 QV 8774).

REFERENCES Anken R., Kappel T., Slenzka K. and Rahmann H. (1993) The early morphogenetic development of the cichlid fish Oreochromis mossambicus. Zool. Anz. 231, 1 10. Anken R. H., Slenzka K., Neubert J. and Rahmann H. (1994) Altered gravity affects succinate dehydrogenase reactivity in specific nuclei in the fish brain. Neuroreport 5, 1313 1316. Bagdasarian G. and Hutanicka D. (1965) Glucose-6-phosphate dehydrogenase during development of rat brain. Biochim. Biophvs. Acta 99, 367 369. Baquer N. Z., Hothersall J. S. and McLean P. (1988) Function and regulation of the pentose phosphate pathway in brain. Curr. Topics Cell. Reg. 29, 265 289. Baquer N. Z., McLean P. and Greenbaum A. L. (1975) Systems relationship and the control of metabolism pathways in developing brain. In Normal and Pathological Detelopment o1' Energy Metabolism (Hommes F. A. and Van den Berg C. J. eds), pp. 109-132. Academic Press, London. Baquer N. Z., Hothersall J. S., McLean P. and Greenbaum A. L. 11977) Aspects of carbohydrate metabolism in developing brains. De~,. Med. Child Neurol. 19, 81-104. yon Baumgarten R. J., Simmonds R. C., Boyd J. F. and Garriott O. K. (1975) Effects of prolonged weightlessness on the swimming pattern of fish aboard Skylab 3. Arieu. Space Era,iron. Med. 46, 902-.-906. Briegteb W. (1967) Ein Modell zur Schwerelosigkeits-Simulation an Mikroorganismen. Naturwissenschq;fien 54, 167. Briegleb W. (1988) Ground-borne methods and results in gravitational cell biology. The Physiologist 31, 4447. Catalano E. W., Johnson G. F. and Solomon H. M. (1975) Measurement of erythrocyte glucose-6-phosphate dehydrogenase with a centrifuge analyser. Clin. Chem. 21, 143 138. Chain W. S. and Stevens J. C. (1989) Uniformity of olfactory Joss in ageing. Ann. N.Y. Acad. Sci. 561, 29-38. Croute F., Gaubin Y., Pianezzi B. and Soleilhavoup J. P. (1994) Effects of hypergravity on growth rate and enzymes involved in carbohydrate metabolism in leukaemic murine cells. In Proc. 5th Eur. Svmp. L(/i, Sci. Res. Space, Arcachon, France. Doty, R. L. (1989) Influence of age and age-related diseases on olfactory function. Ann. N~ Y. Acad. Sci. 561, 7(~86. Easter S. S. and Stuermer C. A. O. (1984) An evaluation of the hypothesis of shifting terminals in goldfish optic tectum. J. Neurosci. 4, 1052 1063. Flohr H. (1988) Post-lesion Neural Plasticity. Springer, Berlin. Gaubin Y.. Croute F., Hartmann D., Pianezzi B. and Sole-

Brain glucose-6-phosphate dehydrogenase and influence of gravity ilhavoup J.-P. (1990) Human dermal fibroblast sensitivity to hypergravity. In Proc. 4th Eur. Symp. Life Sci. Res. Space. Trieste, Italy. Hoffman R. B., Salinas G. A., Boyd J. F., von Baumgarten R. J. and Baky A. A. (1978) Effect of prehatching weightlessness on adult fish behaviour in dynamic environments. Aviat. Space Environ. Med. 49, 576-581. HothersaU J. S., Greenbaum A. L. and McLean P. (1982) The functional significance of the pentose phosphate pathway in synaptosomes: protection against peroxidative damage by catecholamines and oxidants. J. Neurochem. 39, 1325-1332. Hothersall J. S., E1-Hassan A., McLean P. and Greenbaum A. L. (1981) Age-related changes in enzymes of rat brain. 2. Redox-systems linked to NADP and glutathione. Enzymes 26, 271-276. Kesslak J. P., Cotman C. W., Chui H. C., Van den Noort S., Fang H., Pfeffer R. and Lynch G. (1988) Olfactory tests as possible probes for detecting and monitoring Alzheimer's disease. Neurobiol. Ageing 9, 399-403. Krasnov I. B. (1975) Quantitative cyto- and histochemical studies of the Deiters' nucleus and nodular cortex of the cerebellum in rats exposed to weightlessness. Aviat. Space Environ. Med. 46, 1119-1122. Krasnov I. B. (1977) Quantitative histochemistry of the vestibular cerebellum of the fish Fundulus heterocolitus flown aboard the Biosatellit Cosmos-782. Aviat. Space Environ. Med. 48, 808-811. Krasnov I. B., Olenev S. N., Babichenko I. I. and Kesarev V. S. (1987) Morphogenesis of the brains of rats developing in weightlessness. Kosmichesk. Biol. Aviakosmiehesk. Medits. 21, 16-22. Lehrer G. M., Bornstein M. B., Wiess C. and Silides D. J. (1970) Interaction of lipid synthesis and embryonic development. Exptl Neurol. 26, 595-606. Leong S. F., Lai J. C. K., Kim L. and Clark J. B. (1981) Energy-metabolising enzymes in brain regions of adult and ageing rats. J. Neurochem. 37, 1548-1556. Lowry O. H., Rosebrough H. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. Maker H. S. and Hauser G. (1967) Embryonic development of rats and related brain enzyme activities. J. Neurochem. 14, 457-464. Miller S., Coopersmith R. and Leon M. (1991) Biochemical quantification and histochemical localisation of glucose6-phosphate dehydrogenase activity in the olfactory system of adult and aged rats. Neurochem. Res. 16, 475-481. Mizuno Y. and Ohta K. (1986) Regional distribution of thiobarbituric acid-reactive products, activities of enzymes regulating the metabolism of free radicals, and some of the related enzymes in adult and aged brains. J. Neurochem. 46, 1344-1352. Murakami D. M., Miller J. D. and Fuller C. A. (1985)

585

Changes in functional metabolism in the rat central nervous system following space flight. The Physiologist 28, 143-144. Paulus U., Krrtje K. H. and Rahmann H. (1993) Effects of development and altered gravity conditions on cytochrome oxidase activity in a vestibular nucleus of the larval teleost brain : a quantitative electronmicroscopical study. J. Neurobiol. 24, 1131-1141. Rahmann H., Slenzka K., Krrtje K. H. and Hilbig R. (1992) Synaptic plasticity and gravity: ultrastructural, biochemical and physico-chemical fundamentals. Adv. Space. Res. 12, 63-72. Rahmann H., Slenzka K., Anken R., Appel R., B~iuerle A., Flemming J., Hilbig R., Kappel T., Krrtje K. H., Paulus U., Neubert J., Briegleb W., Schatz A. and Bromeis B. (1995) Behavioral, histochemical, biochemical and ultrastructural investigations on the influence of hyper- and hypo-gravity on the early ontogenetic development of fish and toad larvae. Acta Astronautica. In press. Scheld H. W., Boyd J. F., Bozarth G. A., Conner J. A., Eichler V. B., Fuller P. M., Hoffman R. B., Keefe J. R., Kuchnow K. P., Oppenheimer J. M., Salinas G. A. and von Baumgarten R. J. (1977) Killifish hatching and orientation: experiment MA-161. Apollo Sojus Test Project Preliminary Scientific Report, NASA Document TM X58173, 19-1-19-13. Slenzka K., Appel R. and Rahmann H. (1991) Brain Ca2+/Mg2÷-ATPase activity and seasonal adaptation of the djungarian dwarf hamster Phodopus sungorus. Comp. Biochem. Physiol. 100A, 937-941. Slenzka K., Appel R. and Rahmann H. (1993) Brain creatine kinase activity during ontogeny of the cichlid fish Oreochromis mossambicus and the clawed toad Xenopus laevis, influence of gravity? Neurochem. Int. 22, 405-411. Slenzka K., Appel R., Hilbig R., Kappel T., Vetter S., FreischOtz, B. and Rahmann H. (1994) Behavioural and biochemical investigations of the influence of altered gravity on the CNS of aquatic vertebrates during ontogeny. Adv. Space Res. 14, 309-312. Souza K. A., Ross M., Black S. and Wassersug R. (1995) The effects of microgravity on amphibian development. Acta Astronautica. In press. Stuermer C. A. O. (1986) Pathways of regenerated retinotectal axons in goldfish I. Optic nerve, tract and tectal fascicle layer. J. Embryol. exptl Morph. 93, 1-28. Tabakoff B., Groskopf W., Anderson R. and Alivisatos S. G. A. (1974) Biogenic aldehyde metabolism, relation to pentose shunt in brain. Biochem. Pharmac. 23, 1701-t 719. Zeutzius I., Probst W. and Rahmann H. (1984) Influence of dark-rearing on the ontogenetic development of Sarotherodon mossambicus (Cichlidae, Teleostei). I: Effects on body weight, body growth pattern, swimming activity and visual acuity. II: Effects on allometrical growth relations and differentiation of the optic tectum. Exptl Biol. 43, 77-96.