Influence of altered gravity on brain cellular energy and plasma membrane metabolism of developing lower aquatic vertebrates.

Biochemical analyses of the brain of cichlid fish larvae, exposed for 7 days to increased acceleration of 3g (hyper-g), revealed an increase in energy availability (succinate dehydrogenase activity, SDH), and in mitochondrial energy transformation (creatine kinase, Mia-CK), but no changes in an energy consumptive process (high-affinity Ca(2+)-ATPase). Brain glucose-6-phosphate dehydrogenase (G6PDH) of developing fish was previously found to be increased after hyper-g exposure. Three respectively 5 hours thereafter dramatic fluctuations in enzyme activity were registered. Analysing the cytosolic or plasma membrane-located brain creatine kinase (BB-CK) of clawed toad larvae after long-term hyper-g exposure a significant increase in enzyme activity was demonstrated, whereas the activity of a high affinity Ca(2+)-ATPase remained unaffected.     

Metabolic adaptation to long term changes in gravity environment.

Biochemical analyses of the brain of Cichlid fish larvae, exposed during their very early development for 7 days to an increased acceleration of 3g (hyper-gravity), revealed a decrease in brain nucleoside diphosphate kinase (NDPK) as well as creatine kinase (BB-CK) activity. Using high performance liquid chromatography (HPLC) the concentrations of adenine nucleotides (AMP, ADP, ATP), phospliocreatine (CP), as well as of nicotineamide adenine dinucleotides (NAD, NADP) were analyzed in the brain of hyper-g exposed larvae vs. 1g controls. A slight reduction in the total adenine nucleotides (TAN) as well as the adenylate energy charge (AEC) was found. In parallel a significant increase in the NAD concentration and a corresponding decrease in NADP concentration occurred in larva's hyper-g brains vs. 1 g controls. These results give further evidence for an Influence of gravity on cellular level and furthermore contribute to a clarification of the cellular signal-response chain for gravity perception.     

Synaptic plasticity and gravity: ultrastructural, biochemical and physico-chemical fundamentals.

On the basis of quantitative disturbances of the swimming behaviour of aquatic vertebrates ("loop-swimming" in fish and frog larvae) following long-term hyper-g-exposure the question was raised whether or not and to what extent changes in the gravitational vector might influence the CNS at the cellular level. Therefore, by means of histological, histochemical and biochemical analyses the effect of 2-4 x g for 9 days on the gross morphology of the fish brain, and on different neuronal enzymes was investigated. In order to enable a more precise analysis in future-microgravity-experiments of any gravity-related effects on the neuronal synapses within the gravity-perceptive integration centers differentiated electron-microscopical and electronspectroscopical techniques have been developed to accomplish an ultrastructural localization of calcium, a high-affinity Ca2(+)-ATPase, creatine kinase and cytochrome oxidase. In hyper-g animals vs. 1-g controls, a reduction of total brain volume (15%), a decrease in creatine kinase activity (20%), a local increase in cytochrome oxidase activity, but no differences in Ca2+/Mg(2+)-ATPase activities were observed. Ultrastructural peculiarities of synaptic contact formation in gravity-related integration centers (Nucleus magnocellularis) were found. These results are discussed on the basis of a direct effect of hyper-gravity not only on the gravity-sensitive neuronal integration centers but possibly also on the physico-chemical properties of the lipid bilayer of neuronal membranes in general.     

Influence of long-term altered gravity on the swimming performance of developing cichlid fish: including results from the 2nd German Spacelab Mission D-2.

This study presents qualitative and quantitative data concerning gravity-dependent changes in the swimming behaviour of developing cichlid fish larvae (Oreochromis mossambicus) after a 9 resp. 10 days exposure to increased acceleration (centrifuge experiments), to reduced gravity (fast-rotating clinostat), changed accelerations (parabolic aircraft flights) and to near weightlessness (2nd German Spacelab Mission D-2). Changes of gravity initially cause disturbances of the swimming performance of the fish larvae. With prolonged stay in orbit a step by step normalisation of the swimming behaviour took place in the fish. After return to 1g earth conditions no somersaulting or looping could be detected concerning the fish, but still slow and disorientated movements as compared to controls occurred. The fish larvae adapted to earth gravity within 3-5 days. Fish seem to be in a distinct early developmental stages extreme sensitive and adaptable to altered gravity; However, elder fish either do not react or show compensatory behaviour e.g. escape reactions.     

Neuroplastic reactivity of fish induced by altered gravity conditions: a review of recent results.

A review is being presented concerning behavioural, biochemical, histochemical and electronmicroscopical data on the influence of altered gravitational forces on the swimming performance and on the neuronal differentiation of the brain of cichlid fish larvae and adult swordtail fish that had been exposed to hyper-gravity (3g in laboratory centrifuges), hypo-gravity (>10(-2) g in a fast-rotating clinostat) and to near weightlessness (10(-4) g aboard the Spacelab D-2 mission). After long-term alterations of gravity (and parallel light deprivation), initial disturbances in the swimming behaviour followed by a stepwise regain of normal swimming modes are induced. Parallel, neuroplastic reactivities on different levels of investigation were found, such as adaptive alterations of activities of various enzymes in whole brain as well as in specific neuronal integration centers and an intraneuronal reactivity on ultrastructural level in individual brain parts and in the sensory epithelia of the inner ear. Taken together, these data reveal distinct adaptive neuroplastic reactions of fish to altered gravity conditions.     

Influence of altered gravity on the cytochemical localization of cytochrome oxidase activity in central and peripheral gravisensory systems in developing cichlid fish.

Cichlid fish larvae were reared from hatching to active free swimming under different gravity conditions: natural environment, increased acceleration in a centrifuge, simulated weightlessness in a clinostat and near weightlessness during space flight. Cytochrome oxidase activity was analyzed semiquantitatively on the ultrastructural level as a marker of regional neuronal activity in a primary, vestibular brainstem nucleus and in gravity receptive epithelia in the inner ear. Our results show, that gravity seems to be positively correlated with cytochrome oxidase activity in the magnocellular nucleus of developing fish brain. In the inner ear the energy metabolism is decreased under microgravity concerning utricle but not saccule. Hypergravity has no effect on cytochrome oxidase activity in sensory inner ear epithelia.     

Fish otolith growth in 1g and 3g depends on the gravity vector.

Size and asymmetry (size difference between the left and the right side) as well as calcium (Ca) content of inner ear otoliths of larval cichlid fish Oreochromis mossambicus were determined after a long-term stay at hypergravity conditions (3g; centrifuge). Both utricular and saccular otoliths (lapilli and sagittae, respectively) were significantly smaller after hyper-g exposure as compared to parallely raised 1g-control specimens and the absolute amount of otolith-Ca was diminished. The asymmetry of sagittae was significantly increased in the experimental animals, whereas the respective asymmetry concerning lapilli was markedly decreased. In the course of another experiment larvae were raised in aquarium hatch baskets, from which one was placed directly above aeration equipment which resulted in random water circulation shifting the fish around ("shifted" specimens). The lapillar asymmetry of the "stationary" specimens showed a highly significant increase during early development when larvae were forced to lay on their sides due to their prominent yolk-sacs. In later developmental stages, when they began to swim freely, a dramatic decrease in lapillar asymmetry was apparent. Taken together with own previous findings according to which otolith growth stops after vestibular nerve transaction, the results presented here suggest that the growth and the development of bilateral asymmetry of otoliths is guided by the environmental gravity vector, obviously involving a feedback loop between the brain and the inner ear.     

Effect of hypergravity on carboanhydrase reactivity in inner ear ionocytes of developing cichlid fish.

It has been shown earlier that hypergravity slows down inner ear otolith growth in developing fish. Otolith growth in terms of mineralization mainly depends on the enzyme carboanhydrase (CA), which is responsible for the provision of the pH-value necessary for calcium carbonate deposition. Larval siblings of cichlid fish (Oreochromis mossambicus) were subjected to hypergravity (3 g, hg; 6 h) during development and separated into normally and kinetotically swimming individuals following the transfer to 1 g (i.e., stopping the centrifuge; kinetotically behaving fish performed spinning movements). Subsequently, CA was histochemically demonstrated in inner ear ionocytes (cells involved in the endolymphatic ion exchange) and enzyme reactivity was determined densitometrically. It was found that both the total macular CA-reactivity as well as the difference in reactivities between the left and the right maculae (asymmetry) were significantly lower (1) in experimental animals as compared to the 1 g controls and (2) in normally swimming hg-animals as compared to the kinetotically behaving hg-fish. The results are in complete agreement with earlier studies, according to which hypergravity induces a decrease of otolith growth and the otolithic calcium incorporation (visualized using the calcium-tracer alizarin complexone) of kinetotically swimming hg-fish was higher as compared to normally behaving hyper-g animals. The present study thus strongly supports the concept that a regulatory mechanism, which adjusts otolith size and asymmetry as well as otolithic calcium carbonate incorporation towards the gravity vector, acts via activation/deactivation of macular CA.     

Effects of altered gravity on the swimming behaviour of fish.

Humans taking part in parabolic aircraft flights (PAFs) may suffer from space motion sickness-phenomena (SMS, a kinetosis). It has been argued that SMS during PAFs might not be based on microgravity alone but rather on changing accelerations from 0 g to 2 g. We test here the hypothesis that PAF-induced kinetosis is based on asymmetric statoliths (i.e., differently weighed statoliths on the right and the left side of the head), with asymmetric inputs to the brain being disclosed at microgravity. Since fish frequently reveal kinetotic behaviour during PAFs (especially so-called spinning movements and looping responses), we investigated (1) whether or not kinetotically swimming fish at microgravity would have a pronounced inner ear otolith asymmetry and (2) whether or not slow translational and continuously changing linear (vertical) acceleration on ground induced kinetosis. These latter accelerations were applied using a specially developed parabel-animal-container (PAC) to stimulate the cupular organs. The results suggest that the fish tested on ground can counter changing accelerations successfully without revealing kinetotic swimming patterns. Kinetosis could only be induced by PAFs. This finding suggests that it is indeed microgravity rather than changing accelerations, which induces kinetosis. Moreover, we demonstrate that fish swimming kinetotically during PAFs correlates with a higher otolith asymmetry in comparison to normally behaving animals in PAFs.     

Readaptation of fish to 1g after long-term microgravity: behavioural results from the STS 89 mission.

The swimming behaviour of adult and neonate swordtail fish Xiphophorus helleri was qualitatively analysed from video recordings taken throughout the STS 89 spaceshuttle mission from launch to landing and thereafter. After the flight, the swimming behaviour of neonate samples was quantitatively assessed in the course of the readaptation to 1g earth gravity at days 0, 1 and 4 after recovery. Regarding the swimming behaviour during the mission, the adult fish swam thigmotactically (i.e., responding to tactile stimuli) along the walls of their aquarium, but like the neonates, they did not show any aberrant behavioural patterns. This indicates that they could easily adapt themselves to microgravity. On mission day 9, however, looping responses (most probably initiated by mechanical disturbances) occurred indicating a continuously performed "C-start" escape response (the respective body bend looks like the letter "C"). Immediately after landing (observed in videos recorded onboard the space shuttle), the adults performed a head-up swimming beating heavily with the caudal and pectoral fins; this aberrant behaviour gradually decreased during the first hours after recovery.     

Survey of the vestibulum, and behavior of xenopus laevis larvae developed during a 7-days space flight

Aquatic animals have almost no body weight related proprioception for spatial orientation. larvae, like fish, maintain their attitude in water by continuous correction with their fin(s). For these reasons a special performance of the equilibrium system compared to terrestrial animals is necessary. Evidently fish therefore have more compact (dense) otoliths; larvae have less dense otolith (membranes) similar to land vertebrates; but their sacculus-otoliths are vertically positioned, which also may lead to a higher g-sensitivity.

For plausibility reasons gravity should influence the embryonic development of gravity receptors. Yet, evaluations of photographs taken from the surface of cut deep-frozen objects by incident light show no aberration of the shape of the whole vestibulum and of the shape, density, size and position of the otolith membrane in larvae developed under near-zero g (NEXPA-BW-STATEX in D1-Mission).

The further evaluation of the “weightless-larvae” revealed a probably not yet described statolith-like formation in the dorsal wall of the vestibulum. In the weightless larvae this formation outnumbers, also qualitatively, strongly the 1-g controls.

An extra result is the lack of striking effects of cosmic radiation on the embryonic development of the flown eggs.

The swimming behavior of the larvae which was observed about one hour after landing of the Space Shuttle showed a typical anomaly (loop swimming), which is known from larvae developed on the clinostat or from fish flown aboard Apollo capsules.     

Swimming behavior of Paramecium--first results with the low-speed centrifuge microscope (NIZEMI).

The low-speed centrifuge microscope NIZEMI (= Nieder-Geschwindigkeits-Zentrifugen-Mikroskop) is an excellent tool with which to investigate the effects of slightly increased gravity in the fields of biology and material sciences. We investigated the swimming behavior of Paramecium in the NIZEMI, by aid of a computer-controlled image analysis system. In the range of acceleration (1 g to 5 g), cells retained their swimming capability, did not sediment, and even increased the precision of their negative gravitaxis but reduced their mean swimming velocity.     

Effects of gravity on early development.

The development of embryonic and larval stages of the South African Toad Xenopus laevis D, was investigated in hyper-g up to 5 g (centrifuge), in simulated 0 g (fast-rotating clinostat), in alternating low g, hyper-g (parabolic flights) and in microgravity (Spacelab missions D1, D-2). The selected developmental stages are assumed to be very sensitive to environmental stimuli. The results showed that the developmental reaction processes run normal also in environments different to 1 g and that aberrations in behavior and morphology normalize after return to 1 g. Development, differentiation, and morphology of the gravity perceiving parts of the vestibular system (macula-organs) had not been affected by exposure to different g-levels.     

Influence of hypergravity on fish inner ear otoliths: II. Incorporation of calcium and kinetotic behaviour.

Larval siblings of cichlid fish (Oreochromis mossambicus) were subjected to hypergravity (hg; 3 g, 14 days) during development. Following the transfer to 1 g (i.e., stopping the centrifuge) they were separated into normally and kinetotically swimming individuals (the latter performed spinning movements). During hg, the animals were maintained in aquarium water containing alizarin-complexone (AC), a fluorescent calcium tracer. Densitometric measurements of AC uptake into inner ear otoliths (optical density of AC/micrometers2) revealed that the kinetotic individuals had incorporated significantly more AC/calcium than the normally behaving fish. Since the amount of otolithic calcium can be taken as an approximation for otolith weight, the present results indicate that the otoliths of kinetotically swimming samples were heavier than those of the normally behaving larvae, thus exhibiting a higher absolute weight asymmetry of the otoliths between the right vs. the left side of the body. This supports an earlier concept according to which otolith (or statolith) asymmetry is the cause for kinetoses such as human static space sickness.     

Early development in aquatic vertebrates in near weightlessness during the D-2 Mission STATEX project.

Aboard the German-Spacelab-Mission D-2 the project "Gravity Perception and Neuronal Plasticity (STATEX II)" was performed. STATEX is for STATolith EXperiment. Objects were growing tadpoles of the South African Toad (Xenopus laevis D.) and a juvenile cichlid fish (Oreochromis mossambicus). The results give a broader base for the understanding of how environmental stimuli (e.g. linear accelerations) affect the development and function of the gravity perceiving systems in these two vertebrates. These systems are accepted as models for the human vestibulum. Results of experiments in hyper-g (up to 5 g), simulated weightlessness (Fast-rotating-clinostat) and parabolic flights are compared and discussed.     

Histochemical investigations on the influence of long-term altered gravity on the CNS of developing cichlid fish: results from the 2nd German Spacelab Mission D-2.

The effect of long-term (10 days) altered gravitational conditions upon succinate dehydrogenase (SDH) reactivity in total brains as well as in individual brain nuclei of developing cichlid fish larvae had been investigated by means of semiquantitative histochemical methods (densitometric grey value analysis). Increasing accelerations from near weightlessness (spaceflight) via 1g controls to 3g hyper gravity (centrifuge) resulted in slightly increasing "all over the brain" (total brain) SDH reactivity. When focusing on distinct neuronal integration centers within the same brains in order to find the anatomical substratum of the gross histochemical data, significant effects of altered gravity only within vestibulum related brain parts were obtained.     

Swimming behaviour of the upside-down swimming catfish (Synodontis nigriventris) at high-quality microgravity – A drop-tower experiment

The catfish Synodontis nigriventris often shows a unique swimming behaviour in being oriented upside-down. When swimming near a (e.g., vertical) substrate, however, the animals orient themselves with their ventral side towards this substrate. This tendency is called ventral substrate response (VSR). The VSR does not only override the upside-down swimming behaviour but also the dorsal light response and the ventral light response.     

鱼类摆动推进的双涡模型及实验研究

通过对鱼类摆动推进的观测、实验和分析研究、从仿生学、运动学和涡动力学等方面综合研究了鱼类游动的机理，提出双涡结构模型，理论分析认为“８”字涡的存在与相互作用是一种动力机制，是鱼类摆动产生推力的原因，以此模型为指导设计了刚性仿鱼尾摆动翼的有关实验，实验结果得到启发性的有力证据，在不同流速的水洞中摆动翼均产生一定的推力，并且在水介质中的耗功率明显低于在空气中的耗功率，由此得了出结论：“８”字涡具有储能     

Behavioural changes in Paramecium and Didinium exposed to short-term microgravity and hypergravity.

The swimming behaviour of two ciliate species, Paramecium caudatum and Didinium nasutum was analyzed under microgravity and hypergravity. In Paramecium the differences between former upward and downward swimming rates disappeared under weightlessness. At microgravity the swimming rates equalled those of horizontally swimming cells at 1g. In contrast, the swimming rates of Didinium increased under microgravity conditions, being larger than horizontal swimming rates at 1g. These findings are in accordance with a hypothesis of gravireception in ciliates based on electrophysiological data, which considers the different topology of mechanoreceptor channels in theses species. The hypothesis received further support by data recorded under hypergravity conditions.     

Swimming behavior of larval Medaka fish under microgravity.

Fish exhibit looping and rolling behaviors when subjected to short periods of microgravity during parabolic flight. Strain-differences in the behavioral response of adult Medaka fish (Oryzias latipes) were reported previously, however, there have been few studies of larval fish behavior under microgravity. In the present study, we investigated whether microgravity affects the swimming behavior of larvae at various ages (0 to 20 days after hatching), using different strains: HNI-II, HO5, ha strain, and variety of different strains (variety). The preliminary experiments were done in the ground laboratory: the development of eyesight was examined using optokinetic response for the different strains. The visual acuity of larvae improved drastically during 20 days after hatching. Strain differences of response were noted for the development of their visual acuity. In microgravity, the results were significantly different from those of adult Medaka. The larval fish appeared to maintain their orientation, except that a few of them exhibited looping and rolling behavior. Further, most larvae swam normally with their backs turning toward the light source (dorsal light response, DLR), and the rest of them stayed with their abdomen touching the surface of the container (ventral substrate response, VSR). For larval stages, strain-differences and age-differences in behavior were observed, but less pronounced than with adult fish under microgravity. Our observations suggest that adaptability of larval fish to the gravitational change and the mechanism of their postural control in microgravity are more variable than in adult fish.