Behavioural and biochemical investigations of the influence of altered gravity on the CNS of aquatic vertebrates during ontogeny.

Quantitative data are presented on the influences of hyper-gravity (3 +/- 1g) and of simulated weightlessness (approximately 0g) during early ontogeny of cichlid fish (Oreochromis mossambicus) and clawed toad (Xenopus laevis, Daudin) demonstrating changes in the swimming behaviour and the brain energy and plasma membrane metabolism. After return to 1g conditions, hyper-g reared fish and toads express the well known "loop-swimming" behaviour. By means of a computer based video analyzing system different types of swimming movements and velocities were quantitatively determined. Analyses of the brain energy and plasma-membrane metabolism of hyper-g fish larvae demonstrated an increase in energy availability (glucose 6Pi dehydrogenase, G-6P-DH), a decrease of cellular energy transformation (creatine kinase activity, CK) but no changes in energy consumptive processes (e.g. ATPases) and cytochrome oxidase activity (Cyt.-Ox). In contrast hypo-g fish larvae showed a slight increase in brain CK activity. In addition, unlike 1g controls, hyper-g fish larvae showed pronounced variations in the composition (=polarity) of sialoglycosphingolipids (=gangliosides), typical constituents of the nerve cell membranes, and a slight increase in the activity of sialidase, the enzyme responsible for ganglioside degradation.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Influence of cosmic radiation and/or microgravity on development of Carausius morosus.

Eggs of Carausius morosus were exposed to spaceflight conditions in two spaceflight missions, the German 7 day Spacelab Mission D1 and the Soviet 12.56 day Biosatellite Mission "COSMOS 1887". During spaceflight the eggs continued their development. Eggs of five different ages representing different sensitivity to radiation and different capacity to regeneration were used to investigate the influence of cosmic radiation and/or microgravity on insect development. Using the Biostack concept--eggs in monolayers sandwiched between nuclear track detectors--and the 1 g reference centrifuge of BIORACK in D1 we were able to separate effects of heavy ions of the cosmic radiation from microgravity effects and also from combined effects of these two factors in space. After retrieval, hatching rates, embryonic and larval growth kinetics and anomaly frequencies were determined. Microgravity leads to a reduced hatching rate of eggs exposed in the early stages of development. Hatching was normal in eggs which were exposed on the 1 g reference centrifuge. Hits by heavy ions caused body anomalies. The combined action of heavy ions and microgravity resulted in an unexpectedly high frequency of anomalies. These results obtained from the Spacelab Mission D1, were confirmed in an experiment onboard of COSMOS 1887. In addition to the previous analysis, embryonic development before hatching was followed which showed no major difference between flight and the ground control specimens. Since a reconfirmation of reduced hatching rates was observed in COSMOS 1887, too, the above results suggest some microgravity induced functional impairment of the hatching activity, rather than blockage in embryonic development.     

Peculiarities of genital organ formation in Arabidopsis thaliana (L) Heynh. under spaceflight conditions.

An experiment was carried out ahoard the Salyut 6 research orbital station on Arabidopsis thaliana cultivations. The seeds were sprouted in the Svetoblok 1 device which provides for plant growth in the agar medium under sterile conditions and at 4000 lux illumination. The experimental plants, as well as the controls, reached approximately the same developmental stages: both flowered and began to bear fruit. A microscopic examination of the generative organs in the control and experimental plants shows that in normally formed (by appearance) flower buds and flowers of the experimental plants, as distinct from the controls, there were no fertile elements of the adroecium and gynoecium. Degeneration of the latter occurred at different stages of generative organ development. Possible reasons for this phenomenon in plants grown under weightless conditions are considered.     

Life sciences flight hardware development for the International Space Station.

During the construction phase of the International Space Station (ISS), early flight opportunities have been identified (including designated Utilization Flights, UF) on which early science experiments may be performed. The focus of NASA's and other agencies' biological studies on the early flight opportunities is cell and molecular biology; with UF-1 scheduled to fly in fall 2001, followed by flights 8A and UF-3. Specific hardware is being developed to verify design concepts, e.g., the Avian Development Facility for incubation of small eggs and the Biomass Production System for plant cultivation. Other hardware concepts will utilize those early research opportunities onboard the ISS, e.g., an Incubator for sample cultivation, the European Modular Cultivation System for research with small plant systems, an Insect Habitat for support of insect species. Following the first Utilization Flights, additional equipment will be transported to the ISS to expand research opportunities and capabilities, e.g., a Cell Culture Unit, the Advanced Animal Habitat for rodents, an Aquatic Facility to support small fish and aquatic specimens, a Plant Research Unit for plant cultivation, and a specialized Egg Incubator for developmental biology studies. Host systems (Figure 1A, B: see text), e.g., a 2.5 m Centrifuge Rotor (g-levels from 0.01-g to 2-g) for direct comparisons between g and selectable g levels, the Life Sciences Glovebox for contained manipulations, and Habitat Holding Racks (Figure 1B: see text) will provide electrical power, communication links, and cooling to the habitats. Habitats will provide food, water, light, air and waste management as well as humidity and temperature control for a variety of research organisms. Operators on Earth and the crew on the ISS will be able to send commands to the laboratory equipment to monitor and control the environmental and experimental parameters inside specific habitats. Common laboratory equipment such as microscopes, cryo freezers, radiation dosimeters, and mass measurement devices are also currently in design stages by NASA and the ISS international partners.     

Embryogenesis and organogenesis of Carausius morosus under spaceflight conditions

The influence of cosmic radiation and/or microgravity on insect development was studied during the 7 day German Spacelab Mission D1. Eggs of Carausius morosus of five stages differing in sensitivity to radiation and in capacity to regeneration were allowed to continue their development in the BIORACK 22°C incubator, either at microgravity conditions or on the 1 g reference centrifuge. Using the Biostack concept - eggs in monolayers were sandwiched between visual track detectors - and the 1 g reference centrifuge, we were able to separate radiation effects from microgravity effects and also from combined effects of these two factors in space. After retrieval, hatching rates, growth kinetics and anomaly frequencies were determined in the different test samples. The early stages of development turned out to be highly sensitive to single hits of cosmic ray particles as well as to the temporary exposure to microgravity during their development. In some cases, the combined action of radiation and microgravity even amplified the effects exerted by the single parameters of space. Hits by single HZE particles caused early effects, such as body anomalies, as well as late effects, such as retarded growth after hatching. Microgravity exposure lead to a reduced hatching rate. A synergistic action of HZE particle hits and microgravity was established in the unexpectedly high frequency of anomal larvae. However, it cannot be excluded, that cosmic background radiation or low LET HZE particles are also causally involved in damage observed in the microgravity samples.     

垂直旋转圆盘边缘液体形态

为了解重力对旋转圆盘表面液体流动的影响,利用高速摄影,对垂直旋转圆盘边缘液体形态进行了试验研究。结果表明,与水平旋转圆盘边缘液体分为直接液滴、液柱和液膜3种形态不同,垂直旋转圆盘边缘液体分为液柱、液膜和柱膜纠缠3种形态。垂直旋转圆盘底部与顶部液体形态并不一致。底部未出现液膜形态,当流量不大于24 g/s时,为液柱形态;当流量大于等于30 g/s时,为柱膜纠缠形态。当流量为12~21 g/s、转速为1 000~2 100 r/min,顶部出现液膜形态;当流量小于12 g/s时,顶部为液柱形态;当流量大于12 g/s时,液柱形态消失,由柱膜纠缠形态取代。由于重力影响,垂直旋转圆盘边缘液体形态变化程度远大于水平旋转圆盘;在流量大到一定程度后,圆盘底部形成液柱形态需要的转速会大大增加。      

Electronmicroscopic investigations on the role of vesicle-like bodies in inner ear maculae for fish otolith growth.

The presence, morphology and possible origin of vesicle-like bodies (VBs) within the inner ear otolithic membrane of developmental stages of cichlid fish Oreochromis mossambicus and adult swordtail fish Xiphophorus helleri was analysed by means of transmission and scanning electron microscopy (TEM and SEM, respectively) employing various fixation procedures. The VBs are believed to be involved in the formation of the otolith (or statolith in birds and mammals) regarding the supply of the otolith's organic material. Increasing the osmolarity of the fixation medium decreased the number of VBs seen. Decalcification ended up in a complete disappearance of the VBs. Whilst a fixation with glutaraldehyde followed by OSO4 fixation yielded numerous VBs, only few of them were observed when the tissue was fixed with glutaraldehyde and OSO4 simultaneously. Therefore, the results strongly suggest that the VBs are fixative (i.e., glutaraldehyde) induced artifacts, so-called blisters. With this, the supply of an oto- or statolith's organic material remains obscure. Possibly, it is provided by secretion from the supporting cells as has been hypothesized earlier.     

Ultrastructural aspects of otoliths and sensory epithelia of fish inner ear exposed to hypergravity.

The present electron microscopical investigations were directed to the question, whether alterations in the gravitational force might induce structural changes in the morphology of otoliths or/and inner ear sensory epithelia of developing and adult swordtail fish (Xiphophorus helleri) that had been kept either under long-term moderate hypergravity (8 days; 3g) or under short-time extreme hypergravity (10 minutes up to 9g). The otoliths of adult and neonate swordtail fish were investigated by means of scanning electron microscopy (SEM). Macular epithelia of adult fish were examined both by SEM and transmission electron microscopy (TEM). The saccular otoliths (sagittae) of normally hatched adult fish revealed an enormous inter- (and even intra-; i.e. left vs. right) individual diversity in shape and size, whereas the otoliths of utricles (lapilli) and lagenae (asterisci) seemed to be more constant regarding morphological parameters. The structural diversity of juvenile otoliths was found to be less prominent as compared to the adults, differing from the latter regarding their peculiar crystalline morphology. Qualitative differences in the fine structure (SEM) of otoliths taken from adult and larval animals kept under 3g in comparison to 1g controls could not be observed. The SEM and TEM investigations of sensory epithelia also did not reveal any effects due to 3g stimulation. Even extreme hypergravity (more than 7g) for 10 minutes did not result in distinct pathological changes.     

The influence of gravity on the structure and functions of plant material

Growth process generate plant form and relate to most physiological functions. The Earth's gravity force affects plant growth in both obvious and subtle ways. It is a major environmental influence on morphology and physiology of plants. Gravity is less important as an agent for plant stress than as an environmental signal to guide growth. The plant's bioaccelerometers are remarkably sensitive, especially in hypogravity. Simulation (clinostat) studies and experiments in satellite laboratories are needed to understand the sensing, transduction, and response characteristics of g related mechanisms. By examining how plants alter growth processes to accomplish developmental or physiological “objectives” we may find it pragmatically desirable to ask ourselves how we might design a plant to achieve such responses to environmental influences. Examples of this design engineering approach for gravity related effects are described as an aid to experimentation.     

The reaction of Xenopus laevis Daudin (South African toad) to linear accelerations.

Preparing the German Spacelab Mission D-2 project "Gravity Perception and Neuronal Plasticity"--STATEX II--ground based experiments have been performed with larvae of the amphibian vertebrate Xenopus laevis Daud. to study the reactions to different levels of acceleration forces and profiles. The larvae have been exposed to accelerations of up to 5 g for different time periods using a modified laboratory centrifuge and the NIZEMI (Niedergeschwindigkeits-Zentrifugen-Mikroskop) which allows direct observation and video documentation. The results will be discussed and compared with those of the D1-Mission, parabolic flights, and simulated weightlessness.     

飞机板金件冲压模CAD系统的研究与开发

以AutoCAD2000软件作为开发平台,使用其提供的二次开发工具Visual Lisp 和VBA,并以Sybase作为后台数据库管理系统,在飞机板金成形数据库Ashbase的基础上,结合生产实际,研究了飞机板金件冲压模CAD系统.实现了冲压模标准件和常用件参数化绘图,开发了拉深模设计模块.本系统融二维和三维于一体,提高了模具设计效率,实用性很强.      

Light conditions affect the roll-induced vestibuloocular reflex in Xenopus laevis tadpoles

In Xenopus laevis tadpoles, effects of asymmetrical light conditions on the roll-induced vestibuloocular reflex (rVOR) were tested for the developmental period between stage 47 and 49. For comparison, the rVOR was tested in dim- and high-symmetrical light environments. Test parameters were the rVOR gain and rVOR amplitude. Under all light conditions, the rVOR increased from tadpole stage 47 to 49. For all stages, the asymmetrical light field induced the strongest response, the dim light field the weakest one. The response for the left and right eye was identical, even if the tadpoles were tested under asymmetrical light conditions. The experiments can be considered as hints (1) for an age-dependent light sensitivity of vestibular neurons, and (2) for the existence of control systems for coordinated eye movements that has its origin in the proprioceptors of the extraocular eye muscles.     

微重力下层流扩散火焰碳烟生成过程

根据详细的燃料氧化机理和多环芳烃生成机理，对乙烯同轴射流火焰在重力变化下碳烟生成情况进行计算.认为碳烟的初始成核是由两个较大的多环芳烃（PAH）二聚而成，碳烟的表面生长机理为HACA，凝结过程主要考虑PAH与碳烟的碰撞吸附，碳烟生长和氧化过程耦合在分节气溶胶模型中.计算结果表明，微重力条件下乙烯同轴射流火焰峰值温度下降230K，碳烟浓度显著增加，且浓度峰值在微重力条件下更加偏离中心线.分析重力变化对碳烟前驱体乙炔和多环芳烃的分布、初始成核速率、表面生长速率及凝结速率的影响.结果表明碳烟在中心轴线上主要是通过凝结过程生成的，且微重力条件下PAH在碳烟表面的凝结更加重要.由于微重力条件下停留时间更长，导致碳烟直径更大.