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 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.     

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.     

Experimental drop tube of the metallurgy department of Grenoble

The drop tube which will be available in the “Centre d'Etudes Nucléaires de Grenoble” is described. Its main features are the following: - Dimensions : Drop height : 47.1 m Drop time : 3.1 s Tube inside diameter : 0.2 m - Experimental atmosphere : 1 Ultra-vacuum : 10⁻⁶ to 10⁻⁷ Pa - Residual gravity level : 10⁻⁸ to 10⁻⁹ g according to the vacuum level and drop diameter.

This facility is unique insofar as it enables experiments to be performed under ultra-vacuum conditions which, by delaying the formation of surface oxides, should contribute to improving maximum undercooling values.

The techniques used for obtaining small metallic drops (0.5 to 3 mm) are described. The availability of this instrument for the scientific community is also foreseen by the french sponsoring organizations (CEA, CNES, CNRS) ; some practicle informations will be given to potential experimenters.     

Efficacy of an ototoxic aminoglycoside (gentamicin) on the differentiation of the inner ear of cichlid fish.

Previous investigations revealed that the growth of fish inner ear otoliths depends on the amplitude and the direction of gravity, thus suggesting the existence of a (negative) feedback mechanism. In the course of these experiments, it was shown that altered gravity both affected otolith size (and thus the provision of the proteinacious matrix) as well as the incorporation of calcium. It is hitherto unknown, as of whether sensory hair cells are involved either in the regulation of otolith growth or in the provision of otolithic material (such as protein or inorganic components) or even both. The ototoxic aminoglycoside gentamicin (GM) damages hair cells in many vertebrates (and is therefore used for the treatment of Meniere's disease in humans). The present study was thus designed to determine as of whether vestibular sensory cells are needed for otolith growth by applying GM in order to induce a (functionally relevant) loss of these cells. Developing cichlid fish Oreochromis mossambicus were therefore immersed in 120 mg/l GM for 10 or 21 days. At the beginning and at the end of the experimental periods, the fish were incubated in the calcium-tracer alizarin complexone (AC). After the experiment, otoliths were dissected and the area grown during GM-exposure (i.e., the area enclosed by the two AC labellings) was determined planimetrically. The results showed that incubating the animals in a GM-solution had no effect on otolith growth, but the development of otolith asymmetry was affected. Ultrastructural examinations of the sensory hair cells revealed that they had obviously not been affected by GM-treatment (no degenerative morphological features observed). Overall, the present results suggest that hair cells are not affected by GM concerning their possible role in (general) otolith growth, but that these cells indeed might have transitionally been impaired by GM resulting in a decreased capacity of regulating otolith symmetry.     

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.     

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.     

Marangoni convection when the surface tension increases with the temperature in normal and low gravity conditions

Thermocapillary convection has been studied in n-heptanol aqueous solutions whose surface tension is increasing with the temperature.

The fluid was confined in a parallelipipedic enclosure and a thermal gradient was imposed parallel to the free liquid/gas interface. The motions induced by the thermocapillary forces have been studied under low gravity conditions during Texus sounding rocket experiments and during the Spacelab D1 mission.

The combined thermocapillary and buoyancy convection have been extensively studied during ground based experiments.

The influence of the aspect ratio and of the alcohol concentration were investigated. Detailed velocity field was determined at the steady state under normal gravity conditions by Laser Doppler Anemometry (LDA).

The aspect ratio was of 0.3 and the temperatures imposed to the lateral sides of the cell were respectively 45°C and 60°C. With these experimental conditions, two superposed contrarotative cells were observed with rapid motions in the surface from the cold to the hot side. This convective pattern was also observed during the normal and high gravity periods of parabolic flights but during the low gravity period of the parabola the motions stopped everywhere in the fluid even in the surface and reappeared immediatly at the pull out of the parabola.     

Formation of otoconia in the Japanese red-bellied newt, Cynops pyrrhogaster.

Pre-mated adult female newts and fertilized eggs will be flown on the International Microgravity Laboratory-2 flight, in 1994. One objective of the flight will be to observe the influence of microgravity on the development of the gravity-sensing organs in the inner ear. These organs contain sensory hair cells covered by a layer of dense stones (otoconia). Gravity and linear acceleration exert forces on these masses, leading to excitation of the nerve fibers innervating the hair cells. If the production of the otoliths is regulated to reach an optimal weight, their development might be abnormal in microgravity. Ground-based control experiments are reported describing the developmental sequence in which both the otoliths and their associated sensory epithelium and the semicircular canals appear and develop. Three-dimensional reconstruction of serial sections through the otic vesicle of newt embryos at stages 31 through 58 demonstrate the first appearance, relative position and growth of the otoliths. Reports of experiments in which fertilized frog eggs were flown on a Russian Cosmos mission conclude that the utricular otolith is increased in volume, whereas the saccular otolith maintains normal size, suggesting that at least in the utricle, the weight of the otolith might be regulated.     

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.     

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.     

Collection of cometary dust

Rendezvous Missions to Comets lead to low velocities at the nucleus of the comet. The resulting impact velocity of the cometary dust on a target will range between 10 and 400 m/s. The dust particle which impacts on a target can be collected for a subsequent in-situ analysis.

The collection efficiency of a target depends in addition to obvious geometrical conditions upon the surface of the target. The surface characteristics can be divided into two groups:

• “dirty” surfaces, covered with silicate or hydrocarbon compounds (for example vacuum grease),

• “clean” surfaces, like gold (with additional sputtering).

This paper deals with the experimental and theoretical investigation of the collection efficiency of “clean” targets. Laboratory experiments are described which were conducted at the Technische Universität München, Lehrstuhl für Raumfahrttechnik, and the Max-Planck-Institut für Kernphysik, Heidelberg. In both experiments an electromagnetic accelerator is used to accelerate different types of dust in vacuum to velocities between 10 and 400 m/s.

The target is then examined under the microscope and a secondary ion mass spectrometer (which is a model of the laboratory carried on board of the spacecraft for “in situ” analysis). The adhesion of the dust grains at the target is evaluated experimentally in an ultracentrifuge.     

Size and cell number of the utricle in kinetotically swimming fish: a parabolic aircraft flight study.

Humans taking part in parabolic aircraft flights (PAFs) may suffer from space motion sickness (SMS, a kinetosis). Since it has been repeatedly shown earlier that some fish of a given batch also reveal a kinetotic behavior during PAFs (especially so-called spinning movements and looping responses) and due to the homology of the vestibular apparatus among all vertebrates, fish can be used as model systems to investigate the origin of susceptibility to motion sickness. Therefore, we examined the utricular maculae (they are responsible for the internalization of gravity in teleosteans) of fish swimming kinetotically at microgravity in comparison with animals from the same batch who swam normally. On the histological level, it was found that the total number of both sensory and supporting cells of the utricular maculae did not differ between kinetotic animals as compared to normally swimming fish. Cell density (sensory and supporting cells/100 micrometers2), however, was reduced in kinetotic animals (p<0.0001), which seemed to be due to malformed epithelial cells (increase in cell size) of the kinetotic specimens. Susceptibility to kinetoses may therefore originate in malformed sensory epithelia.     

A drop-tower experiment to determine the threshold of gravity for inducing motion sickness in fish.

It has been repeatedly shown earlier that some fish of a given batch reveal motion sickness (a kinetosis) at the transition from 1 g to microgravity. In the course of parabolic aircraft flight experiments, it has been demonstrated that kinetosis susceptibility is correlated with asymmetric inner ear otoliths (i.e., differently weighed statoliths on the right and the left side of the head) or with genetically predispositioned malformed cells within the sensory epithelia of the inner ear. Hitherto, the threshold of gravity perception for inducing kinetotic behavior as well as the relative importance of asymmetric otoliths versus malformed epithelia for kinetosis susceptibility has yet not been determined. The following experiment using the ZARM drop-tower facility in Bremen, Germany, is proposed to be carried out in order to answer the aforementioned questions. Larval cichlid fish (Oreochromis mossambicus) will be kept in a camcorder-equipped centrifuge during the microgravity phases of the drops and thus receive various gravity environments ranging from 0.1 to 0.9 g. Videographed controls will be housed outside of the centrifuge receiving 0 g. Based on the video-recordings, animals will be grouped into kinetotically and normally swimming samples. Subsequently, otoliths will be dissected and their size and asymmetry will be measured. Further investigations will focus on the numerical quantification of inner ear supporting and sensory cells as well as on the quantification of inner ear carbonic anhydrase reactivity. A correlation between: (1) the results to be obtained concerning the g-loads inducing kinetosis and (2) the corresponding otolith asymmetry/morphology of sensory epithelia/carbonic anhydrase reactivity will further contribute to the understanding of the origin of kinetosis susceptibility. Besides an outline of the proposed principal experiments, the present study reports on a first series of drop-tower tests, which were undertaken to elucidate the feasibility of the proposal (especially concerning the question, if some 4.7 s of microgravity are sufficient to induce kinetotic behavior in larval fish).     

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.     

Pleurodeles waltl, amphibian, Urodele, is a suitable biological model for embryological and physiological space experiments on a vertebrate.

Pleurodeles waltl (amphibian, Urodele) is an appropriate biological model for space experiments on a vertebrate. One reason for interest in this animal concerns the study of the effects of absence of gravity on embryonic development. First, after mating (on Earth) the females retain live, functional sperm in their cloacum for up to 5 months, allowing normal in vivo fertilisation after hormonal stimulation. Second, their development is slow, which allows analyses of all the key stages of ontogenesis from the oocyte to swimming tailbud embryos or larvae. We have performed detailed studies and analyses of the effects of weightlessness on amphibian Pleurodeles embryos, fertilised and allowed to develop until the swimming larvae stage. These experiments were performed in space during three missions on the MIR-station: FERTILE I, FERTILE II and NEUROGENESIS respectively in 1996, 1998 and 1999. We show that in microgravity abnormalities appeared at specific stages of development compared to 1g-centrifuge control embryos and 1g-ground control embryos. In this report we describe abnormalities occurring in the central nervous system. These modifications occur during the neurulation process (delay in the closure of the neural tube and failure of closure of this tube in the cephalic area) and at the early tailbud stage (microcephaly observed in 40% of the microgravity-embryos). However, if acephalic and microcephalic embryos are not taken into account, these abnormalities did not disturb further morphological, biochemical and functional development and the embryos were able to regulate and a majority of normal hatching and swimming larvae were obtained in weightlessness with a developmental time-course equivalent to that of 1g-centrifuge control embryos (on the MIR station) and 1g-ground control embryos.     

Swimming behaviour and calcium incorporation into inner ear otoliths of fish after vestibular nerve transection.

Previous investigations on neonate swordtail fish (Xiphophorus helleri) revealed that otolithic calcium incorporation (visualized using the calcium tracer alizarin complexone) and thus otolith growth had ceased after nerve transection, supporting a hypothesis according to which the gravity-dependent otolith growth is regulated neuronally. Subsequent investigations on larval cichlid fish (Oreochromis mossambicus) yielded contrasting results, repeatedly depending on the particular batch of cichlids investigated. Like most neonate swordtails, Type I cichlids revealed a stop of calcium incorporation after unilateral vestibular nerve transection. Their behaviour after transection was normal, and the otolithic calcium incorporation in controls of the same batch was symmetric. In Type II cichlids, however, vestibular nerve transection had no effect on otolithic calcium incorporation. They behaved kinetotically after transection (this kind of kinetosis was qualitatively similar to the swimming behaviour exhibited by larval cichlids during microgravity in the course of parabolic aircraft flights). The otolithic calcium incorporation in control animals was asymmetric. These results show that the effects of vestibular nerve transection as well as the efficacy of the mechanism, which regulates otolith growth/otolithic calcium incorporation, are--depending on the particular batch of animals--genetically predispositioned. In conclusion, the regulation of otolithic calcium incorporation is guided neuronally, in part via the vestibular nerve and, in part, via a further pathway, which remains to be addressed in the course of future investigations.     

Neuronal feedback between brain and inner ear for growth of otoliths in fish.

Previous investigations revealed that fish inner ear otolith growth (concerning otolith size and calcium-incorporation) depends on the amplitude and the direction of gravity, suggesting the existence of a (negative) feedback mechanism. In search for the regulating unit, the vestibular nerve was unilaterally transected in neonate swordtail fish (Xiphophorus helleri) which were subsequently incubated in the calcium-tracer alizarin-complexone. Calcium incorporation ceased on the transected head sides, indicating that calcium uptake is neurally regulated.     

Influence of hypergravity on fish inner ear otoliths: I. Developmental growth profile.

Inner ear stones (otoliths) of larval cichlid fish Oreochromis mossambicus were marked with the calcium-tracer alizarin-complexone (AC) at 1 g earth gravity before and after a 3, 7, 14 or 21 days stay of the animals at hypergravity conditions (hg; 3 g, centrifuge). After the experiment, the otoliths' area between the two AC-labellings was measured with regard to size and asymmetry (size difference between the left and the right stones). Both utricular and saccular otoliths (lapilli and sagittae, respectively) continued growing in a linear way at hg, but growth was significantly slowed down as compared to parallely raised 1 g-control specimens. In case of bilateral asymmetry between the corresponding otoliths its formation in hg-animals became reduced as compared to the 1 g controls. The reduction of asymmetry was much more pronounced in the sagittae than in the lapilli. The latter result supports an earlier hypothesis, according to which especially a low sagittal asymmetry has a functional advantage. In general, the results strongly suggest that otolith growth is continuously regulated in dependence of the environmental gravity vector.     

Vestibular and visual contribution to fish behavior under microgravity.

Vestibular and visual information are two major factors fish use for controlling their posture under 1 G conditions. Parabolic flight experiments were carried out to observe the fish behavior under microgravity for several different strains of Medaka fish (Oryzias latipes). There existed a clear strain-difference in the behavioral response of the fish under microgravity: Some strains looped, while other strains did not loop at all. However, even the latter strains looped under microgravity conditions when kept in complete darkness, suggesting the contribution of visual information to the posture control under microgravity. In the laboratory, eyesight (visual acuity) was checked for each strain, using a rotating striped-drum apparatus. The results also showed a strain-difference, which gave a clue to the different degree of adaptability to microgravity among different strains. Beside loopings, some fish exhibited rolling movement around their body axis. Tracing each fish during and between parabolas, it was shown that to which side each fish rolls was determined specifically to each individual fish, and not to each strain. Thus, rolling direction is not genetically determined. This may support the otolith asymmetry hypothesis. Fish of a mutant strain (ha strain, having homozygous recessive of one gene ha) have some malfunction in otolith-vestibular system, and their behavior showed they are not dependent on gravity. Morphological abnormalities of their ear vesicles during the embryonic and baby stages were noted. Their eyesight and dorsal light responses were also studied. Progress in the project of establishing a new strain which has good eyesight and, at the same time, being deficient in otolith-vestibular system was reported. Crosses between the strain of good eyesight and ha strain were made, and to some extent, F2 fish have already shown such characteristics suited for living under microgravity conditions.