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The inner ears of all vertebrates are designed to perceive auditory and vestibular inputs. Although a tremendous diversity in the inner ear can be found even among bony fishes, the morphologies of the utricle and of the semicircular canals are rather conservative among vertebrates. Fish show kinetoses under reduced gravity (spinning movements and looping responses) and are regarded model organisms concerning the performance of the otolithic organs. Otoliths can be analysed easily because they are compact, in contrast to the otoconial masses of other vertebrates. Here, late-larval Oreochromis mossambicus were subjected to 0.0001 × g and 0.04 × g aboard a sounding rocket, their behaviour was observed and morphometrical analyses on otoliths were carried out. Fish swimming kinetotically at 0.0001 × g had a higher asymmetry of utricular otoliths (gravity perception) but not of saccular otoliths (hearing process) than specimens behaving normally at this gravity level (p = 0.0055). Also, asymmetries of lapilli in animals swimming normally at 0.0001 × g were lower than asymmetries in specimens swimming normally at 0.04 × g (p = 0.06). This supports the "otolith asymmetry hypothesis", an explanation for the susceptibility to kinetosis, particularly concerning the utricular otoliths. It would be interesting to identify processes generating asymmetric otoliths, also in regard to human motion sickness.

The gravity-dependent behavior of Paramecium biaurelia and Euglena gracilis have previously been studied on ground and in real microgravity. To validate whether high magnetic field exposure indeed provides a ground-based facility to mimic functional weightlessness, as has been suggested earlier, both cell types were observed during exposure in a strong homogeneous magnetic field (up to 30 T) and a strong magnetic field gradient. While swimming, Paramecium cells were aligned along the magnetic field lines; orientation of Euglena was perpendicular, demonstrating that the magnetic field determines the orientation and thus prevents the organisms from the random swimming known to occur in real microgravity. Exposing Astasia longa, a flagellate that is closely related to Euglena but lacks chloroplasts and the photoreceptor, as well as the chloroplast-free mutant E. gracilis 1F, to a high magnetic field revealed no reorientation to the perpendicular direction as in the case of wild-type E. gracilis, indicating the existence of an anisotropic structure (chloroplasts) that determines the direction of passive orientation. Immobilized Euglena and Paramecium cells could not be levitated even in the highest available magnetic field gradient as sedimentation persisted with little impact of the field on the sedimentation velocities. We conclude that magnetic fields are not suited as a microgravity simulation for gravitactic unicellular organisms due to the strong effect of the magnetic field itself, which masks the effects known from experiments in real microgravity.

Research in microgravity is indispensable to disclose the impact of gravity on biological processes and organisms. However, research in the near-Earth orbit is severely constrained by the limited number of flight opportunities. Ground-based simulators of microgravity are valuable tools for preparing spaceflight experiments, but they also facilitate stand-alone studies and thus provide additional and cost-efficient platforms for gravitational research. The various microgravity simulators that are frequently used by gravitational biologists are based on different physical principles. This comparative study gives an overview of the most frequently used microgravity simulators and demonstrates their individual capacities and limitations. The range of applicability of the various ground-based microgravity simulators for biological specimens was carefully evaluated by using organisms that have been studied extensively under the conditions of real microgravity in space. In addition, current heterogeneous terminology is discussed critically, and recommendations are given for appropriate selection of adequate simulators and consistent use of nomenclature.

Inner ear otolith formation in fish is supposed to be performed by the molecular release of proteinacious precursor material from the sensory epithelia, followed by an undirected and diffuse precipitation of calcium carbonate (which is mainly responsible for the functionally important weight of otoliths). The pathway of calcium into the endolymph, however, still remains obscure. Therefore, the presence of calcium within the utricle of larval cichlid fish Oreochromis mossambicus was analyzed by means of energy filtering transmission electron microscopy (EFTEM). Electron spectroscopic imaging (ESI) and electron energy loss spectra (EELS) revealed discrete calcium precipitations, which were especially numerous in the proximal endolymph as compared to the distal endolymph. A decreasing proximo-distal gradient was also present within the proximal endolymph between the sensory epithelium and the otolith. Further calcium particles covered the peripheral proteinacious layer of the otolith. They were especially pronounced at the proximal surface of the otolith. Other calcium precipitates were found to be accumulated at the macular junctions. These results strongly suggest that the apical region of the macular epithelium is involved in the release of calcium and that calcium supply of the otoliths takes place in the proximal endolymph., (c2003 COSPAR. Published by Elsevier Ltd. All rights reserved.)

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)., (c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.)

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., (c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.)

Since changing gravity (concerning direction and amplitude) strongly affects inner ear otolith growth and otolithic calcium incorporation in developing fish, it was the aim of the present study to locate the site of mineralization in order to gain cues and insights into the provenance of the otoliths inorganic compounds. Therefore, larval cichlid fish (Oreochromis mossambicus) were incubated in the calcium-tracer alizarin complexone (AC; red fluorescence). After maintenance in aquarium water for various periods (1, 2, 3, 6, 9 and 12 h; 1, 2, 3, 5, 6, 7, 15, 29, 36 and 87 d), the animals were incubated in the calcium-tracer calcein (CAL; green fluorescence). AC thus labeled calcium being incorporated at the beginning of the experiment and would subsequently accompany calcium in the course of a possible dislocation, whereas CAL visualized calcium being deposited right at the end of the test. Subsequently, the otoliths were analyzed using a laser scanning microscope and it was shown that the initial site of calcium incorporation was located directly adjacent to the sensory epithelium and the otolithic membrane. Later, calcium deposits were also found on further regions of the otoliths' surface area, where they had been shifted to in the course of dislocation. This finding strongly indicates that the sensory epithelium plays a prominent role in otolithic biomineralization, which is in full agreement with an own electron microscopical study [ELGRA News 23 (2003) 63]., (c2003 COSPAR. Published by Elsevier Ltd. All rights reserved.)

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., (c2003 COSPAR. Published by Elsevier Ltd. All rights reserved.)

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., (c2003 COSPAR. Published by Elsevier Ltd. All rights reserved.)

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 behaviour 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 videorecordings, 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 behaviour in larval fish).

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., (c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.)