Inner Ear Otolith Asymmetry in Late-Larval Cichlid Fish (Oreochromis mossambicus, Perciformes) Showing Kinetotic Behaviour Under Diminished Gravity.

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.

Impact of a high magnetic field on the orientation of gravitactic unicellular organisms--a critical consideration about the application of magnetic fields to mimic functional weightlessness.

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.

Ground-based facilities for simulation of microgravity: organism-specific recommendations for their use, and recommended terminology.

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.

On the role of the central nervous system in regulating the mineralisation of inner-ear otoliths of fish.

Stato- or otoliths are calcified structures in the organ of balance and equilibrium of vertebrates, the inner ear, where they enhance its sensitivity to gravity. The compact otoliths of fish are composed of the calcium carbonate polymorph aragonite and a small fraction of organic molecules. The latter form a protein skeleton which determines the morphology of an otolith as well as its crystal lattice structure. This short review addresses findings according to which the brain obviously plays a prominent role in regulating the mineralisation of fish otoliths and depends on the gravity vector. Overall, otolith mineralisation has thus been identified to be a unique, neuronally guided biomineralisation process. The following is a hypothetical model for regulation of calcification by efferent vestibular neurons: (1) release of calcium at tight junctions in the macular epithelia, (2) macular carbonic anhydrase activity (which in turn is responsible for carbonate deposition), (3) chemical composition of matrix proteins. The rationale and evidence that support this model are discussed.

Hypergravity decreases carbonic anhydrase-reactivity in inner ear maculae of fish.

Previous investigations revealed that fish inner ear otolith growth depends on the amplitude and the direction of gravity. Both otolith total size, otolith bilateral size-asymmetry and the total and bilateral calcium-incorporation are also affected by gravity. Hypergravity, e.g., slows down otolith growth and diminishes bilateral otolith asymmetry as compared to 1 g control specimens raised in parallel. Since the enzyme carbonic anhydrase (CA) plays a prominent role in otolithic calcification, the reactivity of inner ear CA during otolith growth under hypergravity was investigated. CA-reactivity was demonstrated histochemically and densitometrically on sections of inner ear maculae of larval cichlid fish (Oreochromis mossambicus), that were kept for 6 hrs in a 3 g hypergravity centrifuge. The total unilateral macular CA-reactivity and the bilateral difference in CA between the left and the right maculae were significantly lower in 3 g animals than in 1g controls. The result is in complete agreement with previous studies indicating that a regulatory mechanism, which adjusts otolith size and asymmetry towards the gravity vector, acts via activation/deactivation of macular CA.

Histology of the utricle in kinetotically swimming fish: a parabolic aircraft flight study.

OBJECTIVES: Humans taking part in parabolic aircraft flights (PAFs) may suffer from space motion sickness, which is a form of kinetosis. As it has been repeatedly shown that some fish in a given batch also reveal kinetotic behaviour (especially so-called spinning movements and looping responses) during PAFs, and as a result of the homology of the vestibular apparatus of all vertebrates, fish can be used as model systems to investigate the origin of susceptibility to motion sickness. Therefore. we were prompted to examine the utricular maculae, which are responsible for the internalization of gravity in teleosteans of fish swimming kinetotically in microgravity (microg) in comparison with those of animals from the same batch who swam normally. MATERIAL AND METHODS: Larval cichlid fish (Oreochromis mossambicus) were subjected to PAFs. Post-flight, animals which had behaved normally or kinetotically during the microg phases were examined histologically The sizes of the inner ear utricular maculae as well as the numbers of sensory and supporting cells were determined. RESULTS: The total numbers of both sensory and supporting cells of the utricular maculae did not differ between kinetotic and normally swimming fish. Cell density (number of sensory and supporting cells/100 microm2) was, however, reduced in kinetotic animals (p < 0.0001), which seemed to be due to the presence of malformed epithelial cells of increased size in the kinetotic specimens. CONCLUSION: These results indicate that susceptibility to kinetosis may originate from genetically predisposed malformed sensory epithelia.

Neurophysiology of developing fish at altered gravity: background--facts--perspectives.

During the entire evolution of life on Earth, the phylogenetic as well as the individual development of all organisms took place under constant gravity conditions, against which they achieved specific countermeasures for compensation and adaptation. On the one side, gravity represents a factor of physical restriction, which compelled the ancestors of all extant living beings to develop basic achievements to counter the gravitational force (e.g., elements of statics like any kind of skeleton--from actin to bone--to overcome gravity enforced size limits or to keep form). On the other side, already early forms of life possibly used gravity as an appropriate cue for orientation and postural control, since it is continuously present and has a fixed direction. Due to such a thorough adaptation to the Earthly gravity vector, both orientation behaviour as well as the ontogenetic development of animals is impaired, when they have to experience altered gravity (delta g; i.e., hyper- or microgravity). On this background, it is still an open question to which extent delta g affects the normal individual development, either on the systemic level of the whole organism or on the level of individual organs or even single cells. The present review provides information on these questions, focusing on developing fish as model systems. Special emphasis is being laid on the effect of delta g on the developing brain and vestibular system, comprising investigations on behaviour and plastic reactivities of the brain and inner ear. Moreover, clues and insights into the possible basic causes of space motion sickness-phenomena (SMS; a kinetosis) are provided. Overall, the results speak in favour of the following concept: short-term altered gravity (< or = 1 day) can induce transitional aberrant behaviour due to malfunctions of the inner ear, originating from asymmetric otoliths or, generally, from a mismatch between canal and otolith afferents. The vanishing aberrant behaviour is due to a reweighing of sensory inputs and neurovestibular compensation, probably on bioelectrical basis. During long-term altered gravity (several days and more), step by step neuroplastic reactivities on molecular basis (i.e., molecular facilitation) in the brain and inner ears obviously activate feedback mechanisms between the CNS and the vestibular organs for the regain of normal behaviour. Mainly, the following areas of research with animals at altered gravity need to be addressed in the future: (1) Maintenance of animals through two complete life cycles in the space environment (developmental deficiencies?). (2) Investigation of the peripheral and central vestibular system by ground-based studies (mutants, hypergravity experiments...), focusing on plasticity in developing animals as well as in adults. (3) Investigation of the effect of microgravity during critical developmental periods (imprinting phase for graviperception?). Answers to these questions may be of crucial interest for basic gravitational research.

Susceptibility to abnormal (kinetotic) swimming fish correlates with inner ear carbonic anhydrase-reactivity.

Larval cichlid fish (Oreochromis mossambicus) were kept at hypergravity (hg; centrifuge) for 6 h. Following the transfer to 1 g (i.e. stopping the centrifuge), animals were separated into normally and abnormally (kinetotic) swimming individuals (the latter were swimming kinetotically, i.e. performing spinning movements). Subsequently, carbonic anhydrase- (CA-) reactivity was histochemically demonstrated and densitometrically determined in inner ear maculae. It was found that both the total macular CA-reactivity as well as the difference in reactivates between left and right maculae were significantly lower in normally swimming hg-animals as compared to the kinetotically behaving hg-fish (P<0.0001). This result is in complete agreement with closely related studies carried out on the calcium incorporation of inner ear otoliths and indicates that a regulatory mechanism, which adjusts otolithic calcium carbonate incorporation towards the gravity vector, acts via activation/deactivation of macular CA.

On the origin of susceptibility to kinetotic swimming behaviour in fish: a parabolic aircraft flight study.

Humans taking part in parabolic aircraft flights (PAFs) may suffer from motion sickness (SMS, a kinetosis; it comprises a dynamic and a static component). It has been argued that the so-called static variety of SMS during PAFs might be 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 in microgravity. Since it has been repeatedly shown earlier that some fish of a given batch reveal a kinetotic behaviour during PAFs (especially so-called spinning movements and looping responses), we investigated whether fish swimming kinetotically in microgravity have a pronounced inner ear otolith asymmetry. Therefore, the swimming behaviour of larval cichlid fish was video-recorded during PAFs and subsequently, size and asymmetry (size difference between the left and the right side) of inner ear otoliths were determined. The asymmetry of utricular otoliths of kinetotic samples was found to be significantly higher than that of normally behaving experimental specimens. Regarding the asymmetry of saccular otoliths of the two groups, statistically different results were not obtained. The findings strongly support the earlier theoretical concept, according to which otolith asymmetry causes (static) SMS.