Ralf Anken

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.

Morphometry of fish inner ear otoliths after development at 3g hypergravity

Size and asymmetry (size difference between the left and right sides) of inner ear otoliths of larval cichlid fish were determined after a long-term stay in moderate hypergravity conditions (3g; centrifuge), in the course of which the animals completed their ontogenetic development from hatch to freely swimming. Neither the normal morphogenetic development nor the timely onset and gain of performance of swimming behaviour were impaired by the experimental conditions. However, both utricular and saccular otoliths (lapilli and sagittae, respectively) were significantly smaller after hyper-g exposure compared to 1g control specimens raised in parallel. The asymmetry of sagittae was significantly increased in the experimental animals, whereas the respective asymmetry of lapilli was pronouncedly decreased compared with the 1g controls. These findings suggest that growth and development of bilateral asymmetry of otoliths are guided by the environmental gravity vector. Some of the hyper-g animals revealed a kinetotic behaviour on transfer to normal 1g earth conditions, which was similar to the behaviour observed in previous experiments on the transfer from 1g to microgravity (parabolic aircraft flights). The lapillar asymmetry of kinetotic samples was found to be significantly higher than that of normally behaving experimental specimens. No differences in asymmetry of sagittae were obtained between the two groups. This supports an earlier theoretical concept, according to which human static space sickness might be based on asymmetric utricular otoliths.

Fish inner ear otoliths stop calcium incorporation after vestibular nerve transection

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

Effect of hypergravity on the Ca/Sr composition of developing otoliths of larval cichlid fish (Oreochromis mossambicus).

The amounts of calcium and strontium were measured by inductively coupled plasma mass spectrometry (ICP-MS) in saccular and utricular inner ear otoliths (sagittae and lapilli, respectively) of developing cichlid fish. These fish had been maintained for 22 days at 3-g hypergravity conditions within a centrifuge. During this time-span, the animals completed their ontogenetic development from hatch to the free-swimming stage. Neither the morphogenetic development nor the timely onset and gain of performance of the swimming behaviour was impaired by the experimental conditions. Experimental and control animals also did not differ concerning their size (total length). ICP-MS revealed that the otoliths contained significantly less calcium (in microg/otolith) after hyper-g exposure compared to parallelly raised 1-g control specimens (lapilli: 0.74+/-0.21 vs. 1.16+/-0.41; sagittae: 2.09+/-0.49 vs. 2.76+/-0.47). The content of strontium (in microg/otolith: lapilli: 0.0044+/-0.0023 vs. 0.0022+/-0.0013; sagittae: 0.0094+/-0.0026 vs. 0.0081+/-0.0016) and, consequently, the Sr/Ca ratio (Sr/Cax100) was increased (lapilli: 0.607+/-0.267 vs. 0.201+/-0.12; sagittae: 0.439+/-0.093 vs. 0.301+/-0.086). Since the calcium content can be taken as a proxy for otolith weight, and because parallelly undertaken morphometric investigations revealed smaller otoliths (maximum radius and surface area) due to hyper-g exposure, the results suggest that the growth of otoliths at hyper-g is slowed down. Since the concentration of trace elements incorporated into otoliths is likely based on the composition of the respective protein matrix, our findings suggest that the protein metabolism is affected by hypergravity.

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.

Weightlessness during spaceflight results in enhanced synapse formation in a fish brain vestibular nucleus

Synapse counts were undertaken by conventional electron microscopy in primary vestibular integration centers, (i.e. nucleus descendens and nucleus magnocellularis of the brainstem area octavolateralis) and in the diencephalic visual nucleus corticalis of spaceflown neonate swordtail fish Xiphophorus helleri as well as in 1 g control siblings. Spaceflight (16 days microgravity, (microg), STS-90 Neurolab Mission) yielded an increase in synaptic contacts within the vestibular nucleus descendens indicating that lack of input resulted in compensation processes. No effect of microg, however, was observed in the visual nucleus corticalis and in the vestibular nucleus magnocellularis which is situated in the close vicinity of the nucleus descendens. In contrast to the latter, the nucleus magnocellularis does not receive exclusively vestibular input, but inputs from the lateral line as well, possibly providing sufficient input at microgravity.

Altered gravity affects succinate dehydrogenase reactivity in specific nuclei of fish brain.

The effect of long-term (10 days) altered gravitational conditions upon succinate dehydrogenase (SDH) reactivity in total brain as well as in individual brain nuclei of developing cichlid fish larvae has been investigated by means of semiquantitative histochemical methods (densitometric grey value analysis). Increasing acceleration from near weightlessness (spaceflight) via 1 g controls to 3 g hypergravity (centrifuge) resulted in slightly increased total brain SDH reactivity. When focusing on distinct neuronal integration centres within the same brains in order to find the anatomical substratum of the gross histochemical data, significant effects of altered gravity on vestibulum-related brain parts were obtained. The total brain results may therefore represent the sum of such particular indirect effects but may also comprise in addition a non vestibular-related general and therefore direct influence of altered gravitational conditions, possibly on all cells.

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.

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.

Gravitational neurobiology of fish.

In vertebrates (including man), altered gravitational environments such as weightlessness can induce malfunctions of the inner ears, based on irregular movements of the semicircular cristae or on dislocations of the inner ear otoliths from the corresponding sensory epithelia. This will lead to illusionary tilts, since the vestibular inputs are not confirmed by the other sensory organs, which results in an intersensory conflict. Vertebrates in orbit therefore face severe orientation problems. In humans, the intersensory conflict may additionally lead to a malaise, commonly referred to as space motion sickness (SMS), a kinetosis. During the first days at weightlessness, the orientation problems (and SMS) disappear, since the brain develops a new compensatory interpretation of the available sensory data. The present review reports on the neurobiological responses--particularly of fish--observed at altered gravitational states, concerning behaviour and neuroplastic reactivities. Recent investigations employing microgravity (spaceflight, parabolic aircraft flights, clinostat) and hyper-gravity (laboratory centrifuges as ground based research tools) yielded clues and insights into the understanding of the respective basic phenomena.