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.     

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.     

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.     

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.     

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.     

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.     

Histochemical localisation of carbonic anhydrase in the inner ear of developing cichlid fish,Oreochromis mossambicus

Inner ear otolith growth in terms of mineralisation mainly depends on the enzyme carbonic anhydrase (CAH). CAH is located in specialised, mitochondria-rich macular cells (ionocytes), which are involved in the endolymphatic ion exchange, and the enzyme is responsible for the provision of the pH-value necessary for otolithic calcium carbonate deposition.     

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.     

Calcium gradients in the fish inner ear sensory epithelium and otolithic membrane visualized by energy filtering transmission electron microscopy (EFTEM).

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.     

Mass and mechanical sensitivity of otoliths.

It has been suggested that the changes of otolith mass during the otolith development in altered gravity conditions as well as the growth of otoliths in fishes in normal conditions are determined by the feedback between the otolith dynamics and the processes that regulate otolith growth. This hypothesis originates from the pendulum model of an otolith (de Vries, 1950), in which otolith mass is a parameters. The validity of this hypothesis is tested by comparing the pendulum model with a simplified spatially distributed model of an otolith. It was shown that when the otolith plate (otoconial layer) was spatially distributed and fixed to the macular surface, the mechanical sensitivity of the otolith does not depend on the total otolith mass and its longitudinal dimensions. It is determined by otolith thickness, Young's modulus, and the viscosity of the gel layer of the growing otolith. These parameters may change in order to secure otolith sensitivity under altered dynamic conditions (e.g., in microgravity). Possible hypotheses regarding the relationship between the otolith growth, otolith dynamics and animal growth are proposed and discussed here.     

New model of hair cell bundle functioning in otoliths.

Transformation of the mechanical input in the chain: acceleration of otolithic membrane (OM)-displacement of the OM gel layer -deflection of hair cell bundle (HCB) -deformation of the system of tip-links- formation of temporal pattern of polarization was studied using simplified analytical models of these stages of conversion of mechanical stimulus into the HCB electrical response. The process of transformation of information in this chain was considered for two extreme cases of OM gel-HCB interaction: 1) the HCBs exactly follow the gel displacement; 2) stiff stereocilia and weak surrounding gel allow the relative motion of the bundle with regard to the gel. The analysis of a simplified model of cell polarization based on threshold triggering of the HCB tip-links allows to hypothesize that spatially nonhomogeneous HCB structure with a set of stereocilia of varying heights is designed to perceive spatially nonhomogeneous gel displacements caused by external acceleration. Thus, the HCB-OM gel interaction in the first case leads to formation of temporal pattern of depolarization that corresponds to the temporal pattern of gel displacement. In the second case the kinetics of depolarization reflects time dependence of gel displacement velocity.     

No correlation between multilamellar bodies in the inner ear and further organs of mutant (backstroke, bks) and wildtype zebrafish embryos.

The origin of the proteinacious matrix of the inner ear stones (otoliths) of vertebrates has not yet been clarified. Using the backstroke mutant (bks) of the zebrafish Danio rerio, which is characterized by a complete lack of otoliths, we searched for possibly missing or aberrant structural components within the macular epithelia of the inner ears of embryos on the ultrastructural level. Numerous multilamellar bodies (MLBs) were found. The MLBs were, however, not restricted to the inner ears of mutants but were also found in wildtype individuals and in further organs such as brain and liver. MLBs have hitherto never been described from the inner ear of fish and are generally estimated to be rare structures. Their occurrence in fish liver can, however, be induced by using particular chemical substances, which seem to effect adaptive compensatory processes on the cellular level. Such a chemical treatment also affects the ultrastructure of further organelles. Since the occurrence of MLBs in the liver of zebrafish was not accompanied by an alteration of the morphology of other organelles, their occurrence seems not to be due to environmental stress. The findings indicate that the MLBs cannot be correlated with bks-inherent features as well as with missing otolith development/growth. Since the occurrence of MLBs was independent from the developmental stage of a specimen and its overall tissue preservation, it can moreover be excluded that these MLBs merely represent fixation artifacts. Their presence more likely indicates cellular remodelling processes of hitherto unknown significance.     

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.     

Cosmic-ray diffusion in the inner heliosphere

For about the last 40 years, we have been trying to understand the propagation of cosmic rays and other energetic charged particles through the interplanetary medium. Identification of the basic processes affecting the propagation, namely diffusion, convection by the solar wind, adiabatic deceleration, and gradient and curvature drifts, was attained early on, but reaching detailed physical understanding, particularly of the roles of diffusion and gradient and curvature drifts, continues as an active topic of research to this day. Particularly unclear is the nature of the cross-field propagation. Many observations seem to require more efficient cross-field propagation than theoretical propagation models can easily produce. At the same time, there are other observations that seem to show strong guidance of the particles by the interplanetary magnetic field. With current measurements from spacecraft near Earth and from the Ulysses spacecraft, which samples nearly the complete range of heliographic latitudes in the inner heliosphere, critical tests of the ways in which cosmic rays and other energetic charged particles propagate through the interplanetary medium are possible. I briefly review the status of observations that are relevant to the characterization of diffusive propagation in the inner heliosphere and will present evidence for a possibly previously overlooked contribution from transport along magnetic flux tubes that deviate dramatically from the average interplanetary spiral configuration.     

Substorms in the inner plasma sheet

Thin Current Sheets (TCS) are regularly formed prior to substorm breakup, even in the near-Earth plasma sheet, as close as the geostationary orbit. A self-consistent kinetic theory describing the response of the plasma sheet to an electromagnetic perturbation is given. This perturbation corresponds to an external forcing, for instance caused by the solar wind (not an internal instability). The equilibrium of the configuration of this TCS in the presence of a time varying perturbation is shown to produce a strong parallel thermal anisotropy (T T) of energetic electrons and ions (E>50keV) as well as an enhanced diamagnetic current carried by low energy ions (E<50keV). Both currents tend to enhance the confinement of this current sheet near the magnetic equator. These results are compared with data gathered by GEOS-2 at the geostationary orbit, where the magnetic signatures of TCS, and parallel anisotropics are regularly observed prior to breakup. By ensuring quasi-neutrality everywhere we find, when low frequency electromagnetic perturbations are applied, that although the magnetic field line remains an equipotential to the lowest order in T_e/T_i, a field-aligned potential drop exists to the next order in (T_e/T_i). Thus the development of a TCS implies the formation of a field-aligned potential drop ( few hundred volts) to ensure the quasi-neutrality everywhere. For an earthward directed pressure gradient, a field-aligned electric field, directed towards the ionosphere, is obtained, on the western edge of the perturbation (i.e. western edge of the current sheet). Thus field aligned beams of electrons are expected to flow towards the equatorial region on the western edge of the current sheet. We study the stability of these electron beams and show that they are unstable to “High Frequency” (HF) waves. These “HF” waves are regularly observed at frequencies of the order of the proton gyrofrequency (f_H+) just before, or at breakup. The amplitude of these HF waves is so large that they can produce a strong pitch-angle diffusion of energetic ions and a spatial diffusion that leads to a reduction of the diamagnetic current. The signature of a fast ion diffusion is indeed regularly observed during the early breakup; it coincides with the sudden development of large amplitude transient fluctuations, ballooning modes, observed at much lower frequencies (fH+). These results suggest that the HF waves, generated by field-aligned electron beams, provide the dissipation which is necessary to destabilize low frequency (ballooning) modes.     

Modeling the time-intensity profile of solar flare generated particle fluxes in the inner heliosphere.

It is possible to model the time-intensity profile of solar particles expected in space after the occurrence of a significant solar flare on the sun. After the particles are accelerated in the flare process, if conditions are favorable, they may be released into the solar corona and then into space. The heliolongitudinal gradients observed in the inner heliosphere are extremely variable, reflecting the major magnetic structures in the solar corona which extend into space. These magnetic structures control the particle gradients in the inner heliosphere. The most extensive solar particle measurements are those observed by earth-orbiting spacecraft, and forecast and prediction procedures are best for the position of the earth. There is no consensus of how to extend the earth-based models to other locations in space. Local interplanetary conditions and structures exert considerable influence on the time-intensity profiles observed. The interplanetary shock may either reduce or enhance the particle intensity observed at a specific point in space and the observed effects are very dependent on energy.     

Two-velocity structure observed in the inner solar wind

Measurements of the motion of plasma density inhomogeneities in the inner solar wind are presented. The speeds were estimated using a cross-correlation analysis of radio frequency fluctuations of the Galileo spacecraft measured simultaneously at widely spaced ground stations. The radial projections of the correlation baselines on the pattern plane were of the order of several thousand kilometers. For cross-correlation functions calculated with comparatively short averaging times, we find that a pronounced two-velocity configuration is occasionally observed over the range of heliocentric distances 20 R < R < 40 R. The typical mean speed for such observations is about 300–400 km/s and the difference between the two predominant speeds is about 150–200 km/s. These results may indicate that the density fluctuations are associated with slow magnetosonic waves propagating in opposite directions at the local speed of sound in the reference frame moving with the mean solar wind speed. Quite reasonable estimates of the solar wind speed and speed of sound are obtained from this model. Another possible explanation of the two-velocity structures is that two independent solar wind streams are present simultaneously along different segments of the radio ray path.     

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.