M. Ibsch

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.

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.

Neurobiology of fish under altered gravity conditions

In vertebrates (including man), an altered gravitational environment such as weightlessness can induce malfunction of the inner ear, based on an irregular dislocation of the otoliths from the corresponding sensory epithelia. This dislocation leads to an illusionary tilt, since the otolithic inputs are not in register with other sensory organs. This 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). During the first days in weightlessness, the orientation problems (and SMS) disappear, since the brain develops a new compensatory interpretation of the available sensory data. The present review reports the neurobiological responses-particularly in 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. The possible sources of human space sickness (a kinetosis) and of the space adaptation syndrome (when a sensory reinterpretation of gravitational and visual cues takes place) are particularly highlighted with regard to the functional significance of bilaterally asymmetric otoliths (weight, size).

Endolymphatic calcium supply for fish otolith growth takes place via the proximal portion of the otocyst

The presence of calcium within the utricle of larval cichlid fish Oreochromis mossambicus was analysed by means of energy-filtering transmission electron microscopy. Electron-spectroscopic imaging and electron energy loss spectra revealed discrete calcium precipitations that were more numerous in the proximal endolymph than in the distal endolymph, clearly indicating a decreasing proximo-distal gradient. This decreasing proximo-distal gradient was also present within the proximal endolymph between the sensory epithelium and the otolith. Further calcium particles covered the peripheral proteinaceous layer of the otolith. They were especially pronounced at the proximal surface of the otolith indicating that otolithic calcium incorporation takes place here. Other calcium precipitates accumulated at the macular junctions clearly supporting an earlier assumption according to which the endolymph is supplied with calcium via a paracellular pathway. The present results clearly show that the apical region of the macular epithelium is involved in the release of calcium and that the calcium supply of the otoliths takes place via the proximal endolymph.

Vesicular bodies in fish maculae are artifacts not contributing to otolith growth.

The presence, morphology and possible origin of vesicle-like bodies (VBs) within the inner ear macular otolithic membrane of developmental stages of cichlid fish Oreochromis mossambicus and neonate (i.e. functionally fully developed except the reproductive organs) swordtail fish Xiphophorus helleri were analyzed by means of transmission and scanning electron microscopy (TEM and SEM, respectively) employing various fixation procedures. Some authors believe that these VBs are involved in the formation of the organic phase of inner ear otoliths (or statoliths in birds and mammals). Decreasing the osmolarity of the fixation medium from a value rather close to that of native fresh water fish tissue (i.e. 250 mOsm and 290--300 mOsm, respectively) to a value of fixatives mostly employed in TEM studies (ca. 190 mOsm), the amount of VBs increased and the components of sensory inner ear tissue increasingly dilated. Whilst a conventional prefixation with aldehydes followed by osmium tetroxide postfixation yielded numerous VBs, only few of them were observed when the tissue was fixed with aldehydes and osmium tetroxide simultaneously. Therefore, the results demonstrate that inner ear sensory epithelia are extremely sensitive to altering fixation media. On this background it must be concluded that VBs are fixative (i.e. glutaraldehyde) induced artificial structures, so-called membrane blisters. Thus, the protein matrix of otoliths (and possibly that of statoliths in higher vertebrates) is rather provided by secretion processes than by the release of vesicles.

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.

Microgravity (STS-90 neurolab-mission) influences synapse formation in a vestibular nucleus of fish brain

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

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 otoliths 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 statoliths organic material remains obscure. Possibly, it is provided by secretion from the supporting cells as has been hypothesized earlier.