Eberhard R. Horn

Altered gravitational experience during early periods of life affects the static vestibulo-ocular reflex of tadpoles of the Southern Clawed Toad, Xenopus laevis Daudin

The effects of altered gravitational forces (AGF) on the development of the static vestibulo-ocular reflex (VOR) were investigated in Xenopus laevis tadpoles exposed to hypergravity (1.4g; 3g) or microgravity conditions (German spacelab mission D-2) for 9–10 days. The effects of light conditions during development were also tested by exposing tadpoles to either complete darkness (DD) or 12∶12 h light-dark conditions (LD). The static VOR was induced by lateral roll. The efficacy of the VOR circuit after termination of AGF conditions was described by the peak-to-peak amplitude of the sinusoidal VOR characteristics (i.e. amplitude). The static VOR was first observed at stage 41 for both LD and DD tadpoles. Its further development was retarded in the DD tadpoles compared with the LD tadpoles up to stage 48. Microgravity as well as hypergravity exposure caused a significant (P<0.05, at least) decrease in the static VOR amplitude during the first week after termination of the AGF period. The decreases were 39.4% in the microgravity group, 16.2% in the 1.4g group and 24.9–42.9% in the 3g group compared with the 1g ground-reared siblings at the same developmental stages. The response deficits usually disappeared but persisted for at least 2 weeks in animals whose development was retarded by hypergravity exposure. It is postulated (i) that gravity exerts an important influence on the normal development of the roll-induced static VOR; (ii) that hypergravity exposure decreases the sensitivity of the gravity-sensitive system so that recordings under 1g conditions cause a weaker static VOR; and (iii) that the vestibulo-spinal pathway possesses a higher degree of plasticity than the vestibulo-ocular pathway.

Altered gravitational forces affect the development of the static vestibuloocular reflex in fish (Oreochromis mossambicus)

Young fish (Oreochromis mossambicus) were exposed to microgravity (micro g) for 9 to 10 days during space missions STS-55 and STS-84, or to hypergravity (hg) for 9 days. Young animals (stages 11-12), which had not yet developed the roll-induced static vestibuloocular reflex (rVOR) at micro g- and hg-onset, and older ones (stages 14-16), which had already developed the rVOR, were used. For several weeks afterwards, the rVOR was recorded after termination of mug and hg. Here are the main results: (1) In the stage 11-12 fish, the rVOR gain (response angle/roll angle) measured for roll angles 15 degrees, 30 degrees, and 45 degrees was not affected by microgravity if animals were rolled from the horizontal to the inclined posture, but was increased significantly if animals were rolled in the opposite manner. The rVOR amplitude (maximal eye movement during a complete 360 degrees roll) of micro g animals increased significantly by 25% compared to 1g controls during the first postflight week, but decreased to the control level during the second postflight week. Microgravity had no effect in stage 14-16 fish on either rVOR gain or amplitude. (2) After 3g exposure, both rVOR gain and amplitude were significantly reduced for both stage 11-12 and stage 15 fish. One g readaptation was completed during the second post-3g week. Hypergravity at 2 or 2.5 g had no effect. (3) Hypergravity at all three levels tested (2g, 2.5g, and 3g) accelerated the morphological development as assessed by external morphological markers. Exposure to micro g- or 3g-periods during an early developmental period modifies the physiological properties of the neuronal network underlying the static rVOR; in susceptible developmental stages, these modifications include sensitization by microgravity and desensitization by hypergravity.

The minimum duration of microgravity experience during space flight which affects the development of the roll induced vestibulo-ocular reflex in an amphibian (Xenopus laevis)

In tadpoles of Xenopus laevis, the effects of microgravity on the development of the roll-induced vestibuloocular reflex (rVOR) was investigated. Special attention was focused on sensitive periods and the minimum duration of microgravity exposure by which the development of the rVOR is affected. The peak-to-peak excursion (rVOR amplitude) of the rVOR characteristic for a lateral 360 degrees roll was used to describe microgravity effects. Fertilization of all eggs was performed 40 h before launch. Tadpoles were exposed to microgravity either during the first (MC-group) or second half of the mission (CM-group), or throughout the 9-day mission (MM-group). Inflight, 1G-gravity was simulated by a centrifuge (CC-group). After termination of the mission, the rVOR amplitude was only reduced in the MM-group with respect to the 1 G-inflight and 1 G-ground control by approximately 20-30% while both the MC- and CM-groups were not affected by the 4-day and 5-day microG exposure, respectively. However, CM-tadpoles like MM-tadpoles showed malformation of their body characterized by a dorsal bended tail. It disappeared in both groups within 2 weeks after landing. The difference between the rVOR amplitudes of the experimental groups disappeared within 5 weeks after landing. The results demonstrate that microgravity retards the development of the rVOR if it lasted longer than 4 days but that tadpoles are susceptible even for shorter periods as shown by the malformation of the body.

Microgravity-induced modifications of the vestibuloocular reflex in Xenopus laevis tadpoles are related to development and the occurrence of tail lordosis.

SUMMARY During space flights, tadpoles of the clawed toad Xenopus laevis occasionally develop upward bended tails (tail lordosis). The tail lordosis disappears after re-entry to 1g within a couple of days. The mechanisms responsible for the induction of the tail lordosis are unknown; physical conditions such as weight de-loading or physiological factors such as decreased vestibular activity in microgravity might contribute. Microgravity (μg) also exerts significant effects on the roll-induced vestibuloocular reflex (rVOR). The rVOR was used to clarify whether tail lordosis is caused by physiological factors, by correlating the occurrence ofμ g-induced tail lordosis with the extent of μg-induced rVOR modifications. Post-flight recordings from three space flights (D-2 Spacelab mission, STS-55 in 1993; Shuttle-to-Mir mission SMM-06, STS-84 in 1997; French Soyuz taxi flight Andromède to ISS in 2001) were analyzed in these experiments. At onset of microgravity, tadpoles were at stages 25-28, 33-36 or 45. Parameters tested were rVOR gain (ratio between the angular eye movement and the lateral 30° roll) and rVOR amplitude (maximal angular postural change of the eyes during a 360° lateral roll). A ratio of 22-84% of tadpoles developed lordotic tails, depending on the space flight. The overall observation was that the rVOR of tadpoles with normal tails was either not affected by microgravity, or it was enhanced. In contrast, the rVOR of lordotic animals always revealed a depression. In particular, during post-flight days 1-11, tadpoles with lordotic tails from all three groups (25-28, 33-36 and 45) showed a lower rVOR gain and amplitude than the 1g-controls. The rVOR gain and amplitude of tadpoles from the groups 25-28 and 33-36 that developed normal tails was not affected by microgravity while the rVOR of μg-tadpoles from the stage-45 group with normal tails revealed a significant rVOR augmentation. In conclusion: (1) the vestibular system of tadpoles with lordotic tails is developmentally retarded by microgravity; (2) after a critical status of vestibular maturation obtained during the appearance of first swimming, microgravity activates an adaptation mechanism that causes a sensitization of the vestibular system.

Light-dependent suppression of the vestibulo-ocular reflex during development

In the fish Oreochromis mossambicus, light conditions affect the development of the roll-induced vestibuloocular reflex (rVOR). During development under continous light-dark conditions the rVOR amplitude, which is the maximum eye movement during a complete 360 degrees lateral roll, shows a secondary drop after a first peak at stage 17 by 64% (36.3 degrees at stage 17; 13.0 degrees at stage 20). This drop was shifted by 2 stages to older postembryonal stages and was 33% (29.2 degrees at stage 20; 19.5 degrees at stage 22) less pronounced in animals which were exposed to complete darkness for several days. Because the period of rVOR diminution is sensitive to light conditions, it is likely that outgrowing visual projection fibres reorganize the neuronal network underlying visual-vestibular behavior thus transiently suppressing the rVOR.

An age-dependent sensitivity of the roll-induced vestibuloocular reflex to hypergravity exposure of several days in an amphibian (Xenopus laevis).

In tadpoles of the Southern Clawed Toad (Xenopus laevis), the effects of an exposure to hypergravity of several days duration on the development of the roll-induced static vestibuloocular reflex (rVOR) were investigated. Special attention was given to the onset of the 9 or 12 days lasting 3G-period during early life. First recordings of the rVOR characteristics for complete 360 degrees rolls of the tadpoles were performed 24 hrs after the end of the 3G-period. The rVOR peak-to-peak amplitudes as well as the VOR-gain for a roll angle of 15 degrees from 3G-and 1G-samples recorded at the 2nd and 3rd day after 3G-termination agreed for the youngest group, but were reduced by approx. 30% in the older tadpoles. Long-term observations lasting up to 8 weeks after termination of the 3G-period, demonstrated (i) an early retardation of the development, and (ii) a developmental acceleration in all groups so that after 2 weeks in the stage 6/9- and 33/36-samples and after 8 weeks in the stage 45-tadpoles, the rVOR-amplitude as well as the rVOR-gain for a 15 degrees roll were at the same level in both the 3G- and the 1G-samples. The results support the existence of a sensitive period for the rVOR development, and additionally demonstrate the importance of the period of the first appearance of the rVOR for the development of adaptive properties of the underlying neuronal network. They also demonstrate the dominant efficiency of genetic programs in the functional development of the vestibular system. Methodological approaches are discussed which will be useful in the further description of the critical period. They include studies on the neuronogenesis and synaptic maturation within the vestibular pathways as well as on the fundamentals of buoyancy control during swimming. A modular but closed mini-system for experimental use is described which allows survival periods lasting many weeks and multiple types of treatments of developing aquatic animals in orbit, controlled automatically.

The development of gravity sensory systems during periods of altered gravity dependent sensory input.

Gravity related behavior and the underlying neuronal networks are the most suitable model systems to study basic effects of altered gravitational input on the development of neuronal systems. A feature of sensory and motor systems is their susceptibility to modifications of their adequate physical and/or chemical stimuli during development. This discovery led to the formulation about critical periods, which defines the period of susceptibility during post-embryonal development. Critical periods can be determined by long-lasting modifications of the stimulus input for the gravity sensory system (GSS). Techniques include: (1) destruction of the gravity sense organ so that the gravity cannot be detected any longer and the central neuronal network of the GSS is deprived of gravity related information, (2) loading or deloading of parts of the body by weights or counterweights, respectively, which compensates for the gravitational pull, and (3) absence or augmentation of the gravitational environment per se by the exposure of organisms to microgravity during spaceflights or to hypergravity by centrifugation. Most data came from studies on compensatory eye or head movements in the clawed toad Xenopus laevis, the cichlid fish Oreochromis mossambicus, and crickets (Acheta domesticus, Gryllus bimaculatus). The responses are induced by a roll or pitch stimulation of the gravity sense organs, but are also affected by sensory inputs from proprioreceptors and eyes. The development of these compensatory eye and head responses reveals species-specific time courses. Based on experiments using spaceflights, centrifugation, lesion and loading or deloading, all species revealed a significant susceptibility to modifications of the gravity sensory input during development. Behavioral responses were depressed (Xenopus) or augmented (Xenopus, Oreochronis) by microgravity, and depressed by hypergravity except in crickets. In Acheta, however, the sensitivity of its position sensitive neuron PSI was reduced by microgravity. After termination of the period of modified gravity sensory input, all behavioral and physiological modifications disappeared, in some preparations such as the PSI of Acheta or the eye response in Xenopus, however, delayed after exposure to hypergravity. Irreversible modifications were rare; one example were malformations of the body of Xenopus tadpoles caused by lesion induced deprivation. Several periods of life such as the period of hatching or first appearance of gravity related reflexes revealed a specific sensitivity to altered gravity. Although all studies gave clear evidences for a basic sensitivity of developing GSSs to long-lasting modifications of the gravity sensory input, clear arguments for the existence of a critical period in the development of the sense of gravity are still missing. It has to take into consideration that during long-term exposures, adaptation processes take place which are guided by central physiological and genetically determined set points. The International Space Station (ISS) is the necessary platform of excellence if biological research is focussed on the analysis of long-term space effects on organisms.

A hypergravity related sensitive period during the development of the roll induced vestibuloocular reflex in an amphibian (Xenopus laevis)

In tadpoles of Xenopus laevis, the effects of an exposure to hypergravity on the development of the roll-induced static vestibuloocular reflex (rVOR) were investigated. Special attention was given to the onset of the 9 or 12 days lasting 3 g period during early life. Recordings of rVOR characteristics for complete 360 degrees rolls of the tadpoles started 24 h after the end of the 3 g period. The rVOR peak-to-peak amplitudes from the 3 g samples recorded at the 2nd and 3rd day after termination of the 3 g exposure agreed with that recorded from the 1 g reared tadpoles for the youngest group, but were reduced by 30% in the older tadpoles. During further development under 1 g condition, the rVOR amplitude of tadpoles with 3 g experience did not change if the 3 g exposure started before the first appearance of the rVOR, but increased if it had started thereafter, albeit on a lower level than that of the 1 g reared siblings. The results support the existence of a sensitive period for the rVOR development, and additionally demonstrate that the period during which the rVOR appeared for the first time is an important milestone for the development of adaptive properties of the underlying neuronal network.

Features of vestibuloocular reflex modulations induced by altered gravitational forces in tadpoles (Xenopus laevis).

In Xenopus laevis tadpoles, we studied the static vestibuloocular reflex (rVOR) in relation to modifications of the gravitational environment to find basic mechanisms of how altered gravitational forces (AGF) affect this reflex. Animals were exposed to microgravity during space flight or hypergravity (3g) for 4 to 12 days. Basic observations were that (1)the development of the rVOR is significantly affected by altered gravitational conditions, (2) the duration of 1g-readaptation depends on the strength of the test stimulus, (3) microgravity induces malformations of the body which are related to the rVOR depression. Future studies are based on the hypotheses (1) that the vestibular nuclei play a key roll in the adaptation to AGF conditions, (2) that the stimulus transducing systems in the sense organ are affected by AGF conditions, and (3) that fertilized eggs will be converted to normal adults guided by physiological and morphological set points representing the genetic programs. Developmental retardation or acceleration, or otherwise occurring deviations from standard development during embryonic and postembryonic life will activate genes that direct the developmental processes towards normality.

The "AGEING" Experiment in the Spanish Soyuz Mission to the International Space Station

Human exploration of outer space will eventually take place. In preparation for this endeavour, it is important to establish the nature of the biological response to a prolonged exposure to the space environment. In one of the recent Soyuz Missions to serve the International Space Station (ISS), the Spanish Soyuz mission in October 2003, we exposed four groups of Drosophila male imagoes to microgravity during the almost eleven days of the Cervantes mission to study their motility behaviour. The groups were three of young flies and one of mature flies, In previous space experiments, we have shown that when imagoes are exposed to microgravity they markedly change their behaviour by increasing their motility, especially if subjected to these conditions immediately after hatching. The constraints of the current Soyuz flights made it impossible to study the early posthatching period. A low temperature cold transport was incorporated as a possible way out of this constraint. It turned out that on top of the space flight effects, the cold treatment by itself, modifies the motility behaviour of the flies. Although the four groups increased their motility, the young flies did it in a much lower extent than the mature flies that had not been exposed to the low temperature during transportation. Nevertheless, the flies flown in the ISS are still more active than the parallel ground controls. As a consequence of the lower motility stimulation in this experiment, a likely consequence of the cold transport step, no effects on the life spans of the flown flies were detected. Together with previous results, this study confirms that high levels of motility behaviour are necessary to produce significant decreases in fly longevity.