BIOLOGY / NEUROBIOLOGY/ DEVELOPMENTAL BIOLOGY / TECHNOLOGY and EUROPEAN SOUNDING ROCKET PROGRAMME

EXPERIENCE FROM COMPARATIVE STUDIES ON THE POSTEMBRYONIC DEVELOPMENT OF GRAVITY RELATED BEHAVIOR IN ANIMALS – CONSEQUENCES FOR FUTURE EXPERIMENTS

E. R. Horn
Gravitational Physiology, Department of Neurobiology, University of Ulm, D-89081 Ulm, Germany

The fact that the development of nervous and muscular systems depends on neuronal activation implies that long-lasting deprivation incurs the risk of permanent morphological and/or physiological defects. Life, which will sooner or later develop in space, might be affected by the lack of the neurotrophic effect of gravity induced vestibular activity. Consequences might be less severe during permanent life in space but might have a dramatic impact on organisms after re-entry in 1g-Earth conditions. Experiments on effects of microgravity on young fish, amphibia and crickets (STS-55, STS-84, STS-90) revealed that gravity is necessary for normal postembryonic development of the sense of gravity. Our experiments were based on the ocular counter-roll (fish, amphibians), and compensatory head movements and activity modulations of central neurons (crickets) during static lateral roll. We showed that during early periods of life, microgravity and hypergravity activate mechanisms of central adaptation which modify vestibular reflexes recorded after re-entry in 1g-conditions (Horn et al. 1995, Naturwiss 82:289; Sebastian et al. 1995, Acta Astronautica 36:487; Sebastian et al. 1996, Exp Brain Res 112:213; Horn and Sebastian 1996, Neurosci Lett 216:25; Sebastian et al. 1998, Acta Astronautica 42:419; Sebastian and Horn 1998, Neurosci Lett 253:171; Sebastian and Horn 1999, Neuroreport 10:171). We considered the period of life during which these reflexes appeared for the first time as critical for the development of the ocular counter roll in fish and amphibian. In fish, exposure to microgravity starting before this period was effective while exposure starting thereafter was ineffective (Horn and Sebastian 1999, ESA SP-1222:127). In the amphibians, this period seems to be important for the development of neuroplastic properties in the underlying neuronal network (Horn and Sebastian 1996, Neurosci Lett 216:25). Crickets revealed a remarkable sensitivity of central neurons to gravity deprivation, but not in their behaviour. They probably take advantage of their multi-channel gravity sensory system and other sensory inputs for complete normalization of their behaviour after deprivation. Our experiments also demonstrated the dominance of genetic programs for development in case of exposure to modified gravitational conditions. However, several observations revealed developmental modifications, which might become irreversible. They were obviously related to specific periods of life. We recorded a retardation of reflex development in slowly developing animals with respect to fast developing ones (crickets, amphibians). We also observed microgravity-induced mal-formations of the body, which were strongly coupled with the extent of modifications of ocular counter roll (amphibians). Furthermore, the development of compensatory reflexes was also retarded after termination of the space flight in animals, which hatched during flight (crickets), or after termination of a hypergravity exposure (amphibian). The duration of spaceflight or hypergravity exposures used in these experiments was between 9 and 16 days, and therefore considerablely shorter the than normal development of these species.
These observations prompted the necessity for studies on the vestibular development under the influence of long-term microgravity or hypergravity exposures as well as for an analysis of central adaptive mechanisms during long-term exposures and of the stability of microgravity or hypergravity induced effects after re-entry to 1g-conditions. First choice species for these studies are those, which provide access to a high number of free-living, and clearly defined stages after birth or hatching, and from which a significant number of data are available determined during and/or after short-term exposures. For statistical reasons, species should also give access to large numbers of specimen. Long-term exposures need the development of animal holding facilities to rear animals from the egg to an adult. Estimations of crew times necessary for animal care compared to the costs needed for the development of fully automatically controlled survival systems demonstrate the advantage of crew support. Experiences from the Neurolab mission have also demonstrated that fine neurophysiological procedures can be performed in space. Therefore, experiments in individual animals have to be extended to record the neuronal activity as mass potentials from connectives (crickets) or central nuclei (fish, amphibia, rodents) during the next years; this is not possible without the development of proper hardware and its miniaturization for application in space. These experiments will show whether development under space conditions produces sensory-motor and neuronal mechanisms with high functional stability, which enables animals to live a "normal" life in weightlessness. I postulate that the development from a fertilized egg to an adult animal will be completed under the supervision of physiological and morphological set-points. If developmental retardation or acceleration or other deviations from normal structural and physiological development occurs during exposure of specific stages to micro- or hypergravity, these genetically defined set-points activate adaptive mechanisms towards the level of normality. An orbit-stabilized organism will be dramatically disturbed after return to Earth. Because this failure will need support to reach neuronal stability, research has also to focus on the analysis of mechanisms, which might be able to overcome these disturbances. This research will open the door to an application because it forms the bridge to human medicine with its analytical research on control procedures over neuropathological mechanisms.


HOW HYPERGRAVITY AFFECTS THE DEVELOPMENT OF A BDELLOID ROTIFER EMBRYO

U. Fascio, C. Ricci, C. Sotgia
Department of Biology, Milan University, Via Celoria 26, I-20133 Milan, Italy

Embryos of a species of Rotifera Bdelloidea (microscopic aquatic invertebrates with spiral segmentation pattern) will be produced under space environment in the International Space to study the influence of cytoskeleton perturbations on the pattern of development.  Here we present preliminary data on life-history traits and morphology of embryos produced by mothers exposed to increased gravity (hypergravity). Because the egg cytoplasm is totally provided by the mother and synthesized during ovogenesis in the rotifers, the experiment was designed to test whether cytoskeleton alterations occurring during ovogenesis can affect further development. The experiments were performed in a ‘centrifuge’ designed to culture small aquatic invertebrates under increased gravity for several days, changing culture medium and food daily without the need to interrupt the experiment.
At room temperature (23-25°C), we submitted to 3, 4, 5 g pre-reproductive rotifers for 3 days and then collected the eggs that were produced during this time or immediately after. Such eggs were produced under hypergravity, but most of the development occurred under normal conditions.  We recorded viability, developmental time and morphological integrity of the newborns by inspecting the juveniles, and visualized the cytoskeleton of the embryos at different ages by marking microtubules (tubulin) and microfilaments (F-actin) of eggs, for observation with a Confocal Laser Microscope. The results suggest that hypergravity affects both life history and cytoskeleton of developing embryos. Development after centrifugation was faster, the embryos were viable and hatched into morphologically and physiologically normal animals. Cytoskeleton disposition was altered by centrifugation, most nuclei appeared clumped on one side and the microfilaments and microtubules on the other side, but any alteration was not permanent since the embryos developed into normal newborns. This result revealed that the normal microtubule and microfilament arrangement can be recovered. Whether the effects of hypergravity can be identical to those of microgravity, remains to be determined.


How do Ciliates perceive Gravity?

R. Bräucker¹ and R. Hemmersbach^2
1Rheinische Friedrich-Wilhelms University, Institute of Zoology, D-53115 Bonn, Germany
²Institute of Aerospace Medicine, DLR, D-51147 Cologne, Germany


Ciliates are free-swimming unicellular organisms, which arose on the earth more than 1.5 billion years ago. In free-swimming organisms, the capability of orientation is a great benefit in evolution, and ciliates react to a wide variety of stimuli. Among the physical and chemical parameters of an aquatic environment, the gravity vector is unique, because it is constant in its value and direction; thus being the most reliable cue for orientation. We have evidence that ciliates, like Paramecium, sense the gravity stimulus and react with a change in swimming velocity and swimming direction. In order to identify the signal transduction chain of graviperception in ciliates, several strategies are possible:
1. Stimulus level
Although the gravity vector is indispensable for studies on graviperception, it is necessary to do control experiments under conditions of weightlessness (drop facilities, sounding rockets, parabolic flights, spaceflights). Many fruitful results have been derived using increased acceleration as in centrifuges. Up to 6g, the graviresponses in most ciliates are functions of acceleration; i.e. the graviresponses are increased.
2. Receptors
Due to our hypothesis, gravireceptors are (specialised) mechanoreceptors. By determination of response times and thresholds they can be precislier characterised. From step experiments to µg conditions, we assume the involvement of assisting structures, such as the cytoskeleton, in graviperception. Comparing known species-specific differences in the distribution of mechanoreceptors (e.g. Paramecium versus Didinium) we proved our hypothesis of graviperception. Modification of the cell shape and, if possible, the use of specific channel blockers, may elucidate more of the nature of gravisensors.
3. Membrane potential
It is well known that the behaviour of ciliates is under the strict control of membrane potential. All sensory processes are summarised in potential changes. Hence, we are trying to directly measure a gravireceptor potential by means of intercellular electrophysiological recording.
4. Second messenger
The role of second messengers (such as cAMP and cGMP, calmodulin) has been demonstrated in ciliary activity and thus swimming behaviour. Currently it is investigated, whether second messengers are involved in the gravitransduction chain.
5. Analysis of behaviour
Most evidences in graviresponses have been given from the analysis of behaviour. While inspection of swimming direction displays gravitaxis, the analysis of gravity dependent swimming velocities reveals gravikinesis. The comparison of different locomotion types (such as swimming, gliding and walking on substrates) may increase our knowledge of gravireceptors. An evolutional question is, whether the distribution and function of mechanoreceptors in ciliates depends on the presence of gravity, which may be examined under the conditions of long-term weightlessness, e.g. on the International Space Station.


GRAVITATIONAL ZOOLOGY USING FISH AS MODEL SYSTEMS –PERSPECTIVES CONCERNING THE UTILISATION OF THE INTERNATIONAL SPACE STATION (ISS)

H. Rahmann, and R. H. Anken
Zoological Institute, University of Stuttgart-Hohenheim, Garbenstr. 30, D-70593 Stuttgart, Germany

BACKGROUND: During the entire evolution of life on Earth, the development of all organisms took place under constant gravity conditions, against which they achieved specific countermeasures for compensation and adaptation. On this background, it is still an open question to which extent altered gravity such as hyper- or microgravity (centrifuge/spaceflight) 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 this topic, focusing on the effects of altered gravity on developing fish as model systems, with special emphasis on the effect of altered gravity on the developing brain and vestibular system.
CONCLUSION: The various data being obtained reveal that the normal development is not significantly impaired by altered gravity. Yet, however, it is unknown as of whether a complete life cycle can be completed, e.g., under weightlessness. Overall, the results speak in favour of the following concept: Short-term altered gravity (£ 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 possibly activate feedback mechanisms between the CNS and the vestibular organs for the regain of normal behaviour.
FUTURE PERSPECTIVES: The following areas of research with animals at altered gravity need to be addressed in the future:
1. Maintenance of – preferably vertebrate - animals through two complete life cycles in the space environment to determine whether or not developmental deficiencies are routinely produced.
2. Investigation of the peripheral and central vestibular system by ground-based studies, involving genetic mutations (mice and fish) that eliminate the peripheral organs and by using hypergravity as a research tool, focusing both 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?) when ISS facilities are available.
4. Studies on the effects of the vestibular system on the development of other neuronal systems (neuronal maps, autonomic nervous system etc.) and vice versa (e.g., effects of efferent systems on the development, maintenance and function of the vestibular apparatus).
5. Investigations on the effects of the vestibular system on the development of the postural and locomotive control systems to gain insights into its role in complex, navigational behaviours including studies to clarify the developmental events that lead to the differential susceptibility to sensorimotor disorders.
Answers to these questions may be of crucial interest for basic gravitational research. They might further be of importance concerning the topic "human in space", especially on the background of long-term stays at reduced gravity (ISS, manned mission to Mars).
ACKNOWLEDGEMENT: This work was financially supported by the German Aerospace Center (DLR) e.V. (FKZ: 50 WB 9)


SAMPLE PRESERVATION, ONE OF THE BIG CHALLENGES FOR THE NEXT PHASE OF SPACE RESEARCH:  PROBLEMS AND POSSIBLE SOLUTIONS

R. Marco*, F. Lería**, D. Husson*, J. Mateos*, A. Villa* and F. Javier Medina**
*: Departamento de Bioquímica-Inst. Invest. Biomédicas "Alberto Sols" (UAM-CSIC), Arzobispo Morcillo 4, E-28029 Madrid, Spain.:**: Centro de Investigaciones Biológicas (CSIC), Velázquez 144, E-28006 Madrid, Spain

A critical methodological problem in Space Research, especially in the new phase of long flight experiments in the International Space Station, is the necessity of preserving biological samples during the long periods between the actual experiments on board and their posterior recovery on the ground where further analysis should be carried out. As in previous cases, this may provide another example of a spin-off of Space Research yielding applications of wider use in ground laboratories. In this presentation we will discuss two problems, a) the preservation of living samples and b) the preservation of fixed samples. Our two groups have been working on two different systems, Drosophila, a key animal model, where developmental and evolutionary questions can be investigated and onion roots, a plant model especially adequate to study cell proliferation.
Drosophila melanogaster has so far been practically refractory to the application of the methodology for the preservation of living embryos and/or other specimens, either based on deep-freezing procedures or on other approaches, successfully applied to mammalian sperm, eggs and/or early embryos preservation. The reason for this failure may reside in the extreme sensitivity to low temperatures of the Drosophila embryos, especially, the early ones. In order to find out more on this phenomenon, we have reinvestigated the problem of this sensitivity, applying usual selection procedures to find out if we could obtain Drosophila strains more resistant to this environmental factor. With our original Oregon-R wild type population, we have established that it is possible to maintain late embryos and larvae at 12ºC for up to one month without losing viability. Once transferred to normal temperatures they complete their growth phase, pupate and produce normal imagoes. The animals actually grow and develop much more slowly than at normal temperatures, but once they initiate pupation at 12C they are irreversibly damaged. One month is still too short a period for the ISS current foreseen refurbishing missions (every 3 months). Thus, we are now looking into the effect of exposing the embryos and larvae to 10ºC. On the other hand, by exposing the embryos to lower temperatures (for example, 7ºC) during different periods of time, it is possible to select animals less susceptible to the deleterious effects of low temperatures. We are hopeful that a combination of the two approaches may lead to the overcoming of this problem and the development of a low-temperature preservation methodology that will allow us to preserve living samples for at least three months.
We have also been working on the development of a procedure of stowage of small hydrated biological samples, fixed or unfixed, capable of keeping unaltered their main structural and functional features during extensive periods of time. The strategy chosen for accomplishing these objectives has been subcooling. The term refers to the cooling of a liquid below its freezing temperature while maintaining its liquid state using a water-oil emulsion produced by mixing one part of the aqueous solution with four parts of a mixture of mineral oil and wax. Using isolated nuclei from onion roots, our results indicate that this procedure is able to maintain the structural features of fixed subcooled nuclei, both at the light and electronmicroscopical level, making them undistinguishable from routinely processed controls. Preservation of unfixed nuclei is also successfully maintained by this subcooling procedure, both at the structural and functional level. These preservation tests have been performed during one week and we are now extending this period to see whether they can provide the solution to the problem. Some evidence on the preservation of protein profiles of Drosophila samples by subcooling methodology will also be discussed.


The long-term adaptation of drosophila melanogaster to the space ENVIRONMENT: design, development and test of experimentS and hardware for the i.s.s.

D. Husson¹, J.M. Medina² and R. Marco^1
1Departamento de Bioquímica de la UAM & Instituto de Investigaciones Biomédicas, "Alberto Sols" CSIC-UAM. Facultad de Medicina. Universidad Autónoma de Madrid. Arzobispo Morcillo 4, E-28220 Madrid, Spain. E.mail : rmarco@iib.uam.es, ²Centro de Investigaciones Biológicas (CSIC). Velázquez 144, E-28006 Madrid, Spain

The establishment of permanent colonies of key model living systems in space will be one of the future challenges in Space Research. This will provide a better understanding of the response or lack of response of organisms, tissues and cells in a weightlessness environment, and its long-term adaptation to the different environments outside our planet. These studies will prepare the future long distance Space exploration of mankind.
Drosophila melanogaster is one of the main model systems in current biological research, especially in relation to evolutionary and development mechanisms. In previous experiments, it has been shown that embryonic and larval development in microgravity produces normal adults, as in a wide range of organisms. In contrast, Drosophila aging is altered as a consequence of the exposure to microgravity and the concomitant increase in motility shown by young fruit flies. Other behavioural traits such as negative geotaxis have been also found altered after exposure to this environment.
To tackle these questions, we are preparing experiments for continuous cultivation of Drosophila in Space, monitoring the adaptation and changes that the organisms may experience during many generations. The Experimental Support Equipment Item 1 (ESE–1) of ESA is an automated culture system for multigenerational experiments with insects, built by NTE SA (Barcelona). This hardware is the output of a study in the framework of the development of generic experiment equipment for the ESA ISS facilities Biolab and EMCS. Based on a concept that maximizes automatization, the prototype has already successfully completed different rounds of testing in our hands. The demonstration of its versatility and suitability will allow to breed Drosophila during as many generations as is wanted inside the new Space facilities in the ISS, opening the way to the establishment of a permanent colony of Drosophila in space.
Taking advantage of the ESE-1 item, and through the monitorization of several adult fruitflies behavioral traits in-flight, we plan to study aging and behavior changes of Drosophila as a consequence of the exposure to microgravity. The behavioural traits of the adults of different generations will be monitored in comparison to controls (ground and in 1g on-flight). On the other hand, through the monitorization of the different steps of Drosophila 's development in space, we will establish how the different processes taking place during development actually proceed qualitatively and quantitatively in comparison to control embryos. We will study processes such as cell cytoskeleton, shape, movements, proliferation and differentiation, some of them known to be affected in cells cultured in space. For this purpose, in addition to observing in vivo how embryos actually develop, we propose to monitor the actual timing and spatial patterns of accumulation of gene markers of different steps and processes. The conclusions will be reached by studying the in vivo development of in-flight collected embryos, as well as by studying them after post-flight recovery of in-flight fixed embryos. We are working to improve the ESE-1 hardware and developing a complementary fixation unit that will allow the satisfactory preservation of fixed samples during several weeks up to several months, again with minimal crew involvement.


FROM (FROZEN) CELLS TO (LIVE) CELL CULTURES: BIOLAB EXPERIMENTS ON THE ISS

E. Brinckmann
ESA, Microgravity Facilities for Columbus Division
ESTEC, Postbus 299, NL-2200 AG Noordwijk, The Netherlands


The permanent availability of the International Space Station (ISS) for research under Space conditions offers a new perspective for experimentation: investigations, e.g. in the field of gravitational cell biology, which are usually completed within a few days, may be repeated several times due to the duration of the mission increments in the order of 3 months. To perform repetitive experiment runs, it is necessary to start a new experiment under defined conditions in Space. However, the absence of gravity urges an approach that may be different to routine procedures on ground. If the effect of microgravity is the subject of investigation, then it is probably not recommended to start from a cell culture, which is adapted to micro-gravity; a new cell culture may only be feasible from cells growing permanently on a 1-g centrifuge.
An alternative to this approach could be the start of fresh cell cultures from deep frozen stock cultures, as it is also done routinely on ground, at least as a back-up procedure, if the permanent culture is lost or if the culture has to be transported in a save way. This transportation phase is also an argument to start from frozen cells in Space: the transport in the Space Shuttle to the ISS lasts in the order of
2 days, and it is therefore likely, that the duration between sample preparation on ground and the start of incubation in orbit may take several (5-7) days. If the cells are able to sense gravity during this phase, the experiment will suffer from uncontrolled accelerations before it has been started: hypergravity during launch and microgravity during transport. It is assumed, that cells in a deep frozen state may not sense these accelerations and are therefore well suited to form new cell cultures for experiments on the ISS. However, the thawing process and the removal of the cryo-preservative again needs to be done under 1-g conditions to prevent exposure to microgravity before the actual start of the experiment.
ESA has performed a feasibility study for an autonomous unit, which shall be able to thaw and wash deep frozen cell cultures automatically under 1-g conditions. This Experiment Preparation Unit (EPU) is now under development for ESA’s Biolab facility on the ISS. The EPU is dedicated to preparing biological samples stored at temperatures as low as -80 ºC, or lower (-196 ºC), for further processing in the Biolab facility. It has the following features:
- 2ml samples in cryo-tubes
- thawing time <5 min
- repetitive washing cycles of 1 to 60 min
- residual cryo-protectant <0.1%
- thawing and washing under 1-g conditions
- temperature after thawing between +4 and +10 ºC or ambient
- no more than 10% cells lost during preparation cycle
- maintained at sterile conditions and no cross-contamination
- ground control by telemetry and telecommand
- viability test to be performed in experiment hardware.
The EPU will be mounted in Biolab’s BioGlovebox allowing sterile transfer of freshly prepared samples into the Experiment Container. This transfer is the only step when cells are exposed uncontrolled to the microgravity environment. Cell cultures will start then in experiment specific hardware, allowing a more or less automatic performance of the cell culture experiment in Biolab or any other biological facility on the International Space Station.


INERTIAL SHEAR FORCES IN GRAVITATIONAL BIOLOGY

J. J.W.A. van Loon 1, E. Folgering 2, J. P. Veldhuijzen 3, C. V.C. Bouten 2

1 Dutch Experiment Support Center (DESC), VU, Amsterdam, Netherlands. 2 Eindhoven University Technology, MATE, Eindhoven, Netherlands. 3 ACTA-VU, Oral Biology, Gr. Oral Cell Biology, Amsterdam

Based on Einstein's equivalent principal, a 1g condition is "generated" in a sample that remains on Earth or that is put into a centrifuge running at an angular velocity of 1g on-board a free falling spacecraft. In gravitational biology it was with facilities like Biorack that one could use an on-board 1g control. The on-board centrifuge was regarded as the best control for an actual microgravity experiment. However, one of the never addressed differences between ground 1g and in-flight 1g is the inertial shear force generated in on-board centrifuges while this shear force could be one of the main artefacts in spaceflight and on-ground studies using centrifuges. Shear forces are generated in two ways. Static shear and fluid shear. Fluid shear is best known from the circulatory system where the endothelium is exposed blood flows. Static shear forces are generated in materials exposed to e.g. accelerations. The latter being the main issue of this numerical study. Although differently shear forces have, in vitro, an impact on both attached and free-floating cells. They may even have a significant effect in animal centrifuge studies.
We calculated that for some experiment set-ups in the past as well as for some future ISS facilities the level of shear force in on-board centrifuges could be as much as 95% of the total force. Some of the differences reported between ground 1g and in-flight 1g centrifuge could have been caused by this phenomenon.
The inertial shear force artefact should be dealt with for future missions and hardware designs as well as for the interpretation of previous data.
This work is supported by SRON and the NIVR combined grant # MG-051


Program Description and User Scenario of Sounding Rockets used for microgravity research

B. Franke and D. Grothe
Astrium GmbH, Space Infrastructure, Post Box 28 61 56, D-28361 Bremen, Germany

The announced paper will give an overview of the capability and performances of the Sounding Rocket Programs, which have been established and are operational since 1977. These Programs were initiated by the German National Agency DLR and by ESA to give the scientific community the possibility to be well prepared for the utilization of the Spacelab missions within the different fields of microgravity disciplines. More than 50 flights with about 500 experiments have been performed successfully. This means that about 60 - 70 % of microgravity experiments, which flew later on Spacelab, have been prepared on Sounding Rockets to optimize technical performance and scientific outputs.
In order to also provide in the future, an optimized service for the scientific community, especially for the upcoming utilization of the "International Space Station" (ISS), Astrium, Swedish Space Corporation, the Kayser - Threde Company and DLR have established an industrial group with the objective to concentrate technical competence for a future Sounding Rocket Program with upgraded technical capabilities and lower flight ticket costs. This upgrading should lead to higher data rates, improved "Telescience capabilities" by using digital TV-systems and a stable flight program with a high degree of reliability and continuity for the scientific community.
In addition to the programmatic aspects of the Sounding Rocket Program, an overview of selected experiment modules will demonstrate the high degree of complexity of the experiment facilities which have been developed for the various disciplines of microgravity research e.g. for crystal growth, biology and centrifuges as well as for combustion and evaporation experiments.