EXPERIMENT RECORD N° 9247
XENOPUS - Cellular Modifications within the Vestibulo-ocular System during Adaptation to Microgravity in a Developing Amphibian (Xenopus laevis)
  1. 2008 • ISS Increment 18
Life Sciences:
  • Developmental Biology
Kubik
Jason Hatton
jason.hatton@esa.int
E. Horn (1), M. Gabriel (1)
(1)  
Ulm University
Institute of Neurobiology - Gravitational Physiology
Albert Einstein Allee 11
89081 Ulm
GERMANY
Tel:  
+49(0)7315024565
Fax:  
+49(0)7315024562
e-mail:  
eberhard.horn@uni-ulm.de
martin.gabriel@uni-ulm.de

AIM
Characterise the effect of microgravity on development of the vestibular ocular systems in Xenopus laevis tadpoles at late development stages:

SPECIFIC GOALS
1. To determine the critical period in the developing vestibulo-ocular reflex (rVOR).
2. To determine relations between oculomotor and spinal motor µg-sensitivities (freely swimming).
3.
To correlate tail lordosis with rVOR (roll-induced vestibulo-ocular reflex) modifications.

GENERAL DESCRIPTION
Former space flight experiments on STS-55, STS-84 and Andromède have shown significant modifications of the roll-induced vestibuloocular reflex (rVOR) caused by a 9 to 10 days lasting microgravity exposure. These modifications were age-related and could be correlated with the occurrence of a tail lordosis. Lordotic tadpoles always revealed a rVOR depression after the space flight. Normally shaped tadpoles were not affected if, at launch, they had not yet developed the rVOR while those tadpoles that were just able to do the reflex at launch showed a reflex augmentation after the flight. The so far done studies were limited to those stages that need no regular food supply because they could use their yolk. For the determination of the duration of a critical period for rVOR development, the studies have to be extended to older tadpole stages that have already developed their rVOR at launch of the space craft. Their survival, however, depends on regular food supply. Two stages (45 and 54) will be used because at these stages structural and hormonal modifications occur during further development which are closely related to vestibular function (cerebellum differentiation), or the hormonal control of development (increase of T3/T4-concentrations).

Xenopus is part of the BIO-4 mission (together with BASE-B/C and ROALD) which was launched into space onboard of Soyuz TMA-13 on 12 October 2008. The BIO missions provide flight opportunities for biology experiments requiring a short duration (less than 10 days) and limited on-orbit resources. They make use of the Russian Soyuz spacecraft for both launch and recovery of the experiment samples. Xenopus landed with Soyuz TMA-12 on 24 October 2008.

BACKGROUND
XENOPUS/TADPOLES are follow-up studies of experiments performed on:
STS-55 - German D2 mission, 1993
Graviperception and neuronal plasticity: comparative investigations of weightlessness effects on structural development and function of the gravity perceiving organ of two water living vertebrates (Xenopus laevis, Oreochromis mossambicus)

Graviperception and neuronal plasiticity: structure and functional related neuronal plasticity of the central nervous system of aquatic vertebrates during early ontogenetic development under microgravity

STS-84  - Shuttle-to-Mir mission SMM-06, 1997
A comparison of Normal Vestibulo-Ocular Reflex Development Under Gravity and in the Absence of Gravity

Andromède mission - French Soyuz Taxi Flight to ISS, 2001
AQUARIUS - Embryonic development of amphibians in weightlessness

Experiment TADPOLES is also related to AMPHIBODY from LDM BIO#1:

In Xenopus, the development of the rVOR and the underlying neuronal and sensory system occurs during the first days of microgravity exposure while in Pleurodeles, the species used during AMPHIBODY, the rVOR occurs for the first time immediately after termination of a 10-days flight. Thus, comparison between both species will give an answer to the question to which extent vestibular experience during microgravity (that is possible due to linear acceleration stimulation during swimming) has an impact on the development of the rVOR under gravity deprivation conditions.

Fig. 1: Development of tadpoles during Xenopus and Pleurodeles experiment.  

μg-Exposure Periods for Critical Period´s Analysis
Old tadpoles in microgravity after fertilization on ground (periods 2 and 3 will be used.
Embryonic and young stages in microgravity after fertilization in rnicrogravity (cf. 1).

Fig. 2: Xenopus development.

Precursor flights:
- STS-55 (German D-2 mission, 1993; Experiment STATEX-VOR)
- STS-84 (Shuttle-to-Mir SMM-06, 1997; Experiment TADPOLE)
- Soyuz Taxi flight Andromède to ISS (2001; Experiment AQUARIUS-XENOPUS)

Fig. 3: Experiment Concept Diagram.

The experiment container will be experiment specific, compatible with the KUBIK Centrifuge Insert (CI) interface, but no contained with Typ-I containers. Two containers will be accommodated inside of one KUBIK.

Fig. 4: Mission Work Flow.

Biological Samples
- Xenopus laevis tadpoles - medium & late development stages (stages 45, & 54)

GENERAL EXPERIMENT PROCEDURE
- L-7 to L-5days: Collection & transport of tadpoles from science team lab to Baikonour (21°C condition temperature transport).
- L-36h final selection of tadpoles, loading into experiment cassette & activation of cassette osmotic pump.

Experiment scenario #1

  • Installation of ECs into KUBIK-CI @ L-15h (ambient temperature, 18°C to 28°C, minimise exposure to temperatures >25°C if possible, consider >25°C only as a worst case condition).
  • Incubation of ECs in KUBIK-CI from L-8h until FD9 on ISS at 21°C (+/-1°C).
  • Transfer of ECs to soft stowage for download on Soyuz on FD9, no earlier than 24h before undocking.

Experiment scenario #2 (Baseline for Bio#4 Implementation)

  • Installation of ECs into soft pouch at L-15h.
  • Launch of ECs in soft pouch until docking (FD3) at ambient temperature (18°C to 22°C).
  • Install ECs in KUBIK-CI from as soon as possible after docking (FD3) until FD9 at 21°C (+/-1°C).
  • Transfer of ECs to soft stowage for download on Soyuz on FD9, no earlier than 24h before undocking.

- Return of ECs in soft pouch on-board Soyuz at ambient temperature (18°C to 22°C, avoid any area with high risk for higher temperatures).
- Landing site video observations of tadpole swimming behaviour desirable (R+2h), approx. 40´ time required.
- H/O at R+12h (not later than R+18h) in Moscow Starcity.
- VOR recordings between R+15h to R+120h in Moscow, preferably Starcity.
- Transport of tadpoles to investigators lab at R+4 to R+5 days.
- VOR recordings at R+8 to R+10 days.
- VOR recordings at R+4 weeks.

Parameters measured
Inflight parameters measured:
- Temperature profile during experiment (0.5°C accuracy, 10 minute time resolution.
- Time of experiment steps (1 minute time accuracy).
Planned analyses
- Vestibular ocular reflex, post flight by video recordings (R+15h to R+120h, R+4d, R+8d, R+28d).
- Swimming abilities, post flight by video recordings (R+2h if possible, R+15h to R+120h, R+TBD days).

Ground reference experiment(s)

  • Simultaneous ground reference experiment with 1g samples in identical hardware to flight experiment.
  • Experiment run at 21°C (+/- 2°C).
  • Postflight ground reference experiment may also be run, if off nominal temperature conditions are encountered inflight.

Fig. 5: Detailed Experiment Timeline and associated Functional Objectives

Fig. 6: Flow Diagram with Functional Objectives (FO) indicated.

Fig. 7: Functional Objectives (FO) related to pre-flight/in-flight/post-flight timeline.

Science deliverables
- Live Xenopus Tadpoles, launched at development stages 45 and 54 and maintained onboard Soyuz / ISS for approximately 10 days.
- Recorded temperature data during entire mission profile.
- Time of experiment activities.

HYPOTHESES
If the development of the rVOR is characterized by a CRITICAL PERIOD, exposure of old tadpoles will cause no rVOR modification.
After in-flight fertilization, the complete development reveals adaptation to the microgravity environment despite of transiently occurring modifications of development (gastrula, neurula). Thus, rVOR after landing is normal.

EXPECTED RESULTS

  • If the sensitivity of rVOR and swimming is limited to a specific period of life, stage-54 tadpoles will not be affected, while stage 45-tadpoles will be affected by microgravity.
  • Affected tadpoles will not or only partially recover their rVOR within the observation period if the critical period exist in its most restrictive definition (irreversibility of response modifications).
  • Modification of swimming include increased activity for flight animals; modifications of rVOR include sensitization, i.e., augmentation of the rVOR.
  • Tail lordosis is absent if, under normal conditions, its occurrence is related to the spread of growth factors responsible for axis formation.

Stimulus deprivation or stimulus augmentation can induce long‐lasting modifications to sensory and motor systems. If deprivation is effective only during a limited period of life this phase is called “critical period.” A critical period was described for the development of the roll‐induced vestibule-ocular reflex (rVOR) of Xenopus laevis using spaceflights. Spaceflight durations and basic conditions of Xenopus’ development did not make it possible to answer the question whether exposure of the immature vestibular organ to weightlessness affects rVOR development. The embryonic development of Pleurodeles waltl is slow enough to solve this problem because the rVOR cannot be induced before 15 dpf. Stage 20-21 embryos (4 dpf) were exposed to microgravity during a 10‐day spaceflight, or to 3g hypergravity following the same time schedule. After termination of altered gravity, the rVOR was recorded twice in most animals. The main observations were as follows: (1) after the first rVOR appearance at stage 37 (16 dpf), both rVOR gain and amplitude increased steadily up to saturation levels of 0.22 and 20◦, respectively. (2) Three days after termination of microgravity, flight and ground larvae showed no rVOR; 1 day later, the rVOR could be induced only in ground larvae. Differences disappeared after 3 weeks. (3) For 10 days after 3g exposure, rVOR development was similar to that of 1g‐controls but 3 weeks later, 3g‐larvae showed a larger rVOR than 1g‐controls. These observations indicate that the immature vestibular system is transiently sensitive to microgravity exposure and that exposure of the immature vestibular system to hypergravity leads to a slowly growing vestibular sensitisation.

[1]  
B. Rayer, E. Cagol, E. Horn, (1983), "Compensation of vestibular-induced deficits in relation to the development of the Southern Clawed Toad, Xenopus laevis Daudin", Journal of Comparative Physiology, 151, pp. 487-498.
[2]  
E. Horn, H.G. Lang, B. Rayer, (1986), "The development of the static vestibulo-ocular reflex in the Southern Clawed Toad, Xenopus laevis Daudin: I. Intact animals", Journal of Comparative Physiology, 159A, pp. 869-878.
[3]  
E. Horn, R. Mack, H.G. Lang, (1986), "The development of the static vestibulo-ocular reflex in the Southern Clawed Toad, Xenopus laevis Daudin: II. Animals with acute vestibular lesions", Journal of Comparative Physiology, 159A, pp. 879-885.
[4]  
B. Rayer, E. Horn, (1986), "The development of the static vestibulo-ocular reflex in the Southern Clawed Toad, Xenopus laevis Daudin: III. Chronic hemilabyrinthectomized tadpoles", Journal of Comparative Physiology, 159A, pp. 887-895.
[5]  
C. Sebastian, K. Eßeling, E. Horn, (1996), "Altered gravitational experience during early periods of life affects the static vestibulo-ocular reflex of tadpoles of the Southern Clawed Toad, Xenopus laevis", Experimental Brain Research, 112, pp. 213-222.
[6]  
E. Horn, C. Sebastian, (1996), "A hypergravity related sensitive period during the early development of the roll induced vestibulo-ocular reflex in the Southern Clawed Toad, Xenopus laevis", Neuroscience Letters, 216, pp. 25-28.
[7]  
C. Sebastian, E. Horn, (1998), "The minimum duration of microgravity experience during spaceflight which affects the development of the roll induced vestibulo-ocular reflex in an amphibian", Neuroscience Letters, 253, pp. 171-174.
[8]  
C. Sebastian, K. Eßeling, E. Horn, (2001), "Altered gravitational forces affect the development of the static vestibulo-ocular reflex in fish (Oreochromis mossambicus)", Journal of Neurobiology, 46, pp. 59-72.
[9]  
E. Horn, (2004), ""Critical periods" in vestibular development or adaptation of gravity sensory systems to altered gravitational conditions?", Archives Italiennes de Biologie, 142, pp. 155-174.
[10]  
S. Böser, E. Horn, (2006), "Hypergravity susceptibility of ventral root activity during fictive swimming in tadpoles (Xenopus laevis)", Archives Italiennes de Biologie, 144, pp. 99-113.
[11]  
E. Horn, (2006), "Microgravity-induced modifications of the vestibulo-ocular reflex in Xenopus Laevis tadpoles are related to development and the occurrence of tail lordosis", Journal of Experimental Biology, 209, pp. 2847-2858.
[12]  
E. Horn, S. Böser, H. Membre, C. Dournon, D. Husson, L. Gualandris-Parisot, (2006), "Morphometric investigations of sensory vestibular structures in tadpoles (Xenopus laevis) after a space flight - Implications for microgravity induced alterations of the vestibulo-ocular reflex", Protoplasma, 229, pp. 193-203.
[13]  
E.R. Horn, C. Dournon, J.P. Frippiat, R. Marco, S. Böser, U. Kirschnick, (2007), "Development of neuronal and sensorimotor systems in the absence of gravity: Neurobiological research on four soyuz taxi flights to the international space station", Microgravity Science and Technology, 19, 5-6, DOI: 10.1007/BF02919474, pp. 164-169.
[14]  
S. Böser, C. Dournon, L. Gualandris-Parisot, E. Horn, (2008), "Altered Gravity Affects Ventral Root Activity During Fictive Swimming and The Static Vestibuloocular Reflex In Young Tadpoles (Xenopus Laevis)", Archives Italiennes de Biologie, 146, pp. 1-20.
[15]  
E. Horn, M. Gabriel, (2011), "Gravity‐related critical periods in vestibular and tail development of Xenopus laevis", Journal of Experimental Zoology, 315, DOI: 10.1002/jez.698, pp. 505-511.
[16]  
M. Gabriel, J.P. Frippiat, H. Frey, E. Horn, (2012), "The sensitivity of an immature vestibular system to altered gravity", Journal of Experimental Zoology, 317A, DOI: 10.1002/jez.1727, pp. 333-346.
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Fig. 1: Development of tadpoles during Xenopus and Pleurodeles experiment.

Fig. 2: Xenopus development.

Fig. 3: Experiment Concept Diagram.

Fig. 4: Mission Work Flow.

Fig. 5: Detailed Experiment Timeline and associated Functional Objectives.

Fig. 6: Flow Diagram with Functional Objectives indicated.

Fig. 7: Functional Objectives (FO) related to pre-flight/in-flight/post-flight timeline.

Fig. 8: Xenopus tadpoles in Experiment Container - The Xenopus experiment studies the development of cane toad tadpoles in spaceflight. After their stay in space, scientists will measure the effect of spaceflight exposure on the development of the tadpoles' sense of balance; this is controlled by the vestibular ocular system (inner ears and eyes). Xenopus is part of the BIO-4 mission which was launched into space with the Soyuz TMA-13 on 12 October 2008. The BIO missions provide flight opportunities for biology experiments requiring a short duration (less than 10 days) and limited on-orbit resources. They make use of the Russian Soyuz spacecraft for both launch and recovery of the experiment samples.

Fig. 9: Kubik Experiment Container.
 
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