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Hypergravity exposure decreases gamma-aminobutyric acid immunoreactivity in axon terminals contacting pyramidal cells in the rat somatosensory cortex: a quantitative immunocytochemical image analysis.

Quantitative evaluation of gamma-aminobutyric acid immunoreactivity (GABA-IR) in the hindlimb representation of the rat somatosensory cortex after 14 days of exposure to hypergravity (hyper-G) was conducted by using computer-assisted image processing. The area of GABA-IR axosomatic terminals apposed to pyramidal cells of cortical layer V was reduced in rats exposed to hyper-G compared with control rats, which were exposed either to rotation alone or to vivarium conditions. Based on previous immunocytochemical and behavioral studies, we suggest that this reduction is due to changes in sensory feedback information from muscle receptors. Consequently, priorities for muscle recruitment are altered at the cortical level, and a new pattern of muscle activity is thus generated. It is proposed that the reduction observed in GABA-IR of the terminal area around pyramidal neurons is the immunocytochemical expression of changes in the activity of GABAergic cells that participate in reprogramming motor outputs to achieve effective movement control in response to alterations in the afferent information.

Animals↗

Hypergravity can reduce but not enhance the gravitropic response of Chara globularis protonemata.

The relationship between the position of the statoliths and the direction and rate of tip growth in negatively gravitropic protonemata of Chara globularis was studied with a centrifuge video microscope. Cells placed perpendicularly to the acceleration vector (stimulation angle 90 degrees) showed a gradual reduction of the gravitropic curvature with increasing accelerations from 1 g to 8 g despite complete sedimentation of all statoliths on the centrifugal cell flank. It is argued that the increased weight of the statoliths in hypergravity impairs their acropetal transport which is induced when the cell axis deviates from the normal upright orientation. When the statoliths were centrifuged deep into the apical dome at 6 g and a stimulation angle of 170 degrees the gravitropic curvature after 1 h was identical to that determined for the same cells at 1 g and the same stimulation angle. This indicates that gravitropism in Chara protonemata is either independent of the pressure exerted by the statoliths on an underlying structure or is already saturated at 1 g. When the statoliths were moved along the apical cell wall at 8 g and the stimulation angle was gradually increased from 170 degrees to 220 degrees the gravitropic curvature reverted sharply when the cluster of statoliths passed over the cell pole. This experiment supports the hypothesis that in Chara protonemata asymmetrically distributed statoliths inside the apical dome displace the Spitzenkorper and thus the centre of growth, resulting in gravitropic bending. In contrast to the positively gravitropic Chara rhizoids, no modifications either in the transport of statoliths during basipetal acceleration (6 g, stimulation angle 0 degree, 5 h) or in the subsequent gravitropic response could be detected in the protonemata. The different effects of centrifugation on the positioning of statoliths in Chara protonemata and rhizoids indicate subtle differences in the function of the cytoskeleton in both types of cells.

Centrifugation↗

Halotolerance is enhanced in carrot callus by sensing hypergravity: influence of calcium modulators and cytochalasin D.

Carrot callus was centrifuged at 10 g and compared to callus growing at 1 g on agar in the presence of increasing sodium chloride concentrations. Growth after 14 days was enhanced in the centrifuged samples versus samples kept at 1 g. This effect was not found when the samples were grown on potassium chloride. At 50 mM NaCl, the calcium ionophore ionomycin was applied to centrifuged and noncentrifuged callus samples. In both experiments, the growth of callus increased with increasing ionomycin concentrations but under 10 g this increase was more enhanced. As inhibitors of calcium influx, lanthanum and gadolinium chloride were chosen in the presence of 50 mM NaCl. Both inhibitors inhibited growth at 1 g at low concentrations of around 2 microM, whereas the centrifuged samples were not or much less so inhibited. We tested an involvement of actin by application of cytochalasin D to callus grown in the presence of 50 mM NaCl. In both types of samples, growth at 1 g and growth at 10 g, cytochalasin D enhanced growth but the effect was clearly stronger at 10 g than at 1 g. As increased halotolerance was only observed in the presence of increased sodium ions, not potassium ions, and as halotolerance is known to be induced by an influx of calcium, the data suggest that a calcium influx induced by hypergravity and possibly modulated by actin caused the observed increase in halotolerance at 10 g.

Actin Cytoskeleton↗

Effects of microgravity and hypergravity on the cell: investigations on Paramecium tetraurelia.

Previous space CYTOS experiments have shown that space flights resulted in an increase in growth of Paramecia cultures. Microgravity is the major factor responsible of this response: indeed the stimulatory effect disappeared in inflight cultures placed on a 1 g centrifuge aboard the Spacelab. On the other hand, exposure to different levels of hypergravity on Earth resulted in an opposite response, i.e. to a reduced cell growth rate. A possible mechanism of microgravity on paramecia is discussed.

Animals↗

Effects of unilateral selective hypergravity stimulation on gait.

The purpose of this work is to analyse the neural mechanisms of human motor perturbations induced by dynamic changes in gravity. A unilateral selective hypergravity stimulation (USHS) was produced by stretching an elastic band between the right shoulder and foot. The consequences of the extensor muscle tone change due to the positioning (increased muscular loading) and to its removal (decreased muscular loading) by the elastic band were observed on motor gait skill. Gait spatio-temporal parameters (horizontal displacement of both feet) and lower limb functional length variations (efficiency of flexion and extension movements of the lower limbs) were measured. The latter measure was performed using a device specially designed for that purpose. The main results were: (1) during and after USHS, gait perturbations appeared on the left--the body side not directly stimulated, (2) just after the end of USHS, perturbations were present on the right (homolateral) side evidencing a post treatment effect which caused a decrease in functional shortening of the lower limb during extension and an increase of functional shortening of the lower limb during stance (opposite in sense to the modification observed during swing). Such results afford evidence that, in addition to vestibular receptors, the mechanoreceptors of extensor muscles are involved in determining the changes in motor skills observed at the beginning and at the end of space flights.

Adaptation, Physiological↗

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.

Animals↗

Core temperature is regulated, although at a lower temperature, in rats exposed to hypergravic fields.

1. In rats acclimated to 23 degrees C (RT rats) or 5 degrees C (CA rats), core temperature (Tc), tail temperature (Tt) and oxygen consumption (VO2) were measured during exposure to a hypergravic field. 2. Rats were exposed for 5.5 h to a 3 g field while ambient temperature (Ta) was varied. For the first 2 h, Ta was 25 degrees C; then Ta was raised to 34 degrees C for 1.5 h. During this period of warm exposure, Tc increased 4 degrees C in both RT and CA rats. Finally, Ta was returned to 25 degrees C for 2 h, and Tc decreased toward the levels measured prior to warm exposure. 3. In a second experiment at 3 g, RT and CA rats were exposed to cold (12 degrees C) after two hours at 25 degrees C. During the one hour cold exposure, Tc fell 1.5 degrees C in RT and 0.5 degree C in CA rats. After cold exposure, when ambient temperature was again 25 degrees C, Tc of RT and CA rats returned toward the levels measured prior to the thermal disturbance. 4. Rats appear to regulate their temperature, albeit at a lower level, in a 3 g field.

Acclimatization↗

Effects of hypergravity environments on amphibian development, gene expression and apoptosis.

This study investigates how rearing under conditions of hypergravity affects amphibian development, Xotx2 and Xag1 gene expression and apoptosis. Uncleaved Xenopus laevis eggs 20 min after insemination, 2 cell stage embryos, and gastrula stage embryos were raised at 2G and 5G, while controls were raised in normal gravity. Apoptosis in brain and eye inner structures of hatching embryos was scored using the TUNEL staining method, and gene expression in tail-bud embryos was analyzed by whole-mount in situ hybridization. Results showed that: (1) 5G retarded the development of eggs and embryos and induced microcephaly and microphthalmia. (2) 5G suppressed the expression of the two genes, Xotx2 (involved in fore- and midbrain and eye development) and Xag1 (regulating cement gland formation). (3) Eggs and 2 cell stage embryos raised at 5G showed a greater extent of brain and eye apoptosis compared with controls, while those raised at 2G showed no significant difference. These findings suggest that high gravity suppresses certain gene functions and induces abnormal apoptosis in brain and eyes, resulting in developmental retardation and various morphological abnormalities.

Animals↗

Long-term hypergravity induces plastic alterations in vestibulo-cardiovascular reflex in conscious rats.

To test the hypothesis that an altered gravitational environment induces plastic changes in the vestibulo-cardiovascular reflex, arterial pressure (AP) and hypothalamic glutamate concentration were examined in 2 groups of conscious rats, i.e., a 3-G group and a 1-G group, in which rats were maintained under a 3-G and 1-G environment for 2 weeks, respectively. The vestibulo-cardiovascular reflex was stimulated by a gravitational change induced by a parabolic flight that consisted of 3 phases: "pull-up", during which the G load gradually increased to 2G; a 20s "push-over" into microgravity; and "pull-out", during which the G load increased to 1.8. In the 1-G group, the AP increased by 11.9+/-1.2 mmHg during the pull-up hypergravity period. The AP response was significantly attenuated in the 3-G group (4.0+/-0.8 mmHg). During the push-over microgravity period, the AP decreased from the peak level in the pull-up period and recovered to the pre-parabolic control level (-1.8+/-2.4 mmHg). In rats of the 3-G group, the AP was not altered by push-over microgravity. These AP responses were associated with a significant increase in the glutamate concentration in the hypothalamus (4.4+/-0.7%). The glutamate response was also significantly attenuated in the 3-G group compared with that in the 1-G group. These results indicate that an altered gravitational environment induces plastic alterations in the vestibulo-cardiovascular reflex.

Animals↗

Effects of perfusion on the mechanical behavior of the brain-exposed to hypergravity.

In certain flight configurations, fighter pilots are exposed to high Gz acceleration which may induce inflight loss of consciousness (G-LOC). In order to study the mechanical effects induced by these accelerations on the cerebral structures, an experimental model has been developed in vitro. Fresh bovine brains were excised and placed in a transparent mold modeling the inside of the skull. Half of these brains were perfused during the experiment. This assembly was placed into the gondola of a centrifuge, in front of a camera lens. Displacements and deformations of the brains were filmed and recorded at different onset rates. Measurements were made after off-line digitalization of images. Experimental data were incorporated into a finite element calculation code whose mesh represented the brain. The applied behavior law was elastic, the structure being considered as homogeneous and isotropic. The first results concerned the elastic properties of the brains under hypergravity. The mean value of the Young's modulus of the nonperfused brain was 46.8 kPa, which corresponded to the values published in reference literature. For the perfused brains, the mean value of the Young's modulus was higher. The mean value of the equivalent Poisson's ratio was 0.35. In fact, contrary to impacts, the mechanical stimulation is long enough to allow fluid displacements. The mean value of the equivalent Poisson's ratio calculated in the present study should probably be increased since this study was performed post mortem.

Aerospace Medicine↗

HSP70 induction may explain the long-lasting resistance to heat of Drosophila melanogaster having lived in hypergravity.

In this study, we showed that in flies kept for 2 weeks at 1 (terrestrial gravity), 3 or 5 x g (hypergravity, HG) before transfer to 1 x g, resistance to heat remained higher in HG flies for several weeks after the transfer. The measurement of heat shock protein 70 (hsp70) indicated no induction of the protein in HG, but the study revealed that flies living in HG expressed more hsp70 only after being submitted to severe stress. The higher induction of hsp70 may explain the higher thermotolerance of these HG-treated young flies. Finally, an unknown protein was observed only in females. This protein may belong to a class of higher molecular weight hsp (hsp110), which have not previously been observed in Drosophila.

Age Factors↗

Resistance to stress as a function of age in Drosophila melanogaster living in hypergravity.

Male and female fruitflies (Drosophila melanogaster) living at different gravity levels [1g: terrestrial gravity; 3 and 5g: hypergravity (HG)] were used to investigate the age-specific (young: 7 days; middle-aged: 28 days; and old: 49 days) resistance to various stresses (starvation, desiccation, and cold). The experiment showed that the resistance of the flies to the studied stresses decreased with age, except in the case of females submitted to starvation which was increased. These variations were explained by the amount of lipid. Variation in desiccation resistance was not explained by the amount of water. As a function of gravity, no or slight differences were observed for the studied stresses. The resistance to heat of young flies increased with the gravity level. This resistance was not explained by a decreased locomotor activity of HG-living flies during heat stress, nor by the water and lipid contents.

Aging↗

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.

Adaptation, Physiological↗

The asymmetrical growth of otoliths in fish is affected by hypergravity.

Size and asymmetry (size difference between the left and the right side) of inner ear otoliths of larval cichlid fish were determined after a long-term stay at moderate hypergravity conditions (3g; centrifuge), in the course of which the animals completed their ontogenetic development from hatch to freely swimming. Both the normal morphogenetic development as well as the timely onset and gain of performance of the swimming behaviour was not impaired by the experimental conditions. However, both utricular and saccular otoliths (lapilli and sagittae, respectively) were significantly smaller after hyper-g exposure as compared to parallely raised 1g control specimens. The asymmetry of sagittae was significantly increased in the experimental animals, whereas the respective asymmetry con-cerning lapilli was pronouncedly decreased in comparison to the 1g controls. These findings suggest, that the growth and the development of bilateral asymmetry of otoliths is guided by the environmental gravity vector.

Animals↗

Differential impact of hypergravity on maturating innervation in vestibular epithelia during rat development.

Over the past decades, the new opportunity of space flights has revealed the importance of gravity as a mechanical constraint for terrestrial organisms as well as its influence on the somatosensory system. The lack of gravitational reference in orbital flight induces changes in equilibrium, with major modifications involving neuromorphological and physiological adaptations. However, few data have illustrated the putative effect of gravity on sensory vestibular epithelial development. We asked if gravity, the primary stimulus of utricles could act as an epigenetic factor. As sensorial deprivation linked to weightlessness is technically difficult, we used a ground-based centrifuge to increase the gravitational vector, in order to hyperstimulate the vestibule. In this study, 3 days after mating, pregnant females were submitted to hypergravity, 2 g (HG). Their embryos were raised, born and postnatally developed under HG. The establishment of connections between primary vestibular afferent neurons and hair cells in the utricle of these young rats was followed from birth to postnatal day 6 (PN6) and compared to embryos developed in normogravity (NG): Immunocytochemistry for neurofilaments and microvesicles revealed the differential effects of gravity on the late neuritogenic and synaptogenic processes in utricles. Taking type I hair cell innervation as a criterion of maturation, we found that primary afferent fibres reached the vestibular epithelium and enveloped hair cells in the same way, both under NG and HG. Thus, this phenomenon of leading growth cones to their epithelial target appears to be dependent on intrinsic genetic properties and not on an external stimulus. In contrast, the maturation of connection processes between type 1 hair cells and the afferent calyx, concerning specifically the microvesicles at their apex, was delayed under HG. Therefore, gravity appears to be an epigenetic factor influencing the late maturation of utricles. These differential effects of altered gravity on the development of the vestibular epithelium are discussed.

Animals↗

Effects of hypergravity on the photosynthetic flagellate, Euglena gracilis.

Euglena gracilis, a unicellular, photosynthetic flagellate, orients itself by means of gravi- and phototaxis to reach and stay in regions optimal for survival and growth. An improved version of the slow rotating centrifuge microscope, NIZEMI, was used to test wild type and mutant strains for their responses to hypergravity. Wild type cells could actively move against the acceleration vector up to 8.5 gn and were centrifuged down at higher rates. Even at 10.5 gn, the highest value tested, cells were still negative gravitactically oriented as shown by video images. In contrast, all mutant strains as well as Astasia longa, a close relative of Euglena, could move against the acceleration vector under all conditions tested. With increasing accelerations the mean orientation of the populations shifted according to a vectorial addition of gravity and acceleration. The r-value, a statistical measure of the orientation of a population, increased with moderately increased acceleration rates and decreased at higher values. While wild type Euglena and two of the three mutant strains tested were exclusively negative gravitactically, in the third strain as well as in Astasia longa half of the population reacted negative gravitactically and the other half positive gravitactically. This variation of the wild type behavior was observed at moderate acceleration rates. At high accelerations the cells became exclusively positive gravitactic. The obtained results are discussed on the basis of the current model explaining gravitaxis.

Acceleration↗

Effects of hypergravity on the development of cell number and asymmetry in fish brain nuclei.

Larval cichlid fish (Oreochromis mossambicus) siblings were subjected to 3 g hypergravity (hg) and total darkness for 21 days during development and subsequently processed for conventional histology. Further siblings reared at 1 g and alternating light/dark (12h:12h) conditions served as controls. Cell number counts of the visual Nucleus isthmi (Ni) versus the vestibular Nucleus magnocellularis (Nm) revealed that in experimental animals total cell number was decreased in the Ni, possibly due to retarded growth as a result of the lack of visual input whereas no effect was observed in the Nm. Calculating the percentual asymmetry in cell number (i.e., right vs. the left side of the brain), no effects of hg/darkness were seen in the Ni, whereas asymmetry was slightly increased in the Nm. Since the asymmetry of inner ear otoliths is decreased under hg, this finding may indicate efferent vestibular action of the CNS on the level of the Nm by means of a feedback mechanism.

Adaptation, Physiological↗

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.

Animals↗