Chapter 4

The effect of prolonged hypergravity on the vestibular system; a behavioural study

Abstract

Golden hamsters were exposed to conditions of 2.5 times normal gravity (hypergravity, HG) for 4 months. During this period, tests were carried out to study equilibrium maintenance, swimming behaviour and open field behaviour of these hypergravity hamsters and of control hamsters living in a normal gravity environment. The tests proved to be useful devices for detecting differences in perceptive-motor behaviour between HG hamsters and control hamsters. The HG hamsters had more difficulties with balancing on tubes and orientation during swimming. In the open field study, the HG hamsters showed less locomotor activity than control hamsters. However, no differences were observed between the groups in washing, rearing and number of times having defecation.
These findings indicate that the daily transition from 2.5 to 1 G was not experienced as stressful by the hamsters, although performance on several perceptive motor tasks was decreased, especially during the first weeks.

Keywords: Hypergravity, hamsters, equilibrium maintenance, open field, swimming behaviour, spatial orientation.

Introduction

Our knowledge about the effect of sustained higher gravity (hypergravity, HG) on the behaviour of animals is still scarce. Especially the vestibular end organs are susceptible for alterations in gravity. Information coming from the vestibular system is, among others, important in equilibrium maintenance and swimming.
The aim of this study is to develop tasks to test the vestibular function of HG hamsters. The hamsters are exposed to 2.5 times normal gravity by means of an animal centrifuge. Balance tests and swimming tests are applied to detect differences in perceptive motor skills of animals living in a HG environment and of control animals. An open field study is performed to study the question if the daily transition from 2,5 G to 1 G is experienced as stressful.

Methods

Centrifuge

The animal centrifuge consisted of a centrally placed 3.5. kW DC motor drive and 2 horizontally mounted arms (length = 115 cm) with aerated and darkened free-swinging gondolas (length: 110 cm, width: 45 cm, height: 80 cm, length arm + gondola: 194 cm). During centrifugation, the Z-vector was constantly directed towards the floor of the gondola. At a rotation speed of 34,3 rpm a 2.5 G-value was reached at the bottom of the gondola. A video camera was installed inside the gondola to allow animal observation.

Subjects

A group of female golden hamsters (Mesocricetus auratus), born and raised under conditions of normal gravity, were divided into two groups after weaning. The first group of 3 hamsters was placed in a macrolon box (30 cm x 20 cm x 13 cm) inside the centrifuge-gondola in order to live under conditions of 2.5 G (HG hamsters), while the other group was placed in a similar housing under conditions of normal gravity (control hamsters). Food and water was ad lib. available. The light/dark cycle (light on: 19.00 - 7.00) was reversed in order to have the highest locomotor activity during normal daytime. After 1 week, the hamsters were daily tested on their perceptive-motor skills, such as equilibrium maintenance and swimming ability, in an experimental room with dimmed lights during a centrifuge stop of 30 min. Before testing, the animals were observed for overt signs of vestibular dysfunction during normal gravity.

Testing devices
Five days a week, the hamsters were tested on the following tasks:

The acrylate tubes (length 100 cm, diameter 20 mm) were either fixed to standards (fixed-tube task) or were movable connected by elastic cords which were attached to the standards (mobile-tube task). During the mobile-tube task the hamster could move the tube in all directions when crossing it. Both tubes were covered with tape in order to give the hamster a better grip. The tubes were placed ± 20 cm above ground level. A platform (10 x 10 cm) was placed at the end of the tube where the hamster could collect sun-flower seeds for ± 5 sec. On the day preceding the testing days, the animals were trained to walk the full length of the tube. When the hamster fell down from the tube, the time was stopped until the hamster was placed back on the tube. Each hamster had to cross the tube 3 times. When the hamster did not cross the tube within 2.5 min, the task ended and the hamster had to fulfil the next task. The tube tasks preceded the swimming tasks. Testing on the fixed tube started in the second week after the HG hamsters were placed in the centrifuge, whereas testing on the mobile tube started in the third week.

- Swimming behaviour in a basin (fig.1). Testing started in the third week after the HG hamsters were placed in the centrifuge. On the first testing day, the animals were trained to swim from one end to the other end of the basin where a stair was located in the middle of the target wall. Here, they could escape from the water by climbing the stair which lead to a platform. The stair and its surroundings were illuminated to add an extra visual cue. This task consisted of 3 succeeding trials per day. When the hamster did not find the stair within 2.5 min the swimming task ended. The swimming behaviour was recorded on tape for analysis. After 6 weeks (the ninth week after the HG hamsters were placed in the centrifuge), this task was stopped and changed to the next two swimming tasks.

- Swimming speed in a straight alley (fig. 1). The hamsters had to swim to the end of the lane where they could escape from the water by climbing a stair. Three succeeding trials per day were conducted.

- Orientation ability during swimming in a S-maze (fig. 1). The hamsters had to find a stair which was located at the other end of the maze. Three succeeding trials per day were conducted.

Testing procedures for both swimming tasks were the same as for the basin.

Fig. 1. Swimming tasks: Swimming basin (60 cm x 80 cm); A = straight alley (10 cm x 80 cm) with stair and visual cue; B = maze (50 cm x 80 cm) with stair and visual cue; height of the basin = 30 cm; water depth = 20 cm; water-temperature = 30⁰ c.

Finally, an open field study was performed to examine the question whether the daily transition from 2.5 to 1 G was experienced as stressful by the HG hamsters. After 15 weeks of hypergravity, the HG hamsters and controls were placed in the middle of the open square (60 x 60 cm, height of the walls 25 cm) for 5 min. The floor was cleaned before placing the hamsters in the square. The animal behaviour was recorded on tape for further analysis.

Results

Because the data of the second and the third trial of the tube tasks and the swimming tasks were comparable with the data from the first trial, only the data of the first trial of each day are presented. If possible, the data were analyzed with Analysis of Variance (ANOVA) with repeated measurements or with the Mann-Whitney U test (Non-parametric test). We used the statistical software SPSS 6.1 for windows for our analysis (significance: p<0.05).

Time needed to cross the tube and the number of times the hamsters fell down from the tube were registered.

- Equilibrium maintenance on the fixed tube; Especially during the first days, the HG hamsters had more difficulties in staying on the tube than the controls hamsters (F(1,4)= 25.32, p=0.07). Crossing time was the same for both groups. After day 15, Mann-Whitney tests (p<0.05) showed that the control hamsters spent more time on the tube during the following days than the HG hamsters but did not fall from the tube whereas the HG hamsters occasionally fell down (not significant, fig. 2a, b).

- Equilibrium maintenance on the mobile tube; During the first days, the HG hamsters fell down from the tube, whereas the controls almost never fell down (F(1,4)=25.0, p=0.07). Crossing time was the same for both groups. After this period, the control hamsters and HG hamsters did not differ in crossing time or fall frequency (fig. 3a, b).

Fig. 3. Equilibrium maintenance on the mobile tube. Moving averages over three days are shown. First trial per testing day, Y1-axis: fall frequency, Y2-axis: crossing time*HG = hypergravity hamsters.

- Swimming behaviour in the swimming basin; The HG animals needed more time to find the stair than the control animals during all days (F(1,4)=11.6, p=0.024). For both the crossing time decreased (F(29,116)=12.04, p<0.001), however, faster in the HG group (F(29,116)= 2.3, p=0.001; fig. 4a). Video-analysis showed that HG hamsters spent most of the time in the corners of the basin.

- Swimming speed in the straight alley; The swimming speed was measured from the moment the hamster started to cross the alley till the hamster had reached the stair. The time needed to cross the lane was ± 3 s for the HG hamsters and ± 2.5 s (not significant) for the control hamsters during all days.

- Orientation during swimming in the S-maze (only the trials which lasted shorter then 60 s were taken); Although not significant (p=0.057), the HG animals needed more time to find the stair than the control hamsters during all days (fig. 4b).

Fig. 4. Swimming behaviour. Moving averages over three days are shown. (A) crossing time for the basin (time needed to cross the basin); (B) crossing time for the S-maze (time needed to cross the maze).

The following variables were recorded: ambulation - the distance which the hamster covered in the open field, rearing responses - the number of times the animal raised both forepaws from the floor and extended its body and defaecation - the number of faecal boli deposited in the open field. The control hamsters walked a greater distance than HG hamsters (control hamsters = 14.1 m/5 min, HG hamsters = 8.4 m/5min). No differences were observed in rearing responses (control hamsters = 42 times in 5 minutes, HG hamsters = 35 times in 5 minutes) and washing (both groups 3 times in 5 minutes). No faecal boli were left behind by the control hamsters or the HG hamsters in the open field.

Discussion

In the last three decades only a few animal studies were performed to investigate the effect of long-lasting hypergravity on the vestibular perceptive function. The results of these experiments will be compared with our findings.

Clark (1974) investigated the effects of hypergravity on equilibrium behaviour on a rail during normal gravity in rats centrifuged at 2 G for 60 days. The HG rats had more problems with staying on the rails and therefore had more difficulties with maintaining equilibrium than controls, especially dynamic equilibrium (rotating rail). This difference in equilibrium maintenance behaviour disappeared within 3 days. In our experiment, we used tubes in stead of rails to make the equilibrium task more difficult because it was impossible for the hamster to grab the tube to prevent itself from falling down. We also found that the HG hamsters had difficulties with staying on the tubes whereas the control hamsters almost never fell. Video recordings of the performance of the hamsters on the tube tasks revealed that controls sometimes stopped with walking on the tubes and took their time to explore the surroundings by which the crossing time increased after the first days of testing. However, the control hamsters almost never fell from the tube whereas the HG hamsters had difficulties with staying on the rail even after ten days of testing. It is concluded that the hamsters subjected to prolonged hypergravity have more difficulties with equilibrium maintenance than control hamsters which is in accordance with the results of Clark (1974). However, the differences in the rail tasks between hypergravity rats and controls disappeared within a few days, whereas differences in performance on the tube tasks between the hypergravity hamsters and controls were still seen after several days.
Fox et al. (1992) found, among others, differences in the swimming behaviour between rats subjected to 2 G for 4 to 16 days and control rats. The HG rats swam more underwater than the control rats. The control rats held their forelimbs close to their body while swimming, whereas HG rats swam also with their forepaws. Our hamsters did not swam underwater and both groups used their forelimbs and hindlimbs to swim ('dog paddle style"). Except for 1 hamster, no swimming disturbances were found, the HG hamsters almost swam as fast as the controls. Concerning the S-maze task, the crossing time was increased for the HG hamsters when compared to the controls. Video analysis of the maze swimming behaviour showed that the control hamsters swam directly in a straight line to the stair, whereas the HG hamsters swam along the walls of the basin. These results supported the hypothesis that the vestibular sensitivity to gravity is reduced after prolonged exposure to hypergravity and therefore results in a disorientation with return to the 1G environment as was stated by Fox et al. (1992).
One HG hamster showed back-circling underwater and surface-circling. This abnormal swimming behaviour was also observed in rats without otoconia, mice with otolith defects, bilabyrinthectomized rats during normal gravity and in normal rats during weightlessness (Lim and Erway; 1974; Huygen et al. 1986). We hypothesize that the decreased orientation ability and the circling behaviour observed in our HG hamsters are expressions of vestibular dysfunction. Furthermore, the circling behaviour seems to represent an extreme form of disorientation whereas the swimming along the walls represent a milder form of disorientation.
Although the HG hamsters showed less ambulation behaviour in the open field then controls, no differences were found between the groups in washing, rearing and number of times having defaecation. These results support the assumption that the HG hamsters do not experience the transition from 2.5 to 1G as stressful.

It is concluded that the applied tests proved to be useful devices for detecting differences in behaviour between HG hamsters and controls. The results of our study showed that it is possible to study the effect of hypergravity for a longer period of time. During this period we can observe disturbances of some perceptive motor skills which disappear after a few weeks (equilibrium maintenance on the tubes) while disturbances on other tasks are detectable during the whole period of hypergravity (swimming behaviour).

Acknowledgements

The Netherlands Organization for Scientific Research (NWO) is gratefully acknowledged for funding this project. This research was conducted while HNPM Sondag was supported by a grant of the Foundation for Behavioural and Educational Sciences (SGW) of this organization (575-62-049), awarded to Prof. Dr. WJ Oosterveld.