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Keywords:

  • inner ear;
  • balance;
  • vestibular system

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We have determined a critical period for vestibular development in zebrafish by using a bioreactor designed by NASA to simulate microgravity for cells in culture. A critical period is defined as the briefest period of time during development when stimulus deprivation results in long lasting or permanent sensory deficits. Zebrafish eggs were collected within 3 hours of being laid and fertilized. In experiment 1, eggs were placed in the bioreactor at 3, 24, 30, 36, 48, or 72 hours postfertilization (hPF) and maintained in the bioreactor until 96 hPF. In experiment 2, eggs were placed in the bioreactor immediately after they were collected and maintained in the bioreactor until 24, 36, 48, 60, 66, 72, or 96 hPF. Beginning at 96 hPF, all larvae had their vestibulo-ocular reflexes (VOR) evaluated once each day for 5 days. Only larvae that hatched from eggs that were placed in the bioreactor before 30 hPF in experiment 1 or removed from the bioreactor later than 66 hPF in experiment 2 had VOR deficits that persisted for at least 5 days. These data suggest a critical period for vestibular development in the zebrafish that begins before 30 hPF and ends after 66 hPF. To confirm this, zebrafish eggs were placed in the bioreactor at 24 hPF and removed at 72 hPF. VORs were evaluated in these larvae once each day for 5 days beginning at 96 hPF. These larvae had VOR deficits that persisted for at least 5 days. In addition, larvae that had been maintained in the bioreactor from 24 to 66 hPF or from 30 to 72 hPF, had only temporary VOR deficits. In a final experiment, zebrafish eggs were placed in the bioreactor at 3 hPF and removed at 96 hPF but the bioreactor was turned off from 24 hPF to 72 hPF. These larvae had normal VORs when they were removed from the bioreactor at 96 hPF. Taken as a whole, these data support the idea that there is a critical period for functional maturation of the zebrafish vestibular system. The developmental period identified includes the timeframe during which the vestibular primary afferent neurons are born, innervate their central and peripheral targets, and remodel their central projections. © 2002 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The vulnerability of a developing sensory system to environmental modifications has been established for the visual system and auditory system. For instance, partial or total visual deprivation during the postnatal period can cause morphological and electrophysiological abnormalities in the retina (Sosula and Glow, 1971), lateral geniculate bodies (Wiesel and Hubel, 1963), superior colliculus (Lund and Lund, 1972), and visual cortex (Wiesel and Hubel, 1965). Many of the effects were found to be irreversible if the animal was not exposed to normal stimuli during a critical period of development even if the animal is exposed to normal stimuli later in life (Wiesel and Hubel, 1965; Hubel and Wiesel, 1970). Morphological effects have also been produced by sound deprivation during a critical period in the development of the auditory system. These effects include smaller than normal neuron cell bodies in specific acoustic brainstem cell groups (Webster and Webster, 1977), changes in dendritic distribution of specific brainstem nuclei (Gray et al., 1982), and permanent behavioral deficits (Tees, 1967). Normal development of tactile senses has also been shown to be dependent on stimulation during development (Simons and Land, 1987). These findings suggest that stimulus dependence is a general feature of all developing sensory systems.

The control of movement and posture in the three dimensions of space requires sense organs and a reference system that allows equilibrium orientation to be maintained. In this respect, the vestibular system is the sensory system that transduces the direction of gravity into meaningful neurological signals for proper equilibrium orientation. It is likely, therefore, that development of the vestibular system will be affected by exposure to a microgravity environment.

Until recently, it had only been possible to test hypotheses about the development of the vestibular system in a microgravity environment during Space Shuttle missions. With the exception of hydrozoans (Spangenberg, 1991), many of the experiments that have been performed with the intent of determining the effects of microgravity on the development of the vestibular system have used a variety of animals at diverse developmental stages/ages or animals at an age where the vestibular system had already developed (for instance, see: Vinikov et al., 1976; Lychakov et al., 1993; Kenyon et al., 1995). Therefore, the vast majority of these experiments have either yielded conflicting results or have been inconclusive. It would be advantageous to be able to perform these experiments on earth. NASA designed a bioreactor, a Slow-Rotating Perfused Vessel, for culturing cells in a simulated microgravity environment. We replaced the culture medium with fresh water, allowing the bioreactor to accommodate eggs from aquatic vertebrates, such as zebrafish. This change allowed the bioreactor to be used to test the effects of a simulated-microgravity environment on the developing vestibular systems of zebrafish (Moorman et al., 1999).

A “critical period” is thought to be a general feature of developing sensory systems. This certainly is the case for the visual and auditory systems. Experiments to determine a critical period for the vestibular system have been technically very difficult. Recently, vestibular deficits have been reliably induced in embryonic/larval zebrafish by simulated microgravity (Moorman et al., 1999). Although those experiments did not identify a critical period for vestibular development, they demonstrated that the techniques could be used to accomplish that goal. There are no reports of attempts to determine a critical period during development of the vestibular system in earth-based experiments and the time constraints of a Space Shuttle mission make these experiments difficult to complete. By using the bioreactor, we exposed zebrafish eggs to a simulated-microgravity environment at strategic periods during the development of the vestibular system. This method allowed us to perform experiments to determine the critical period for functional development of the zebrafish vestibular system.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Stimulus-Deprivation Caused Long-Lasting Vestibular Deficits

At 96 hPF, control animals maintain a fairly consistent orientation of the eye with respect to the direction of gravity when the body is tilted tail up and tail down (Moorman et al., 1999; Riley and Moorman, 2000). If the eye maintained a consistent orientation with respect to the body, a regression line fit to the data would have a slope approaching 0.0. If the eye maintained a consistent orientation with respect to the direction of gravity, a regression line fit to the data would have a slope approaching 1. The slope of the regression line for the control data was 0.80 (r = 0.9) for animals at 96 hPF (Fig. 1A) and 0.78 (r = 0.9) for animals at 8 days postfertilization (Tables 1, 2). This finding supports the idea that in a zebrafish with an intact vestibular system, the eye maintains a consistent orientation with respect to the direction of gravity.

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Figure 1. Reflex counter-rotation of the eye of zebrafish during tilt (pitch) of the animals around a transverse body axis. A: Control animals 96 hr postfertilization (hPF). B: Experimental animals that had been in the bioreactor from 24 hPF until 72 hPF. Eye movements were analyzed at 96 hPF. C: The same experimental animals as in B 4 days after removal from the bioreactor. (EYE ANGLE and BODY ANGLE are defined in the Experimental Procedures section).

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Table 1. Slopes of the Regression Lines Fit to the Compensatory Eye-Movement Data for Larvae That Were Placed in the Bioreactor at the Time Indicated and Removed at 96 hPFa
Time inSlope @ 4 dPFSlope @ 8 dPFVestibular deficitsN
  • a

    hPF, hours postfertilization; dPF, days postfertilization.

  • *

    P < 0.01 for comparison with slope for controls.

3 hPF0.41* (r = 0.6)0.58* (r = 0.7)Long-lasting∼500
24 hPF0.35* (r = 0.6)0.49* (r = 0.6)Long-lasting50
30 hPF0.44* (r = 0.5)0.72 (r = 0.7)Temporary50
36 hPF0.39* (r = 0.6)0.70 (r = 0.6)Temporary50
48 hPF0.51* (r = 0.7)0.75 (r = 0.7)Temporary30
72 hPF0.83 (r = 0.9)0.79 (r = 0.9)None30
Control0.80 (r = 0.9)0.78 (r = 0.9)None>500
Table 2. Slopes of the Regression Lines Fit to the Compensatory Eye-Movement Data for Larvae That Were Placed in the Bioreactor at 3 hPF and Removed at the Time Indicateda
Time outSlope @ 4 dPFSlope @ 8 dPFVestibular deficitsN
  • a

    hPF, hours postfertilization; dPF, days postfertilization.

  • *

    P < 0.01 for comparison with slope for controls.

24 hPF0.78 (r = 0.9)0.77 (r = 0.9)None40
36 hPF0.77 (r = 0.7)0.78 (r = 0.8)None40
48 hPF0.39* (r = 0.5)0.75 (r = 0.8)Temporary40
60 hPF0.45* (r = 0.6)0.77 (r = 0.9)Temporary50
66 hPF0.41* (r = 0.5)0.72 (r = 0.8)Temporary50
72 hPF0.43* (r = 0.6)0.45* (r = 0.6)Long lasting∼500
96 hPF0.41* (r = 0.6)0.58* (r = 0.7)Long lasting∼500
Control0.80 (r = 0.9)0.78 (r = 0.9)None>500

When embryos were in the bioreactor from 3 hPF until 96 hPF, the larvae showed some signs of compensatory eye rotation, but with a much less clear relationship between the orientation of the eye and the direction of gravity than the age-matched control animals. The slope of the regression line for the 96-hr experimental animals was 0.41 (r = 0.6). This difference is still very obvious 5 days later when the slope of the regression line for the experimental animals was 0.58 (r = 0.7) (Tables 1, 2). This finding supports the idea that stimulus-deprivation during development causes long-lasting vestibular deficits in zebrafish.

Stimulus-Deprivation During a Critical Period Caused Vestibular Deficits

To identify the beginning of the critical period, we delayed putting the eggs into the bioreactor until 24, 30, 36, 48, or 72 hPF. In each case, the eggs were removed at 96 hPF. Only those eggs placed in the bioreactor at or before 24 hPF had long-lasting vestibular deficits (Table 1). To identify the end of the critical period, we placed the eggs in the bioreactor at 3 hPF and removed them at 24, 36, 48, 60, 66, 72, or 96 hPF. Only those eggs that were removed from the bioreactor at 72 hPF or later had long-lasting vestibular deficits (Table 2). These data suggested that the critical period begins around 24 hPF and ends around 72 hPF. To confirm this, we placed eggs in the bioreactor at 24 hPF and removed them at 72 hPF. These larvae had long-lasting vestibular deficits (Table 3; Fig. 1B,C). To determine whether a briefer exposure to simulated-microgravity could induce a similar effect, we delayed putting the eggs in the bioreactor until 30 hPF and removed them at 72 hPF. These larvae had temporary vestibular deficits (Table 3). Likewise, when eggs were placed in the bioreactor at 24 hPF and removed at 66 hPF, the larvae had temporary vestibular deficits (Table 3).

Table 3. Confirmation of the Beginning and Ending Times of the Critical Perioda
Time inTime outSlope @ 4 dPFSlope @ 8 dPFVestibular deficitsN
  • a

    hPF, hours postfertilization; dPF, days postfertilization.

  • *

    P < 0.01 for comparison with slope for controls.

24 hPF72 hPF0.35* (r = 0.5)0.37* (r = 0.7)Long-Lasting40
30 hPF72 hPF0.45* (r = 0.5)0.75 (r = 0.6)Temporary40
24 hPF66 hPF0.41* (r = 0.5)0.73 (r = 0.7)Temporary40

Normal Stimulus During Only the Critical Period Permitted Normal Vestibular Development

To determine whether the 24 hPF to 72 hPF timeframe is indeed a critical period, we exposed embryos to normal gravity only during this timeframe. To do this, we placed eggs in the bioreactor within 3 hr of fertilization and turned the bioreactor on. The bioreactor was then turned off at 24 hPF but the eggs were not removed. The bioreactor was turned back on at 72 hPF. The bioreactor was kept on until 96 hPF at which time the larvae were removed and testing performed. These larvae had no functional vestibular deficits (slope at 4 days, 0.78 [r = 0.8]; slope at 8 days, 0.77 [r = 0.8]).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

A critical period must meet two key criteria. First, stimulus deprivation during only the critical period should result in long-lasting/permanent sensory deficits. Second, exposure to the normal stimulus during ONLY the critical period should result in relatively normal sensory development. The vestibular deficits in 24–72 hPF larvae persisted until the larvae died at 10–14 days postfertilization ([data not shown]. For a discussion of why these larvae die, see Riley and Moorman, 2000). The larvae that were exposed to normal gravity during only the 24–72 hPF timeframe had normal vestibular function and survived to adulthood. Thus, the criteria for identifying a critical period appear to have been met.

Interestingly, the critical period that we have identified for development of vestibular function includes the timeframe during which the vestibular primary afferent neurons are born (Haddon and Lewis, 1996), innervate their central (Chapman and Fraser, 1995) and peripheral targets, and remodel their central projections (Chapman and Fraser, 1995). This finding is in contrast to the visual system, for which the critical period is thought to include only the timeframe where the central connections are established and remodeled (see Harwerth et al., 1986; Chapman and Stone, 1996). The results of experiments performed on the Space Shuttle as part of Neuro-Lab suggest that microgravity can influence proliferation and apoptosis in the developing central nervous system (Hayes and Nowakowski, 1999). We have not yet determined whether the number of neurons in the statoacoustic (VIIIth) ganglion has been affected in our experimental animals. Likewise, we have not yet analyzed vestibular primary afferent projection patterns in our experimental animals, although those experiments are under way.

The vestibular system is evolutionarily conserved (Romer and Parsons, 1986). In fact, the sequence of developmental events for the vestibular system seems to be quite similar in all jawed vertebrates (see Fritzsch et al., 2000). Thus, having identified a critical period for functional vestibular development in zebrafish allows us to predict the existence of a similar period during vestibular development in other animals, including humans. In addition, it should be possible to predict the developmental period(s) during which such a critical period should occur in other animals. This makes it easier to design experiments that will determine the critical period for functional vestibular development in other animals. These experiments must be done on the Space Shuttle or the International Space Station, because no long-duration microgravity-simulator exists for terrestrial animals.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The Bioreactor

To expose embryos and larvae to a decreased net force, we used a bioreactor that NASA designed for simulating microgravity for cells in culture. The bioreactor (Synthecon, Houston, TX) is a 10-cm-diameter clear Lexan cylinder with a solid 5-cm-diameter Teflon central core (Fig. 2, insert). The cylinder is closed at each end by a Teflon cap. One Teflon cap has several access ports for adding/removing water and/or small objects. The other cap is designed to attach to a shaft that is connected to the apparatus base. This shaft is connected to a variable speed motor by a rubber belt. When the vessel is filled with water, there is a 2.5-cm-thick circular wall of water between the clear Lexan outer wall and the white Teflon inner core. Zebrafish eggs are especially suited for use in the bioreactor because each egg is approximately 1.5 mm in diameter. When zebrafish eggs are placed in the vessel, the eggs are visible through the clear Lexan outer wall. The entire vessel is mounted with its axis on a horizontal plane. When the bioreactor is turned on, the cylindrical vessel rotates on its axis. As the vessel rotates, the wall of water rotates at the same speed as the vessel. The speed of rotation can be adjusted to accommodate objects of different sizes and mass.

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Figure 2. A drawing of a cross-section through the bioreactor (inset) with illustrations indicating the orientation of the same 24 hr postfertilization zebrafish egg at the 3, 6, 9, and 12 o'clock positions during the orbit when the speed of rotation is adjusted correctly. The small circles represent the position of the same egg at the different points in the orbit. The small arrows indicate the direction the egg would be moving at each point in the orbit. The pictures of the zebrafish embryo are the same picture rotated to illustrate the orientation of a representative embryo at each of the indicated points in the orbit. The embryo appears to maintain a uniform orientation with respect to the radius of the vessel. The embryo only changes its orientation with respect to the vessel when there are spontaneous movements of the embryo within the chorion. The embryo would then maintain this new orientation with respect to the vessel until the next spontaneous movement.

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The bioreactor was designed on the principle that the net force vector an object is exposed to, at any point in the vessel's rotation, is a combination of force vectors created by the vessel's rotation, the viscosity of the water, and the gravitational vector (Kessler, 1992). If the speed of rotation is adjusted correctly, the force vectors interact in such a way that the zebrafish eggs can be kept in a synchronous, near-circular orbit around the core of the vessel. This can be visually verified because the eggs are visible through the clear Lexan outer wall of the vessel. A circular shape to the orbit is indicative of a net force vector that has the same magnitude at all points in the rotation but whose direction is constantly changing (see Resnick and Halliday, 1977). Intuitively, one would suspect that the gravitational vector would augment the centripetal vector at the 9 o'clock position of a circular orbit and would result in a net vector with an increased magnitude. If this were true, the orbit would have to be elliptical and not circular.

Although we visually verified that the orbit was approximately circular in all of our experiments, we measured the shape of the orbit for a group of eggs 48 hPF (n = 50). The eggs were placed in the bioreactor and video taped for 15 min. We determined the position of individual eggs between the central core and the outside wall of the bioreactor at the 3, 6, 9, and 12 o'clock positions during the orbit (Fig. 2). Each individual egg maintained approximately the same distance from the central core at each of the four orbital positions checked (Table 4). This finding suggests that the orbit was circular for each egg. Because we have verified that the shape of the orbit is approximately circular at 15 rpm, the net force that the eggs “feel” can be approximated by the centripetal acceleration. The centripetal acceleration vector is directed toward the center of the bioreactor and is the primary force responsible for keeping the eggs in a circular orbit. The minimum and maximum centripetal accelerations, based on the minimum and maximum possible radius of the circular orbit, can be calculated in the following manner:

νmin

Minimumt Orbitalt Velocity

νmin

rmin/T

rmin

0.027m

T

4s

νmin

0.0424t m/s

νmax

Maximumt Orbitalt Velocity

νmax

rmax/T

rmax

0.045m

T

4s

νmax

0.0707t m/s

rmin and rmax

the minimum and maximum radius of an orbit inside the bioreactor where the eggs do not touch the walls

T

time for 1 revelution at 15 rpm

αmin

Minimumt Centripetalt Acceleration

αmin

νmath image/rmin

αmin

0.04242/0.027

αmin

0.06658t m/s2 = 0.0068g

αmax

Maximumt Centripetalt Acceleration

αmax

νmath image/rmax

αmax

0.07072/0.045

αmax

0.111 m/s2 = 0.0113g

If the orbit were elliptical, the magnitude of the net force each egg would be subjected to would oscillate between the calculated minimum and maximum, as long as the egg did not touch either the inner core or the outer wall of the vessel.

Table 4. Distance From the Outside Wall (cm) During a Complete Orbit With the Speed of Rotation Adjusted to 15 rpm
 3 o'clock position6 o'clock position9 o'clock position12 o'clock position
Egg 1, orbit 11.11.11.01.1
Egg 1, orbit 21.21.11.11.1
Egg 1, orbit 31.11.11.11.2
Egg 1, orbit 41.11.11.11.2
Egg 1, orbit 51.01.11.11.1
Egg 1 average1.1 ± 0.0711.12 ± 0.0451.1 ± 0.0711.12 ± 0.045
Overall average for 50 eggs1.3 ± 0.181.3 ± 0.221.3 ± 0.211.3 ± 0.19

Animals

Adult zebrafish were purchased from a local pet store and maintained in aquaria at 25–28°C on a 14/10 hr light/dark cycle. Zebrafish eggs were collected once a week for 26 weeks within 3 hr after they were laid and fertilized. Approximately 50% of those eggs were transferred into a beaker with fresh water (60 mg of Instant Ocean [Aquarium systems, Mentor, OH]/L of distilled H2O) and placed in an incubator at 28°C and approximately 50% were placed in the bioreactor. The bioreactor was filled with aerated fresh water, sealed, placed in the incubator at 28°C, and set to rotate at a speed that kept the eggs in a circular orbit without contacting either the central core or the outside wall of the bioreactor during its rotation (15–20 rpm). Both the control and the experimental eggs were kept on a 14/10 hr light/dark cycle with the light source from above. The hatching rate in the bioreactor was >80% and identical to that of the controls.

Compensatory Eye Movements

We measured compensatory counter-rotation of the eyes of zebrafish larvae to assess the functional integrity of the vestibular end-organs and their central connections. An individual zebrafish larva was transferred into a solution of 4% methyl cellulose (M7140; Sigma, St. Louis, MO) in fresh water. The larva was then transferred into a glass capillary tube and one end of the tube was sealed with a piece of modeling clay. We have found that the methyl cellulose makes it easier to maintain the larva in a uniform orientation inside the capillary tube. The capillary tube was placed on a special holder on an ophthalmic microscope. This type of microscope is positioned with the optics and illumination on a horizontal plane for observing the human retina in conscious patients. The special holder for the capillary tube was designed to allow the tube to be tilted in the plane of focus. By using this microscope and holder, the larva remains dorsal-surface-up, is illuminated from the side, and its left or right eye can be observed while tilting the larva around a transverse body axis (pitch). All of the animals were tested under identical conditions at the same time of day, in the same room, with the room lights off, and the fish illuminated from the side through the microscope optics. Under these conditions, the visual cues presented to all of the animals should have been identical.

Each larva was tilted tail up and tail down 3–5 times each. Each time the larva was tilted, a digitized video image of the larva was acquired and saved. By using NIH Image, the angle between the longitudinal body axis and the horizontal plane was measured. A line was then drawn connecting the center of the pupil and the choroid fissure of the iris. The angle between this line and the longitudinal body axis was then measured. All of the measurements for animals in a single group were pooled, and a linear regression line was computed for the data. The slopes and y-intercepts of these lines were compared by using methods described by Sokal and Rohlf (1995). The measured eye-angles for each group were then corrected by subtracting the y-intercept of the regression line from each measured value. The corrected data were then plotted versus the body angle by using SigmaPlot (Jandel Scientific, SPSS, Chicago, IL). If the slope of the linear regression line was significantly lower in the experimental animals compared with the controls, the experimental animals were scored as having vestibular deficits. The effects were determined to be long-lasting if the slope was still significantly lower at the end of the 5-day measurement period.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Charlotte Burress, Mary Cao, and Farris Altaff for conducting some of the experiments; Denise Dehnbostel for editing the manuscript; and Rebekah Danner, Suzanna Lesko, and Illona Gillette-Ferguson for helpful discussions.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
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