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Vestibular decompensation in labyrinthectomized rats placed in weightlessness during parabolic flight
Neuroscience Letters 344 (2003) 122–126 www.elsevier.com/locate/neulet
Vestibular decompensation in labyrinthectomized rats placed in weightlessness during parabolic flight Annie Rebera,*, Jean-Hubert Courjonb, Pierre Denisec, Gilles Cle´mentd a
Laboratoire de Neurosciences et Environnement, EA 2122 UFR Sciences, Universite´ de Rouen, Mont-Saint-Aignan, F-76821, France b Espace et Action, INSERM U534, Bron, F-69500, France c Laboratoire de Physiologie, UFR Medecine, Caen, F-14032, France d Centre de Recherche Cerveau et Cognition, UMR 5549 CNRS-UPS, Toulouse, F-31062, France Received 12 December 2002; received in revised form 2 April 2003; accepted 2 April 2003
Abstract The purpose of this study was to determine whether the absence of gravitational cues during weightlessness could alter the posture and static eye deviation of Earth compensated rats with peripheral vestibular lesions. The responses of bilaterally (BL) and unilaterally (UL) labyrinthectomized rats at a compensated stage (40 – 43 days after lesion) during parabolic flight were compared with those at an acute stage (2 – 7 h after lesion) on Earth. When free-floating in 0 g, UL animals showed the same postural pattern as during water immersion just after surgery. The most striking observation was a continuous roll body motion at about 4 Hz, and a skewed asymmetric posture. When restrained in 0 g, static eye deviation was also comparable to that observed at an acute stage. A return to a compensated posture and gaze was observed within a few seconds following the end of the weightlessness conditions. BL animals were less affected. These results suggest that vestibular compensation after unilateral lesion can be disrupted momentarily and is a fragile state during which the otolith system in the remaining vestibular apparatus presumably plays a continuous role. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vestibular compensation; Labyrinthectomy; Otoliths; Weightlessness
It is well known that after lesion of the vestibular apparatus on one side, there is an asymmetry in posture and eye movements. Within a few days this asymmetry gradually disappears and normal response is restored (see Refs. [6,21] for review). This process is called vestibular compensation and is a progressive dynamic process involving the substitution of the missing vestibular inputs by visual [5, 17], somatosensory [14,19], as well as vestibular inputs from the intact side [16]. Previous studies have shown that head tilt toward the lesioned ear was present again when compensated animals were placed in darkness [4], indicating that vestibular decompensation could occur in the absence of visual cues. Other studies suggest that vestibular decompensation is present after lesions in structures of the central nervous system which are known to relay somatosensory inputs [1]. The objective of this study was to determine whether the changes in otolith, tactile, and *
Corresponding author. Tel.: þ33-2-35-14-67-24; fax: þ33-2-35-14-69-
54. E-mail address: [email protected] (A. Reber).
proprioceptive inputs in weightlessness could also induce a vestibular decompensation. Posture and static eye position were analyzed during parabolic flight 40– 43 days after surgery when static deficits were fully compensated. Changes in head tilt and muscular tone in restrained labyrinthectomized animals during parabolic flight had previously been reported by our group [8]. It was not possible to test the animals in weightlessness just after the lesions. We therefore compared the posture of compensated animals during free-floating in weightlessness with that of lesioned animals at an acute stage (2 –7 h after surgery) during water immersion. Both weightlessness and water immersion alter tactile cues (no cues during freefloating; cues distributed all over the body during water immersion) and challenge processes such as navigation and spatial orientation. We also compared the surface righting response of compensated animals at the end of the weightlessness phase during parabolic flight when the animals hit the ground with that of acute animals after a short free-fall on land (drop test).
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00433-6
A. Reber et al. / Neuroscience Letters 344 (2003) 122–126
This experiment was carried out on nine adult rats (DA/HAN, male, 5 –8 months old, 250 – 280 g). Three animals served as intact control rats. Three had received a labyrinthectomy on both sides (bilaterally, BL) and three on the left side only (unilaterally, UL). Surgery was performed with accreditation according to the guidelines of the French Ministry of Agriculture and of the European Community under deep ether anesthesia. The bulla tympanica was exposed through a ventral approach, the vestibulum was opened and the organs of the inner ear were mechanically destroyed. The wound edge was sutured and repetitively infiltrated with 1% xylocaine for 2 h after the surgery. After the experiments the animals were deeply anesthetized and the complete extension of the lesion was confirmed on decalcified serial head sections. For the water immersion test, the animals were placed in the prone position at the surface of an aquarium (0.85 £ 0.6 £ 0.7 m) filled with water heated to body temperature for a period of about 20 s repeated three times. For the drop test, the animals were held in the prone position about 60 cm above a cushion and then dropped. Each animal was tested three times. For the parabolic flight test, parabolic flight was conducted on-board the CNES airplane during a series of three flights, including 22 parabolas each. In each parabola, a period of level flight (1 g) was followed by a pull-up phase of 1.8 g for about 20 s, a 0-g phase lasting 20 s, a pull-out phase at 1.8 g also lasting 20 s, and a return to 1 g for 1 –2 min. The animals were placed inside a transparent box (0.5 £ 0.5 £ 0.5 m) with a cushioned surface on its bottom for seven consecutive parabolas. Before being introduced in the observation box, the animals were restrained in a smaller enclosure, which prevented them from free-floating. For gaze deviation, the animals were gently restrained in a tissue bag that limited body movements. The head was fixed, pitched 308 nose-down so that the utricle was approximately horizontal. Movements of both eyes were recorded in darkness with a search coil technique. Each animal was tested during seven parabolas. In all other conditions (water immersion, drop, parabolic flight) the experiment was performed in ambient light. For posture analysis, the animal’s swimming or free-floating behavior and surface righting reflexes were videotaped at 50 frames/s. Lesioned animals generally responded by a rotation of their bodies around the longitudinal axis (roll motion). Both the percentage of time spent rotating for each condition and the rotation frequency were measured using frame-by-frame analysis. After the drop test and at the end of the weightless period, the latency required for the animals to ‘right’ themselves and resume an upright posture was measured using slow motion playback of the videotapes. Just after surgery, the UL animals showed the typical symptoms of postural and ocular imbalance after unilateral labyrinthectomy. The postural symptoms were characterized by a tilt of the head and a twist of the body toward the lesioned side, a flexion of both hind- and forelimbs on the
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side ipsilateral to the lesion, and an extension of both limbs on the contralateral side. BL rats had a more symmetrical posture, with a reduced muscular tone compared to control animals [8]. After 40– 43 days following the surgery, when the animals’ posture was tested during the 1-g phase of parabolic flight, there was no difference between the normal and lesioned animals (Fig. 1, left panel). During the 0-g phase, normal and BL animals extended their four limbs and occasionally rolled on themselves at a frequency of about 1 Hz. By contrast, when free-floating the UL animals twisted and turned at a velocity that exceeded 20008/s in one animal. Their head was tilted toward the lesioned side (left) and they rolled around their long body axis in the counter-clockwise direction (Fig. 1, middle panel). When the UL animals tenaciously grasped at the cushion on the bottom of the cage in weightlessness, they adopted the typical asymmetrical static posture observed just after labyrinthectomy, with a deflection of the head on the lesioned side, and a full extension of the contralateral fore- and hindlimbs (Fig. 1, right panel). The same posture was also observed when the airplane began its pull-out maneuver. However, a return to the normal (i.e. compensated) posture was observed after 4.5 s on average (Fig. 2C). By comparison, between 2 and 7 h after surgery, during the water immersion test, the lesioned animals spent most of the time rolling, at a frequency ranging from 1.2 to 3.2 Hz (velocity from 430 to 11508/s) (Fig. 2A,B). UL animals always rotated toward the lesioned side, whereas BL animals rotated either to the right or to the left. After a drop, which simulated a transient weightlessness on Earth, the BL animals resumed a prone posture almost immediately, whereas the latency for the surface righting response was about 3.5 s on average for the UL animals (Fig. 2C). During the parabolas, the UL animals rotated for about half of the duration of a parabola on average (Fig. 2B) because they were able to grasp at the cage bottom surface for the other half (whereas there was no surface to grasp
Fig. 1. Posture of a hemilabyrinthectomized animal on the left side, during the 1-g (left) and 0-g (middle and right) phases of parabolic flight. During the 0-g phase, two images are shown to illustrate the posture of the animal when free-floating and when grasping the floor of the cage. During the 1.8-g pull-out phase immediately following the 0-g phase, the animals maintained the same posture as shown in the right panel for about 4–5 s.
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Fig. 2. Comparison of the mean postural responses of normal, UL, and BL animals tested at an acute stage (2–7 h after surgery) during water immersion and after a drop on land, and at a compensated stage (40– 43 days after surgery) during and after the 0-g phase of parabolic flight. Average rotation frequency (A) and duration (B) during water immersion (WI) and during the 0-g phase of parabolic flight (0 g). Rotation duration is expressed as a percentage of the total duration of the test (some water immersion tests had to be stopped before the 20 s mark to rescue the lesioned animals from drowning). (C) Average latency of surface righting response after a free-fall on land (After fall) and after the 0-g phase of parabolic flight (After 0 g). Latencies were obtained by calculating the period between the time when the animals hit the floor and the return to an upright posture. *P , 0:05 relative to normal animals; **P , 0:05 relative to BL animals. Numbers in parentheses indicate the number of animals used in the different conditions.
during water immersion). It is important to note though that they immediately started to roll over when they released their support. By contrast, both the BL and normal animals could spend a considerable time free-floating without rolling. The latency of the surface righting reflex of the UL animals at the end of the 0-g phase was not significantly different from that measured after the early drop tests (Fig. 2C). The frequency of rotation of the UL animals was also comparable in weightlessness and during the early water immersion test (Fig. 2A). When the UL animals were restrained and the head fixed, during parabolic flight at 0 g and in the dark the left eye was deviated by 26.18 ^ 6.68 downward and the right eye was deviated by 26.48 ^ 4.58 upward compared to their position in normal gravity (mean and SD of three rats during 21 parabolas). The direction and amplitude of these eye deviations are comparable to those observed in UL rats at an acute stage [15]. By contrast, the changes in eye deviation in 0 g compared to 1 g never exceeded 38 in BL and normal animals. Any vertebrate exposed to weightlessness shows, at least temporarily, a spatial disorientation. For example, humans exposed to microgravity suffer inversion illusion and motion sickness [13], and humans, monkeys and fishes exhibit changes in reflexive eye movements [3,7,20]. When free-floating there no longer are tactile contact cues to provide information about the orientational down direction. One commonly observed response of animals in weightlessness is to react as though they are upside-down and to initiate a repetitive righting response. Typically the animal rolls over and over, since in weightlessness there is no vestibular confirmation that the action was successful [23].
This roll movement was casually seen in our normal and BL animals, and was directed toward either the right or the left. However, when the animals grasped at surfaces and tactile cues were present, they eventually came to perceive themselves as upright in an upright aircraft and the roll motion stopped. By contrast, the UL animals showed a continuous roll motion in weightlessness, in the direction corresponding to the lesioned vestibular apparatus. Although the parabolic flight took place late after surgery, when normal posture was observed in a 1-g environment, the acute symptoms of unilateral labyrinthectomy were again apparent in weightlessness and continued for 4 – 5 s after the end of the weightless period. The similarity between the response pattern of UL animals during parabolic flight at a compensated stage and on Earth at an acute stage is suggestive of a vestibular decompensation in weightlessness. Recent electrophysiological studies in frogs have shown that after unilateral section of the VIII nerve, the asymmetry that resulted was compensated by a functional reorganization of the somatosensory map in the vestibular recipient structures. This reorganization was manifested by an expansion of contralateral afferent vestibular signals onto the deprived ipsilateral neurons [9]. It led to an increase of excitatory commissural inputs from the intact side and a decrease of inhibitory commissural responses [10]. In very much the same way, following unilateral labyrinthectomy in rats and guinea pigs, an increase in spontaneous discharge and excitability has been observed in the vestibular neurons on the operated side [2,18]. This increase in the resting discharge could be responsible for the consequent recovery of static vestibular function. During the 20 s of weightlessness, the static gravitational force no longer stimulates the otolith organs of the intact ear, thereby weakening temporarily their excitatory inputs on the vestibular neurons on the operated side. In 0 g, their resting discharges would presumably not be strong enough to allow the activation of the vestibular neurons on the lesioned side, hence a temporary return to the acute vestibular deficits. In the absence of a surface support, a return to an imbalanced posture in decompensated animals would generate rotation in the direction of the lesioned, i.e. flexed, side. Indeed, in normal gravity, the postural tone maintains an attitude or posture in relation to the acceleration of gravity. Postural control is determined by the overall balance of muscle forces acting on the head, limbs, and torso. According to Newton’s first and second laws of biomechanics, an asymmetrical posture with an extension of the right limbs and a flexion of the left limbs creates a momentum as soon as the body is released from the surface support. In weightlessness, this momentum generates a rotation to the left (i.e. toward the lesioned side in our animals) which continues unless new forces (such as grasping reaction) impress on it and rebalance the posture. Postural decompensation has been reported for various
A. Reber et al. / Neuroscience Letters 344 (2003) 122–126
species and experimental conditions. For example, UL animals placed in the dark at a compensated stage exhibit asymmetrical posture, but normal posture is immediately restored when animals are again placed in the light [4]. Postural decompensation also takes place during handling of operated animals [11]. In our study, the fact that the UL animals also present the typical postural asymmetry even when they are in contact with the floor in 0 g (during grasping at the cage surface) suggests that tactile cues are not primarily responsible for the vestibular decompensation. Nevertheless, tactile inputs sensitive to body weight are not stimulated when the animals grasp at a support surface in the weightless condition, and the muscle groups involved during grasping and standing are different (flexor vs. extensor, respectively). It is therefore possible that the vestibular decompensation observed in weightlessness could also be due to the changes in tactile and proprioceptive cues in the free-floating animals. It is interesting to note that the upright posture was restored well before the end of the hypergravity phase (4.5 s compared to 20 s). In addition, no decompensation was observed during the hypergravity phase prior to the 0-g phase. Therefore, it seems that it is not hypergravity per se that caused a decompensation. The decompensation seen during the first few seconds of hypergravity following 0 g presumably is an after-effect of the decompensation triggered by the removal of gravitational information in weightlessness. It is also possible, however, that in our experiment the recovery of a normal posture after the transient vestibular decompensation provoked by weightlessness is even faster because the animals are exposed to hypergravity after the 0-g phase of parabolic flight. Indeed, recent studies have shown that guinea pigs stimulated with 2 g on a centrifuge following unilateral labyrinthectomy showed faster compensation in head deviation than when maintained in normal gravity [12]. Our results support the hypothesis that an imbalance in the otolith system underlies the ocular and postural asymmetry observed after unilateral labyrinthectomy. In the compensated UL rats, weightlessness uncovered an asymmetry in the otolith system, which was previously cancelled on Earth [8]. These results are in agreement with those obtained in perinatal rats gestated during space flight which suggest a direct effect of gravity on the development of the vestibular system due to a reduction of the otolith input [22]. They also suggest that vestibular compensation after unilateral lesion can be disrupted momentarily and is a fragile state during which the otolith system in the remaining vestibular apparatus plays a continuous role. This finding is consistent with the fragility of the vestibular compensation also observed in humans during the early phase of recovery following unilateral vestibular loss [6]. The decompensation is less evident after bilateral lesion, because since there are no longer otolith inputs, vestibular compensation would predominantly be achieved based on proprioceptive, somatosensory and visual cues.
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Acknowledgements This study was supported by grants from the Centre National d’Etudes Spatiales (CNES). We thank G. Bunel for audiovisual assistance, L. Galas and M. Dumarest for animal care, and an anonymous referee for constructive comments.
References [1] G.B. Azzena, Role of the spinal cord in compensating the effects of hemilabyrinthectomy, Arch. Ital. Biol. 107 (1969) 43–53. [2] S.A. Cameron, M.B. Dutia, Cellular basis of vestibular compensation: changes in intrinsic excitability of MVN neurones, NeuroReport 8 (1997) 2595– 2599. [3] G. Cle´ment, Alteration of eye movements and motion perception in microgravity, Brain Res. Rev. 28 (1998) 161 –172. [4] J.H. Courjon, M. Jeannerod, Visual substitution of labyrinthine defects, in: R. Granit, O. Pompeiano (Eds.), Reflex Control of Posture and Movement, Progress in Brain Research, 50, Elsevier, Amsterdam, 1979, pp. 783–792. [5] J.H. Courjon, M. Jeannerod, I. Ossuzio, R. Schmid, The role of vision in compensation of vestibuloocular reflex after hemilabyrinthectomy in the cat, Exp. Brain Res. 28 (1977) 235 –248. [6] I.S. Curthoys, G.M. Halmagyi, Vestibular compensation: a review of the oculomotor, neural, and clinical consequences of unilateral vestibular loss, J. Vestib. Res. 5 (1995) 67–107. [7] M. Dai, T. Raphan, I. Kozlovskaya, B. Cohen, Vestibular adaptation to space in monkeys, Otolaryngol. Head Neck Surg. 119 (1998) 65–77. [8] O. Etard, O. Hardy, A. Reber, G. Quarck, P. Denise, J. Corvisier, Hyper- and hypogravity alter posture in rats compensated on Earth for a vestibular asymmetry, NeuroReport 10 (1999) 669–673. [9] F. Goto, H. Straka, N. Dieringer, Expansion of afferent vestibular signals after the section of one of the vestibular nerve branches, J. Neurophysiol. 84 (2000) 581 –584. [10] F. Goto, H. Straka, N. Dieringer, Postlesional vestibular reorganization in frogs: evidence for a basic reaction pattern after nerve injury, J. Neurophysiol. 85 (2001) 2643–2646. [11] K.F. Hamann, A. Reber, B.J. Hess, N. Dieringer, Long-term deficits in otolith, canal and optokinetic ocular reflexes of pigmented rats after unilateral vestibular nerve section, Exp. Brain Res. 118 (1998) 331–340. [12] T. Ikeda, T. Sekitani, H. Hara, T. Tahara, M. Takahashi, Effect of hypergravity on vestibular compensation in guinea pigs, Acta OtoLaryngol. Suppl. 519 (1995) 173 –175. [13] J. Lackner, P. DiZio, Human orientation and movement control in weightlessness and artificial gravity environments, Exp. Brain Res. 130 (2000) 2 –26. [14] M. Lacour, C. Xerri, Vestibular compensation: new perspectives, in: H. Flohr, W. Precht (Eds.), Lesion-Induced Neuronal Plasticity in Sensorimotor Systems, Springer-Verlag, New York, 1981, pp. 240–253. [15] R. Llinas, K. Walton, Significance of the olivo-cerebellar system in compensation of ocular position following unilateral labyrinthectomy, in: R. Baker, A. Berthoz (Eds.), Control of Gaze by Brainstem Neurons, Elsevier, Amsterdam, 1977, pp. 399 –408. [16] W. Precht, N. Dieringer, Neuronal events paralleling functional recovery (compensation) following peripheral vestibular lesions, in: A. Berthoz, G. Melvill Jones (Eds.), Adaptive Mechanisms in Gaze Control, Elsevier, Amsterdam, 1985, pp. 251–268. [17] P.T.S. Putkonen, J.H. Courjon, M. Jeannerod, Compensation for
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postural effects of hemilabyrinthectomy in the cat. A sensory substitution process?, Exp. Brain Res. 28 (1977) 249 –257. [18] L. Ris, B. Capron, N. Vibert, P.P. Vidal, E. Godaux, Modification of the pacemaker activity of vestibular neurons in brainstem slices during vestibular compensation in the guinea pig, Eur. J. Neurosci. 13 (2001) 2234–2240. [19] K.P. Schaefer, D.L. Meyer, G. Wihelms, Somatosensory and cerebellar influences on compensation of labyrinthine lesions, in: R. Granit, O. Pompeiano (Eds.), Reflex Control of Posture and Movement, Progress in Brain Research, 50, Elsevier, Amsterdam, 1979, pp. 591–598.
[20] C. Sebastian, K. Essling, E. Horn, Altered gravitational forces affect the development of the static vestibuloocular reflex in fish (Oreochromis mossmbicus), J. Neurobiol. 46 (2001) 59–72. [21] P.F. Smith, I.S. Curthoys, Mechanisms of recovery following unilateral labyrinthectomy: a review, Brain Res. Rev. 14 (1989) 155–180. [22] K. Walton, Postnatal development under conditions of simulated weightlessness and space flight, Brain Res. Rev. 28 (1998) 25–34. [23] R. Wassersug, Vertebrate biology in microgravity, Am. Sci. 89 (2001) 46– 53.
Vestibular decompensation in labyrinthectomized rats placed in weightlessness during parabolic flight Annie Rebera,*, Jean-Hubert Courjonb, Pierre Denisec, Gilles Cle´mentd a
Laboratoire de Neurosciences et Environnement, EA 2122 UFR Sciences, Universite´ de Rouen, Mont-Saint-Aignan, F-76821, France b Espace et Action, INSERM U534, Bron, F-69500, France c Laboratoire de Physiologie, UFR Medecine, Caen, F-14032, France d Centre de Recherche Cerveau et Cognition, UMR 5549 CNRS-UPS, Toulouse, F-31062, France Received 12 December 2002; received in revised form 2 April 2003; accepted 2 April 2003
Abstract The purpose of this study was to determine whether the absence of gravitational cues during weightlessness could alter the posture and static eye deviation of Earth compensated rats with peripheral vestibular lesions. The responses of bilaterally (BL) and unilaterally (UL) labyrinthectomized rats at a compensated stage (40 – 43 days after lesion) during parabolic flight were compared with those at an acute stage (2 – 7 h after lesion) on Earth. When free-floating in 0 g, UL animals showed the same postural pattern as during water immersion just after surgery. The most striking observation was a continuous roll body motion at about 4 Hz, and a skewed asymmetric posture. When restrained in 0 g, static eye deviation was also comparable to that observed at an acute stage. A return to a compensated posture and gaze was observed within a few seconds following the end of the weightlessness conditions. BL animals were less affected. These results suggest that vestibular compensation after unilateral lesion can be disrupted momentarily and is a fragile state during which the otolith system in the remaining vestibular apparatus presumably plays a continuous role. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vestibular compensation; Labyrinthectomy; Otoliths; Weightlessness
It is well known that after lesion of the vestibular apparatus on one side, there is an asymmetry in posture and eye movements. Within a few days this asymmetry gradually disappears and normal response is restored (see Refs. [6,21] for review). This process is called vestibular compensation and is a progressive dynamic process involving the substitution of the missing vestibular inputs by visual [5, 17], somatosensory [14,19], as well as vestibular inputs from the intact side [16]. Previous studies have shown that head tilt toward the lesioned ear was present again when compensated animals were placed in darkness [4], indicating that vestibular decompensation could occur in the absence of visual cues. Other studies suggest that vestibular decompensation is present after lesions in structures of the central nervous system which are known to relay somatosensory inputs [1]. The objective of this study was to determine whether the changes in otolith, tactile, and *
Corresponding author. Tel.: þ33-2-35-14-67-24; fax: þ33-2-35-14-69-
54. E-mail address: [email protected] (A. Reber).
proprioceptive inputs in weightlessness could also induce a vestibular decompensation. Posture and static eye position were analyzed during parabolic flight 40– 43 days after surgery when static deficits were fully compensated. Changes in head tilt and muscular tone in restrained labyrinthectomized animals during parabolic flight had previously been reported by our group [8]. It was not possible to test the animals in weightlessness just after the lesions. We therefore compared the posture of compensated animals during free-floating in weightlessness with that of lesioned animals at an acute stage (2 –7 h after surgery) during water immersion. Both weightlessness and water immersion alter tactile cues (no cues during freefloating; cues distributed all over the body during water immersion) and challenge processes such as navigation and spatial orientation. We also compared the surface righting response of compensated animals at the end of the weightlessness phase during parabolic flight when the animals hit the ground with that of acute animals after a short free-fall on land (drop test).
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00433-6
A. Reber et al. / Neuroscience Letters 344 (2003) 122–126
This experiment was carried out on nine adult rats (DA/HAN, male, 5 –8 months old, 250 – 280 g). Three animals served as intact control rats. Three had received a labyrinthectomy on both sides (bilaterally, BL) and three on the left side only (unilaterally, UL). Surgery was performed with accreditation according to the guidelines of the French Ministry of Agriculture and of the European Community under deep ether anesthesia. The bulla tympanica was exposed through a ventral approach, the vestibulum was opened and the organs of the inner ear were mechanically destroyed. The wound edge was sutured and repetitively infiltrated with 1% xylocaine for 2 h after the surgery. After the experiments the animals were deeply anesthetized and the complete extension of the lesion was confirmed on decalcified serial head sections. For the water immersion test, the animals were placed in the prone position at the surface of an aquarium (0.85 £ 0.6 £ 0.7 m) filled with water heated to body temperature for a period of about 20 s repeated three times. For the drop test, the animals were held in the prone position about 60 cm above a cushion and then dropped. Each animal was tested three times. For the parabolic flight test, parabolic flight was conducted on-board the CNES airplane during a series of three flights, including 22 parabolas each. In each parabola, a period of level flight (1 g) was followed by a pull-up phase of 1.8 g for about 20 s, a 0-g phase lasting 20 s, a pull-out phase at 1.8 g also lasting 20 s, and a return to 1 g for 1 –2 min. The animals were placed inside a transparent box (0.5 £ 0.5 £ 0.5 m) with a cushioned surface on its bottom for seven consecutive parabolas. Before being introduced in the observation box, the animals were restrained in a smaller enclosure, which prevented them from free-floating. For gaze deviation, the animals were gently restrained in a tissue bag that limited body movements. The head was fixed, pitched 308 nose-down so that the utricle was approximately horizontal. Movements of both eyes were recorded in darkness with a search coil technique. Each animal was tested during seven parabolas. In all other conditions (water immersion, drop, parabolic flight) the experiment was performed in ambient light. For posture analysis, the animal’s swimming or free-floating behavior and surface righting reflexes were videotaped at 50 frames/s. Lesioned animals generally responded by a rotation of their bodies around the longitudinal axis (roll motion). Both the percentage of time spent rotating for each condition and the rotation frequency were measured using frame-by-frame analysis. After the drop test and at the end of the weightless period, the latency required for the animals to ‘right’ themselves and resume an upright posture was measured using slow motion playback of the videotapes. Just after surgery, the UL animals showed the typical symptoms of postural and ocular imbalance after unilateral labyrinthectomy. The postural symptoms were characterized by a tilt of the head and a twist of the body toward the lesioned side, a flexion of both hind- and forelimbs on the
123
side ipsilateral to the lesion, and an extension of both limbs on the contralateral side. BL rats had a more symmetrical posture, with a reduced muscular tone compared to control animals [8]. After 40– 43 days following the surgery, when the animals’ posture was tested during the 1-g phase of parabolic flight, there was no difference between the normal and lesioned animals (Fig. 1, left panel). During the 0-g phase, normal and BL animals extended their four limbs and occasionally rolled on themselves at a frequency of about 1 Hz. By contrast, when free-floating the UL animals twisted and turned at a velocity that exceeded 20008/s in one animal. Their head was tilted toward the lesioned side (left) and they rolled around their long body axis in the counter-clockwise direction (Fig. 1, middle panel). When the UL animals tenaciously grasped at the cushion on the bottom of the cage in weightlessness, they adopted the typical asymmetrical static posture observed just after labyrinthectomy, with a deflection of the head on the lesioned side, and a full extension of the contralateral fore- and hindlimbs (Fig. 1, right panel). The same posture was also observed when the airplane began its pull-out maneuver. However, a return to the normal (i.e. compensated) posture was observed after 4.5 s on average (Fig. 2C). By comparison, between 2 and 7 h after surgery, during the water immersion test, the lesioned animals spent most of the time rolling, at a frequency ranging from 1.2 to 3.2 Hz (velocity from 430 to 11508/s) (Fig. 2A,B). UL animals always rotated toward the lesioned side, whereas BL animals rotated either to the right or to the left. After a drop, which simulated a transient weightlessness on Earth, the BL animals resumed a prone posture almost immediately, whereas the latency for the surface righting response was about 3.5 s on average for the UL animals (Fig. 2C). During the parabolas, the UL animals rotated for about half of the duration of a parabola on average (Fig. 2B) because they were able to grasp at the cage bottom surface for the other half (whereas there was no surface to grasp
Fig. 1. Posture of a hemilabyrinthectomized animal on the left side, during the 1-g (left) and 0-g (middle and right) phases of parabolic flight. During the 0-g phase, two images are shown to illustrate the posture of the animal when free-floating and when grasping the floor of the cage. During the 1.8-g pull-out phase immediately following the 0-g phase, the animals maintained the same posture as shown in the right panel for about 4–5 s.
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Fig. 2. Comparison of the mean postural responses of normal, UL, and BL animals tested at an acute stage (2–7 h after surgery) during water immersion and after a drop on land, and at a compensated stage (40– 43 days after surgery) during and after the 0-g phase of parabolic flight. Average rotation frequency (A) and duration (B) during water immersion (WI) and during the 0-g phase of parabolic flight (0 g). Rotation duration is expressed as a percentage of the total duration of the test (some water immersion tests had to be stopped before the 20 s mark to rescue the lesioned animals from drowning). (C) Average latency of surface righting response after a free-fall on land (After fall) and after the 0-g phase of parabolic flight (After 0 g). Latencies were obtained by calculating the period between the time when the animals hit the floor and the return to an upright posture. *P , 0:05 relative to normal animals; **P , 0:05 relative to BL animals. Numbers in parentheses indicate the number of animals used in the different conditions.
during water immersion). It is important to note though that they immediately started to roll over when they released their support. By contrast, both the BL and normal animals could spend a considerable time free-floating without rolling. The latency of the surface righting reflex of the UL animals at the end of the 0-g phase was not significantly different from that measured after the early drop tests (Fig. 2C). The frequency of rotation of the UL animals was also comparable in weightlessness and during the early water immersion test (Fig. 2A). When the UL animals were restrained and the head fixed, during parabolic flight at 0 g and in the dark the left eye was deviated by 26.18 ^ 6.68 downward and the right eye was deviated by 26.48 ^ 4.58 upward compared to their position in normal gravity (mean and SD of three rats during 21 parabolas). The direction and amplitude of these eye deviations are comparable to those observed in UL rats at an acute stage [15]. By contrast, the changes in eye deviation in 0 g compared to 1 g never exceeded 38 in BL and normal animals. Any vertebrate exposed to weightlessness shows, at least temporarily, a spatial disorientation. For example, humans exposed to microgravity suffer inversion illusion and motion sickness [13], and humans, monkeys and fishes exhibit changes in reflexive eye movements [3,7,20]. When free-floating there no longer are tactile contact cues to provide information about the orientational down direction. One commonly observed response of animals in weightlessness is to react as though they are upside-down and to initiate a repetitive righting response. Typically the animal rolls over and over, since in weightlessness there is no vestibular confirmation that the action was successful [23].
This roll movement was casually seen in our normal and BL animals, and was directed toward either the right or the left. However, when the animals grasped at surfaces and tactile cues were present, they eventually came to perceive themselves as upright in an upright aircraft and the roll motion stopped. By contrast, the UL animals showed a continuous roll motion in weightlessness, in the direction corresponding to the lesioned vestibular apparatus. Although the parabolic flight took place late after surgery, when normal posture was observed in a 1-g environment, the acute symptoms of unilateral labyrinthectomy were again apparent in weightlessness and continued for 4 – 5 s after the end of the weightless period. The similarity between the response pattern of UL animals during parabolic flight at a compensated stage and on Earth at an acute stage is suggestive of a vestibular decompensation in weightlessness. Recent electrophysiological studies in frogs have shown that after unilateral section of the VIII nerve, the asymmetry that resulted was compensated by a functional reorganization of the somatosensory map in the vestibular recipient structures. This reorganization was manifested by an expansion of contralateral afferent vestibular signals onto the deprived ipsilateral neurons [9]. It led to an increase of excitatory commissural inputs from the intact side and a decrease of inhibitory commissural responses [10]. In very much the same way, following unilateral labyrinthectomy in rats and guinea pigs, an increase in spontaneous discharge and excitability has been observed in the vestibular neurons on the operated side [2,18]. This increase in the resting discharge could be responsible for the consequent recovery of static vestibular function. During the 20 s of weightlessness, the static gravitational force no longer stimulates the otolith organs of the intact ear, thereby weakening temporarily their excitatory inputs on the vestibular neurons on the operated side. In 0 g, their resting discharges would presumably not be strong enough to allow the activation of the vestibular neurons on the lesioned side, hence a temporary return to the acute vestibular deficits. In the absence of a surface support, a return to an imbalanced posture in decompensated animals would generate rotation in the direction of the lesioned, i.e. flexed, side. Indeed, in normal gravity, the postural tone maintains an attitude or posture in relation to the acceleration of gravity. Postural control is determined by the overall balance of muscle forces acting on the head, limbs, and torso. According to Newton’s first and second laws of biomechanics, an asymmetrical posture with an extension of the right limbs and a flexion of the left limbs creates a momentum as soon as the body is released from the surface support. In weightlessness, this momentum generates a rotation to the left (i.e. toward the lesioned side in our animals) which continues unless new forces (such as grasping reaction) impress on it and rebalance the posture. Postural decompensation has been reported for various
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species and experimental conditions. For example, UL animals placed in the dark at a compensated stage exhibit asymmetrical posture, but normal posture is immediately restored when animals are again placed in the light [4]. Postural decompensation also takes place during handling of operated animals [11]. In our study, the fact that the UL animals also present the typical postural asymmetry even when they are in contact with the floor in 0 g (during grasping at the cage surface) suggests that tactile cues are not primarily responsible for the vestibular decompensation. Nevertheless, tactile inputs sensitive to body weight are not stimulated when the animals grasp at a support surface in the weightless condition, and the muscle groups involved during grasping and standing are different (flexor vs. extensor, respectively). It is therefore possible that the vestibular decompensation observed in weightlessness could also be due to the changes in tactile and proprioceptive cues in the free-floating animals. It is interesting to note that the upright posture was restored well before the end of the hypergravity phase (4.5 s compared to 20 s). In addition, no decompensation was observed during the hypergravity phase prior to the 0-g phase. Therefore, it seems that it is not hypergravity per se that caused a decompensation. The decompensation seen during the first few seconds of hypergravity following 0 g presumably is an after-effect of the decompensation triggered by the removal of gravitational information in weightlessness. It is also possible, however, that in our experiment the recovery of a normal posture after the transient vestibular decompensation provoked by weightlessness is even faster because the animals are exposed to hypergravity after the 0-g phase of parabolic flight. Indeed, recent studies have shown that guinea pigs stimulated with 2 g on a centrifuge following unilateral labyrinthectomy showed faster compensation in head deviation than when maintained in normal gravity [12]. Our results support the hypothesis that an imbalance in the otolith system underlies the ocular and postural asymmetry observed after unilateral labyrinthectomy. In the compensated UL rats, weightlessness uncovered an asymmetry in the otolith system, which was previously cancelled on Earth [8]. These results are in agreement with those obtained in perinatal rats gestated during space flight which suggest a direct effect of gravity on the development of the vestibular system due to a reduction of the otolith input [22]. They also suggest that vestibular compensation after unilateral lesion can be disrupted momentarily and is a fragile state during which the otolith system in the remaining vestibular apparatus plays a continuous role. This finding is consistent with the fragility of the vestibular compensation also observed in humans during the early phase of recovery following unilateral vestibular loss [6]. The decompensation is less evident after bilateral lesion, because since there are no longer otolith inputs, vestibular compensation would predominantly be achieved based on proprioceptive, somatosensory and visual cues.
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Acknowledgements This study was supported by grants from the Centre National d’Etudes Spatiales (CNES). We thank G. Bunel for audiovisual assistance, L. Galas and M. Dumarest for animal care, and an anonymous referee for constructive comments.
References [1] G.B. Azzena, Role of the spinal cord in compensating the effects of hemilabyrinthectomy, Arch. Ital. Biol. 107 (1969) 43–53. [2] S.A. Cameron, M.B. Dutia, Cellular basis of vestibular compensation: changes in intrinsic excitability of MVN neurones, NeuroReport 8 (1997) 2595– 2599. [3] G. Cle´ment, Alteration of eye movements and motion perception in microgravity, Brain Res. Rev. 28 (1998) 161 –172. [4] J.H. Courjon, M. Jeannerod, Visual substitution of labyrinthine defects, in: R. Granit, O. Pompeiano (Eds.), Reflex Control of Posture and Movement, Progress in Brain Research, 50, Elsevier, Amsterdam, 1979, pp. 783–792. [5] J.H. Courjon, M. Jeannerod, I. Ossuzio, R. Schmid, The role of vision in compensation of vestibuloocular reflex after hemilabyrinthectomy in the cat, Exp. Brain Res. 28 (1977) 235 –248. [6] I.S. Curthoys, G.M. Halmagyi, Vestibular compensation: a review of the oculomotor, neural, and clinical consequences of unilateral vestibular loss, J. Vestib. Res. 5 (1995) 67–107. [7] M. Dai, T. Raphan, I. Kozlovskaya, B. Cohen, Vestibular adaptation to space in monkeys, Otolaryngol. Head Neck Surg. 119 (1998) 65–77. [8] O. Etard, O. Hardy, A. Reber, G. Quarck, P. Denise, J. Corvisier, Hyper- and hypogravity alter posture in rats compensated on Earth for a vestibular asymmetry, NeuroReport 10 (1999) 669–673. [9] F. Goto, H. Straka, N. Dieringer, Expansion of afferent vestibular signals after the section of one of the vestibular nerve branches, J. Neurophysiol. 84 (2000) 581 –584. [10] F. Goto, H. Straka, N. Dieringer, Postlesional vestibular reorganization in frogs: evidence for a basic reaction pattern after nerve injury, J. Neurophysiol. 85 (2001) 2643–2646. [11] K.F. Hamann, A. Reber, B.J. Hess, N. Dieringer, Long-term deficits in otolith, canal and optokinetic ocular reflexes of pigmented rats after unilateral vestibular nerve section, Exp. Brain Res. 118 (1998) 331–340. [12] T. Ikeda, T. Sekitani, H. Hara, T. Tahara, M. Takahashi, Effect of hypergravity on vestibular compensation in guinea pigs, Acta OtoLaryngol. Suppl. 519 (1995) 173 –175. [13] J. Lackner, P. DiZio, Human orientation and movement control in weightlessness and artificial gravity environments, Exp. Brain Res. 130 (2000) 2 –26. [14] M. Lacour, C. Xerri, Vestibular compensation: new perspectives, in: H. Flohr, W. Precht (Eds.), Lesion-Induced Neuronal Plasticity in Sensorimotor Systems, Springer-Verlag, New York, 1981, pp. 240–253. [15] R. Llinas, K. Walton, Significance of the olivo-cerebellar system in compensation of ocular position following unilateral labyrinthectomy, in: R. Baker, A. Berthoz (Eds.), Control of Gaze by Brainstem Neurons, Elsevier, Amsterdam, 1977, pp. 399 –408. [16] W. Precht, N. Dieringer, Neuronal events paralleling functional recovery (compensation) following peripheral vestibular lesions, in: A. Berthoz, G. Melvill Jones (Eds.), Adaptive Mechanisms in Gaze Control, Elsevier, Amsterdam, 1985, pp. 251–268. [17] P.T.S. Putkonen, J.H. Courjon, M. Jeannerod, Compensation for
126
A. Reber et al. / Neuroscience Letters 344 (2003) 122–126
postural effects of hemilabyrinthectomy in the cat. A sensory substitution process?, Exp. Brain Res. 28 (1977) 249 –257. [18] L. Ris, B. Capron, N. Vibert, P.P. Vidal, E. Godaux, Modification of the pacemaker activity of vestibular neurons in brainstem slices during vestibular compensation in the guinea pig, Eur. J. Neurosci. 13 (2001) 2234–2240. [19] K.P. Schaefer, D.L. Meyer, G. Wihelms, Somatosensory and cerebellar influences on compensation of labyrinthine lesions, in: R. Granit, O. Pompeiano (Eds.), Reflex Control of Posture and Movement, Progress in Brain Research, 50, Elsevier, Amsterdam, 1979, pp. 591–598.
[20] C. Sebastian, K. Essling, E. Horn, Altered gravitational forces affect the development of the static vestibuloocular reflex in fish (Oreochromis mossmbicus), J. Neurobiol. 46 (2001) 59–72. [21] P.F. Smith, I.S. Curthoys, Mechanisms of recovery following unilateral labyrinthectomy: a review, Brain Res. Rev. 14 (1989) 155–180. [22] K. Walton, Postnatal development under conditions of simulated weightlessness and space flight, Brain Res. Rev. 28 (1998) 25–34. [23] R. Wassersug, Vertebrate biology in microgravity, Am. Sci. 89 (2001) 46– 53.