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Calbindin and parvalbumin are early markers of non-mitotically regenerating hair cells in the bullfrog vestibular otolith organs

Earlier studies have demonstrated hair cell regeneration in the absence of cell proliferation, and suggested that supporting cells could phenotypically convert into hair cells following hair cell loss. Because calcium-binding proteins are involved in gene up-regulation, cell growth, and cell differentiation, we wished to determine if these proteins were up-regulated in scar formations and regenerating hair cells following gentamicin treatment. Calbindin and parvalbumin immunolabeling was examined in control or gentamicin-treated (GT) bullfrog saccular and utricular explants cultured for 3 days in amphibian culture medium or amphibian culture medium supplemented with aphidicolin, a blocker of nuclear DNA replication in eukaryotic cells. In control cultures, calbindin and parvalbumin immunolabeled the hair bundles and, less intensely, the cell bodies of mature hair cells. In GT or mitotically-blocked GT (MBGT) cultures, calbindin and parvalbumin immunolabeling was also seen in the hair bundles, cuticular plates, and cell bodies of hair cells with immature hair bundles. Thus, these antigens were useful markers for both normal and regenerating hair cells. Supporting cell immunolabeling was not seen in control cultures nor in the majority of supporting cells in GT cultures. In MBGT cultures, calbindin and parvalbumin immunolabeling was up-regulated in the cytosol of single supporting cells participating in scar formations and in supporting cells with hair cell-like characteristics. These data provide further evidence that non-mitotic hair cell regeneration in cultures can be accomplished by the conversion of supporting cells into hair cells.

Non-NASA Center

The dynamic contributions of the otolith organs to human ocular torsion

We measured human ocular torsion (OT) monocularly (using video) and binocularly (using search coils) while sinusoidally accelerating (0.7 g) five human subjects along an earth-horizontal axis at five frequencies (0.35, 0.4, 0.5, 0.75, and 1.0 Hz). The compensatory nature of OT was investigated by changing the relative orientation of the dynamic (linear acceleration) and static (gravitational) cues. Four subject orientations were investigated: (1) Y-upright-acceleration along the interaural (y) axis while upright; (2) Y-supine-acceleration along the y-axis while supine; (3) Z-RED-acceleration along the dorsoventral (z) axis with right ear down; (4) Z-supine-acceleration along the z-axis while supine. Linear acceleration in the Y-upright, Y-supine and Z-RED orientations elicited conjugate OT. The smaller response in the Z-supine orientation appeared disconjugate. The amplitude of the response decreased and the phase lag increased with increasing frequency for each orientation. This frequency dependence does not match the frequency response of the regular or irregular afferent otolith neurons; therefore the response dynamics cannot be explained by simple peripheral mechanisms. The Y-upright responses were larger than the Y-supine responses (P < 0.05). This difference indicates that OT must be more complicated than a simple low-pass filtered response to interaural shear force, since the dynamic shear force along the interaural axis was identical in these two orientations. The Y-supine responses were, in turn, larger than the Z-RED responses (P < 0.01). Interestingly, the vector sum of the Y-supine responses plus Z-RED responses was not significantly different (P = 0.99) from the Y-upright responses. This suggests that, in this frequency range, the conjugate OT response during Y-upright stimulation might be composed of two components: (1) a response to shear force along the y-axis (as in Y-supine stimulation), and (2) a response to roll tilt of gravitoinertial force (as in Z-RED stimulation).

NASA Discipline Neuroscience

Influence of vestibular activation on respiration in humans

The purpose of this study was to determine the effects of the semicircular canals and otolith organs on respiration in humans. On the basis of animal studies, we hypothesized that vestibular activation would elicit a vestibulorespiratory reflex. To test this hypothesis, respiratory measures, arterial blood pressure, and heart rate were measured during engagement of semicircular canals and/or otolith organs. Dynamic upright pitch and roll (15 cycles/min), which activate the otolith organs and semicircular canals, increased respiratory rate (Delta2 +/- 1 and Delta3 +/- 1 breaths/min, respectively; P < 0.05). Dynamic yaw and lateral pitch (15 cycles/min), which activate the semicircular canals, increased respiration similarly (Delta3 +/- 1 and Delta2 +/- 1, respectively; P < 0.05). Dynamic chair rotation (15 cycles/min), which mimics dynamic yaw but eliminates neck muscle afferent, increased respiration (Delta3 +/- 1; P < 0.05) comparable to dynamic yaw (15 cycles/min). Increases in respiratory rate were graded as greater responses occurred during upright (Delta5 +/- 2 breaths/min) and lateral pitch (Delta4 +/- 1) and roll (Delta5 +/- 1) performed at 30 cycles/min. Increases in breathing frequency resulted in increases in minute ventilation during most interventions. Static head-down rotation, which activates otolith organs, did not alter respiratory rate (Delta1 +/- 1 breaths/min). Collectively, these data indicate that semicircular canals, but not otolith organs or neck muscle afferents, mediate increased ventilation in humans and support the concept that vestibular activation alters respiration in humans.

NASA Discipline Neuroscience

The Influence of Gravito-Inertial Force on Sensorimotor Integration and Reflexive Responses

Sensorimotor responses (e.g.. eye movements, spinal reflexes, etc depend upon the interpretation of the neural signals from the sensory systems. Since neural signals from the otoliths may represent either tilt (gravity) or translation (linear inertial force), sensory signals from the otolith organs are necessarily somewhat ambiguous. Therefore. the neural responses to changing otolith signals depend upon the context of the stimulation (e.g- active vs. passive, relative orientation of gravity, etc.) as well as upon other sensory signals (e.g., vision. canals, etc.). This session will focus upon the -role -played by the sensory signals from the otolith organs in producing efficient sensorimotor and behavioral responses. Curthoys will show the influence of the peripheral anatomy and physiology. Tomko will discuss the influence of tilt and translational otolith signals on eye movements. Merfeld will demonstrate the rate otolith organs play during the interaction of sensory signals from the canals and otoliths. Watt will show the influence of the otoliths on spinal/postural responses. Guedry will discuss the contribution of vestibular information to "path of movement"' perception and to the development of a stable vertical reference. Sensorimotor responses to the ambiguous inertial force stimulation provide an important tool to investigate how the nervous system processes patterns of sensory information and yields functional sensorimotor responses.

Curthoys, Ian S.

Neurophysiological study on visuo-vestibular control of posture and movement in fish during adaptation to weightlessness

We can stand upright and walk smoothly without paying any particular attention to it. This is because we have established in ourselves an integration center that controls our body subconsciously in response to input from eyes, muscles, joints, foot soles, and also from the gravity sensor in the inner ear (the otolith organ). It has been shown that the cerebellum plays an important role for the establishment of the integration center and that the control pattern is comparable to that of a highly sophisticated computer system. The programming for the control, however, may well be acquired for the 1-g ground condition and does not cover the 0-g in space. Although each of the above organs function as it does on the ground, the signal pattern sent to the center must be different under 0-g and, in addition, complementary signals from the otolith organ are missing, leading to confusion in the integration center and causing a variety of symptoms similar to those of car-sickness or sea-sickness. After exposure to microgravity an immediate process of re-programming will begin and be completed in 2-4 days. There is strong supporting evidence for this sensory conflict theory as an explanation for space motion sickness (SMS) episodes. Fish were selected as test organisms for this investigation because they swim around freely in three dimensions and have well-developed organs for vision and gravity detection. They also have an innate nature to orient their back toward a light source. Actually, on the ground, the fish tilts its vertical axis toward the light when illuminated laterally, and the tilt angle is a function of the intensity of light and the magnitude of gravity, while its posture is completely light-dependent in the low-gravity environment produced by aircraft parabolic flight or when the otolith organs are removed. This implies that fish posture is entirely under visual and otolithic control. In this case, the cerebellum will also contribute to the control. In the space shuttle experiment, two fish (one with otolith-removed and the other with intact otoliths) are onboard for 7 days. The arrangement of the experiment is shown. Lamps illuminate each fish alternately from different directions at a duration of 20 seconds for 10 minutes totally, twice a day. The video images and the brain waves from the cerebellum are analyzed later. If the sensory conflict theory is acceptable, then the confusion at the onset and the following recovery process will be manifested both in the light-dependent behavior and also in the cerebellar activity. In addition, there should be some variation in response between the two fish. Even if the results are positive in the present fish experiment, they would not be extensive enough to allow us to understand the complete mechanisms of SMS. However, what is currently needed is a collection of evidence based on animal experiments on which to base future investigations. Countermeasures for SMS are mandatory since SMS-induced vomiting occurring within a space suit could be fatal for a crew member during extravehicular activities. We expect that countermeasures will be developed before the space station era starts, and that the results of these studies will be applied to improving human welfare.

Mori, Shigeo

M.I.T./Canadian vestibular experiments on the Spacelab-1 mission. III - Effects of prolonged weightlessness on a human otolith-spinal reflex

Reflex responses that depend on human otolith organ sensitivity were measured before, during and after a 10 day space flight. Otolith-spinal reflexes were elicited by means of sudden, unexpected falls. In weightlessness, 'falls' were achieved using elastic cords running from a torso harness to the floor. Electromyographic (EMG) activity was recorded from gastrocnemius-soleus. The EMG response occurring in the first 100-120 ms of a fall, considered to be predominantly otolith-spinal in origin, decreased in amplitude immediately upon entering weightlessness, and continued to decline throughout the flight, especially during the first two mission days. The response returned to normal before the first post-flight testing session. The results suggest that information coming from the otolith organs is gradually ignored by the nervous system during prolonged space flight, although the possibility that otolith-spinal reflexes are decreased independent of other otolith output pathways cannot by ruled out.

Watt, D. G. D.

M.I.T./Canadian vestibular experiments on the Spacelab-1 mission: 3. Effects of prolonged weightlessness on a human otolith-spinal reflex

Reflex responses that depend on human otolith organ sensitivity were measured before, during and after a 10 day space flight. Otolith-spinal reflexes were elicited by means of sudden, unexpected falls. In weightlessness, "falls" were achieved using elastic cords running from a torso harness to the floor. Electromyographic (EMG) activity was recorded from gastrocnemius-soleus. The EMG response occurring in the first 100-120 ms of a fall, considered to be predominantly otolith-spinal in origin, decreased in amplitude immediately upon entering weightlessness, and continued to decline throughout the flight, especially during the first two mission days. The response returned to normal before the first post-flight testing session. The results suggest that information coming from the otolith organs is gradually ignored by the nervous system during prolonged space flight, although the possibility that otolith-spinal reflexes are decreased independent of other otolith output pathways cannot be ruled out.

NASA Discipline Neuroscience

Neural readaptation to Earth's gravity following return from space

The consequence of exposure to microgravity on the otolith organs was studied by recording the responses of vestibular nerve afferents supplying the utricular otolith organ to inertial accelerations in four toadfish, Opsanus tau, sequentially for 5 days following two National Aeronautics and Space Administration shuttle orbital flights. Within the first day postflight, the magnitude of response to an applied translation was on average three times greater than for controls. The reduced gravitational acceleration in orbit apparently resulted in an upregulation of the sensitivity of utricular afferents. By 30 h postflight, responses were statistically similar to control. The time course of return to normal afferent sensitivity parallels the reported decrease in vestibular disorientation in astronauts following return from space.

manned

Visual field influence on manual roll and pitch stabilization

Human control performance in nulling perceived tilt angles was investigated for combinations of pseudo-random vestibular disturbances and different waveforms of low frequency wide visual field motions. For both roll and pitch axes, subjects tilted the trainer in which they were seated in the direction of field rotation. This visual bias was much stronger for pitch backwards with upward field rotation. Frequency response analysis showed the dominance of visual cues at low frequencies (below 0.06 Hz) and the reliance on vestibular information in the high frequency range for both axes. Models suggest that operator balancing responses at high frequencies are mainly processed by the semicircular canals rather than the otolith organs. The results also suggest that the subject tends to rely less on the otolith organs for pitch perception than for roll.

Huang, J.-K.

Post-spaceflight orthostatic intolerance: possible relationship to microgravity-induced plasticity in the vestibular system

Even after short spaceflights, most astronauts experience at least some postflight reduction of orthostatic tolerance; this problem is severe in some subjects. The mechanisms leading to postflight orthostatic intolerance are not well-established, but have traditionally been thought to include the following: changes in leg hemodynamics, alterations in baroreceptor reflex gain, decreases in exercise tolerance and aerobic fitness, hypovolemia, and altered sensitivity of beta-adrenergic receptors in the periphery. Recent studies have demonstrated that signals from vestibular otolith organs play an important role in regulating blood pressure during changes in posture in a 1-g environment. Because spaceflight results in plastic changes in the vestibular otolith organs and in the processing of inputs from otolith receptors, it is possible that another contributing factor to postflight orthostatic hypotension is alterations in the gain of vestibular influences on cardiovascular control. Preliminary data support this hypothesis, although controlled studies will be required to determine the relationship between changes in the vestibular system and orthostatic hypotension following exposure to microgravity. Copyright 1998 Elsevier Science B.V.

NASA Discipline Neuroscience

Vestibular control of sympathetic activity. An otolith-sympathetic reflex in humans

It has been proposed that a vestibular reflex originating in the otolith organs and other body graviceptors modulates sympathetic activity during changes in posture with regard to gravity. To test this hypothesis, we selectively stimulated otolith and body graviceptors sinusoidally along different head axes in the coronal plane with off-vertical axis rotation (OVAR) and recorded sympathetic efferent activity in the peroneal nerve (muscle sympathetic nerve activity, MSNA), blood pressure, heart rate, and respiratory rate. All parameters were entrained during OVAR at the frequency of rotation, with MSNA increasing in nose-up positions during forward linear acceleration and decreasing when nose-down. MSNA was correlated closely with blood pressure when subjects were within +/-90 degrees of nose-down positions with a delay of 1.4 s, the normal latency of baroreflex-driven changes in MSNA. Thus, in the nose-down position, MSNA was probably driven by baroreflex afferents. In contrast, when subjects were within +/-45 degrees of the nose-up position, i.e., when positive linear acceleration was maximal along the naso-ocipital axis, MSNA was closely related to gravitational acceleration at a latency of 0.4 s. This delay is too short for MSNA changes to be mediated by the baroreflex, but it is compatible with the delay of a response originating in the vestibular system. We postulate that a vestibulosympathetic reflex, probably originating mainly in the otolith organs, contributes to blood pressure maintenance during forward linear acceleration. Because of its short latency, this reflex may be one of the earliest mechanisms to sustain blood pressure upon standing.

NASA Discipline Neuroscience

FAR and NEAR Target Dynamic Visual Acuity: A Functional Assessment of Canal and Otolith Performance

Upon their return to earth, astronauts experience the effects of vestibular adaptation to microgravity. The postflight changes in vestibular information processing can affect postural and locomotor stability and may lead to oscillopsia during activities of daily living. However, it is likely that time spent in microgravity affects canal and otolith function differently. As a result, the isolated rotational stimuli used in traditional tests of canal function may fail to identify vestibular deficits after spaceflight. Also, the functional consequences of deficits that are identified often remain unknown. In a gaze control task, the relative contributions of the canal and otolith organs are modulated with viewing distance. The ability to stabilize gaze during a perturbation, on visual targets placed at different distances from the head may therefore provide independent insight into the function of this systems. Our goal was to develop a functional measure of gaze control that can also offer independent information about the function of the canal and otolith organs.

Peters, Brian T.

Otolith-Canal Convergence In Vestibular Nuclei Neurons

The current final report covers the period from June 1, 1999 to May 31, 2002. The primary objective of the investigation was to determine how information regarding head movements and head position relative to gravity is received and processed by central vestibular nuclei neurons in the brainstem. Specialized receptors in the vestibular labyrinths of the inner ear function to detect angular and linear accelerations of the head, with receptors located in the semicircular canals transducing rotational head movements and receptors located in the otolith organs transducing changes in head position relative to gravity or linear accelerations of the head. The information from these different receptors is then transmitted to central vestibular nuclei neurons which process the input signals, then project the appropriate output information to the eye, head, and body musculature motor neurons to control compensatory reflexes. Although a number of studies have reported on the responsiveness of vestibular nuclei neurons, it has not yet been possible to determine precisely how these cells combine the information from the different angular and linear acceleration receptors into a correct neural output signal. In the present project, rotational and linear motion stimuli were separately delivered while recording responses from vestibular nuclei neurons that were characterized according to direct input from the labyrinth and eye movement sensitivity. Responses from neurons receiving convergent input from the semicircular canals and otolith organs were quantified and compared to non-convergent neurons.

Dickman, J. David

Neurophysiology Summary

The terrestrial gravitational field serves as an important orientation reference for human perception and movement, being continually monitored by sensory receptors in the skin, muscles, joints, and vestibular otolith organs. Cues from these graviceptors are used by the brain to estimate spatial orientation and to control balance and movement. Changes in these cues associated with the tonic changes in gravity (gravito-inertial force),during the launch and entry phases of space flight missions result in altered perceptions, degraded motor control performance, and in some cases, "motion" sickness during, and for a period of time after, the g-transitions. In response to these transitions, however, physiological and behavioral response mechanisms are triggered to compensate for altered graviceptor cues and/or to adapt to the new sensory environment. Basic research in the neurophysiology discipline is focused on understanding the characteristic features of and the underlying mechanisms for the normal human response to tonic changes in the gravito-inertial force environment. These studies address fundamental questions regarding the role of graviceptors in orientation and movement in the terrestrial environment, as well as the capacity, specificity, and modes for neural plasticity in the sensory-motor and perceptual systems of the brain. At the 2001 workshop basic research studies were presented addressing: neuroanatomical responses to altered gravity environments, the neural mechanisms for resolving the ambiguity between tilting and translational stimuli in otolith organ sensory input, interactions between the vestibular system and the autonomic nervous system , the roles of haptic and visual cues in spatial orientation, mechanisms for training environment-appropriate sensorimotor responses triggered by environment-specific context cues, and studies of sensori-motor control of posture and locomotion in the terrestrial environment with and without recent exposure to space flight. Building on these basic research studies are more applied studies focused on the development of countermeasures to the untoward neurophysiological responses to space flight. At the 2001 workshop, applied research studies were presented addressing issues related to the use of rotational artificial gravity (centripetal acceleration) as a multisystem (bone, muscle, cardiovascular, and, perhaps, neurovestibular) countermeasure. Also presented was a clinical study reporting on a new rating system for clinical evaluation of postflight functional neurological status.

Paloski, William H.

The Effect of Spaceflight on the Ultrastructure of the Cerebellum

In weightlessness, astronauts and cosmonauts may experience postural illusions as well as motion sickness symptoms known as the space adaptation syndrome. Upon return to Earth, they have irregularities in posture and balance. The adaptation to microgravity and subsequent re-adaptation to Earth occurs over several days. At the cellular level, a process called neuronal plasticity may mediate this adaptation. The term plasticity refers to the flexibility and modifiability in the architecture and functions of the nervous system. In fact, plastic changes are thought to underlie not just behavioral adaptation, but also the more generalized phenomena of learning and memory. The goal of this experiment was to identify some of the structural alterations that occur in the rat brain during the sensory and motor adaptation to microgravity. One brain region where plasticity has been studied extensively is the cerebellar cortex-a structure thought to be critical for motor control, coordination, the timing of movements, and, most relevant to the present experiment, motor learning. Also, there are direct as well as indirect connections between projections from the gravity-sensing otolith organs and several subregions of the cerebellum. We tested the hypothesis that alterations in the ultrastructural (the structure within the cell) architecture of rat cerebellar cortex occur during the early period of adaptation to microgravity, as the cerebellum adapts to the absence of the usual gravitational inputs. The results show ultrastructural evidence for neuronal plasticity in the central nervous system of adult rats after 24 hours of spaceflight. Qualitative studies conducted on tissue from the cerebellar cortex (specifically, the nodulus of the cerebellum) indicate that ultrastructural signs of plasticity are present in the cerebellar zones that receive input from the gravity-sensing organs in the inner ear (the otoliths). These changes are not observed in this region in cagematched ground control animals. The specific changes include the formation of lamellar bodies, profoundly enlarged Purkinje cell mitochondria, the presence of inter-neuronal cellular protrusions in the molecular layer, and signs of degeneration in the distal dendrites of the Purkinje cells. Since these morphologic signs are not apparent in the control animals, they are not likely to be due to caging or tissue processing effects. The particular nature of the structural alterations in the nodulus, most notably the formation of lamellar bodies and the presence of degeneration, further suggests that excitotoxicity (damaging overstimulation of neurons) may play a role in the short-term neural response to spaceflight. These findings suggest a structural basis for the neuronal and synaptic plasticity accompanying the central nervous system response to altered gravity and help identify the cellular bases underlying the vestibular abnormalities experienced by astronauts during periods of adaptation and re-adaptation to different gravitational forces. Also, since the short- and long-term changes in neural structure occurring during such periods of adaptation resemble the neuronal alterations that occur in some neurologic disorders such as stroke, these findings may offer guidance in the development of strategies for rehabilitation and treatment of such disorders.

Holstein, Gay R.