The otolith organs, the utricle and saccule, play a role in a number of clinical vestibular disorders and have been hypothesized to play a role in some of the vestibular dysfunction experienced by astronauts during spaceflight and after re-entry. The mammalian saccule, which senses gravity, accelerations, and loud acoustic stimuli, is one of the least studied organs of the inner ear. Although there are morphological changes in the saccular epithelium (to both type I and type II hair cells) as a result of spaceflight, little is understood about how these changes might affect saccular physiology. Improving our understanding of the saccule and its frequency characteristics is of particular interest because measurements of saccular sensitivity in response to loud sounds are starting to be used in the clinic. The goal of this project is to improve our understanding of the mammalian saccule by focusing on the frequency characteristics of the saccular epithelium. The original project had two aims; the first aim was to characterize the frequency tuning of saccular reflexes in response to tones using both vestibular evoked myogenic potentials, and vestibulo-ocular reflexes in adult rats, and the second aim was to evaluate the frequency characteristics of saccular hair cells by receptor potentials and transduction currents from the central (striolar) and peripheral (extrastriolar) zones of the saccular epithelium. Based on preliminary results from both projects, we decided to focus our efforts on aim 2. We hypothesize that within the saccular epithelium hair cell frequency sensitivity will vary with hair cell type and zone, with some cells sensitive to higher frequency stimuli, including acoustic frequencies and others sensitive to lower frequencies, including frequencies of voluntary head motions. To test this hypothesis we compare voltage-gated currents, receptor potentials and transduction currents from type I and type II hair cells from both striolar and extrastriolar zones of the saccular epithelium using whole-cell patch recording methods in the semi-intact rat saccule.

Preliminary results comparing striolar type I hair cells and extrastriolar type II hair cells suggest differences. Extrastriolar type II hair cells have a midpoint voltage for sodium channel inactivation (-74±2mV, n=6) that is significantly more positive for type II extrastriolar hair cells than striolar type I hair cells (-93±1mV, n=19). Additionally, striolar type I hair cells have a faster fast time constant of adaptation (7.8±2ms, n=7) in response to step bundle deflections when fit with a double exponential than the extrastriolar type II hair cells (15±3ms, n=5), a smaller operating range (1.1±0.2µm, n=8 compared to 1.7±0.1µm, n=5) and a higher upper cutoff frequency (60±10Hz, n=9 compared to 29±3Hz, n=4) when stimulated with sinusoidal bundle deflections from 2Hz to 100Hz and fit with a double exponential. These preliminary data are consistent with our hypothesis that frequency filtering may differ between hair cell types and zones, especially when looking at the fast component of adaptation. Evaluating responses to higher frequencies (up to 500 Hz) and additional cell types and zones we will be able to more fully characterize these differences.

Characterization of the frequency tuning of hair cells in the saccular epithelium will give us insight into what drives vestibular afferent firing patterns, improve our understanding of peripheral contributions to vestibular reflexes, may improve and expand our understanding of the frequency characteristics of VEMPs, and may provide insight into how morphological changes in the saccular epithelium resulting from spaceflight contribute to the vestibular dysfunction experienced by astronauts.

Two years ago, each project was at an early stage of development, such that significant start-up time was required. However, my involvement in both projects from such an early stage provided invaluable training and experience in diverse skills, ranging from eye-coil surgeries in squirrel monkeys to the micro-dissection of postnatal rat saccules, and from the development of a unique sound system for delivery of sinusoidal tone bursts (50 Hz – 4 kHz, 60-125 dB SPL) to awake squirrel monkeys to the development of micromechanical stimulators to deflect saccular hair bundles with sinusoidal stimuli (2 Hz – 500 Hz, 0.5 – 2.0 µm) and step stimuli. Much of the first year of the project was devoted to setting up and debugging these two experiments. By year two, data collection and analysis had begun in earnest on both projects. Our preliminary sound-evoked VOR data from the squirrel monkey suggested that variations in eye position at the onset of the tone, and other variations in the baseline noise, would obscure any sound-evoked potentials, leading us to conclude that experiments might better be conducted on subjects that had larger eye movements and which could be trained to fixate on a target during measurements. On the other hand, the project on saccular hair cells was starting to work and we were recording large transduction currents in response to our sinusoidal stimuli. We settled on evaluating contributions of saccular hair cell properties to acoustic and vestibular tuning by comparing frequency dependence of transduction currents and receptor potentials across hair cell types and locations, and by investigating the sources of underlying tuning in properties of transduction and voltage-gated ion channels.

This narrowing of focus accelerated progress. My preliminary data now suggest that frequency filtering by hair cells varies as a function of hair cell type and zone in a manner consistent with our underlying hypothesis. These data are being presented in a talk and a poster at the 2010 meeting of the Association for Research in Otolaryngology.

33rd Annual Midwinter Research Meeting, Abstract Book, February 2010. , Feb-2010

2010 NASA Human Research Program Investigators' Workshop Program, February 2010. , Feb-2010

33rd Annual Midwinter Research Meeting, Association for Research in Otolaryngology, February 2010. , Feb-2010

During spaceflight and after re-entry, astronauts experience a number of symptoms associated with vestibular dysfunction including the disruption of balance, locomotion, eye-head coordination and space motion sickness. These symptoms may result from changes in the otolith organs. Our long-term goal is to develop and refine sound-evoked diagnostic tests associated with otolith function, which will lead to better ways to evaluate vestibular function in the clinic, in-flight and during re-adaptation. More specifically, the goal of this project is to link mammalian saccular tuning to the frequency tuning of sound-evoked vestibular reflexes. This research originally had two aims.

The first aim is to characterize the frequency tuning of saccular reflexes in response to short duration tones using measurements of both vestibular evoked myogenic potentials (VEMPs) and vestibulo-ocular reflexes (VORs) in rat as well as the responses to long duration tones. In the first year we have worked to establish our animal model of sound-evoked VOR responses. We successfully adapted the surgical techniques previously used in the lab for instrumenting guinea pigs to rats and did preliminary tests of sound-evoked VORs in guinea pigs. Preliminary finding in the guinea pig were inconclusive and we opted to carry out the final measurements in squirrel monkeys (because squirrel monkeys are foveate animals with larger eye movements) instead of rats. These measurements have required the design of a new sound source with specialized fittings to reproducibly introduce sound (with minimal artifact) to the monkey sitting within the eyecoil frame. These tests are currently underway and will continue into the coming year.

The second aim is to use whole cell patch clamp recordings to compare hair cell receptor potentials in the cental (striolar) and peripheral (extrastriolar) zones of the saccular epithelium. In the first year I have learned how to do basic whole-cell voltage clamp measurements and have begun to record receptor potentials and transduction currents from both type I and type II hair cells in the saccular striolar and extrastriolar zones. Preliminary results suggest that the tuning in the different cells vary: some cells exhibit transduction currents that appear to be highpass up to frequencies in the hundreds of Hertz and others that appear to be tuned to more specific frequencies (at tens of Hertz). Despite these promising initial findings, more work needs to be done to improve the methods of hair cell stimulation and to collect sufficient data to see if these differences vary between cell types and zones. Over the course of the upcoming year, we will continue to improve the stability of the measurement apparatus and will collect sufficient data to characterize differences between cell types and zones.

Supplementary to the second aim, I am evaluating the developmental morphology of the saccular epithelium to better assess the location of the striolar and extrastriolar zones. To determine the location of the zones we are evaluating the line of polarity reversal (easily observable both in the fixed tissue used here as well as the live tissue used for the physiological studies) and its location relative to the striola, defined morphological as the region with calretinin positive complex calyces, and observable using confocal microscopy. Over the course of the last year I have been trained in the use of the confocal microscope and have learned about immunohistochemisty. Preliminary results suggest that the striola of the rat saccule is located medial to the line of polarity reversal, which is consistent with previous finding in the rat utricle. In the coming year efforts will continue in characterizing the epithelium at the developmental ages for which I am collecting physiological data.

This work is the first step in linking saccular tuning to the frequency tuning of sound-evoked vestibular reflexes. Not only will this help us answer basic science questions pertaining to specializations of the saccule, it may allow us to improve existing clinical measures of saccular function and explore new measures of saccular function.

Aim 2: Frequency characteristics of the saccule: hair cells. We have developed a semi-intact preparation of the rat saccular epithelium and have begun measuring transduction currents and receptor potentials from saccular hair cells both within the striolar (central) and extrastriolar (peripheral) zones of the epithelium. Significant improvements have been made in the stimulus used for the assessment of the frequency characteristics of the cells so that a wider range of frequencies can be assessed in a shorter period of time. Additionally, improvements in tissue maintenance and health have resulted in larger measurable currents in response to stimulation. Preliminary data has been collected suggesting differences in the tuning of saccular hair cells. These results show that some cells appeared to be tuned to specific frequencies and others appear to have highpass characteristics up to hundreds of Hz. Additional recordings are still necessary to fully characterize these responses and determine if they vary by cell type and region.

During spaceflight and after re-entry, astronauts experience a number of symptoms associated with vestibular dysfunction including the disruption of balance, locomotion, eye-head coordination and space motion sickness. These symptoms may result from changes in the otolith organs. Our long-term goal is to develop and refine sound-evoked diagnostic tests associated with otolith function, which will lead to better ways to evaluate vestibular function in the clinic, inflight and during re-adaptation. More specifically, we will evaluate the saccule, which is sensitive to loud sounds both in normal and pathological conditions and undergoes morphological changes as a result of microgravity. In the clinic, loud sounds are used to stimulate the saccule, triggering a sound-evoked vestibular reflex that is measured from the muscles of the neck as a vestibular-evoked myogenic potential (VEMP). Loud sounds can also evoke eye movements in normal animals, probably through vestibulo-ocular reflex (VOR) pathways. In this work, we will evaluate the frequency tuning (f <4kHz) of the saccule both in vitro and in vivo in an animal model. This will enable us to develop a better understanding of the basic science underlying saccular contributions to vestibular reflexes such as the VEMP and VOR and help us evaluate potential diagnostic tests of the saccule.

The development of sound-evoked VOR measurements in particular may be relevant for flight diagnostics as it would require minimal equipment and would be a fast and reliable measure of vestibular function.

Specific Aims

1. We will characterize the frequency tuning of saccular reflexes in response to short-duration tones (100-200ms) using measurements of both VEMPs and VORs in rats. We hypothesize that the VEMP and VOR will have similar thresholds and frequency characteristics in response to these stimuli. We will then measure the VOR in response to long-duration tones, which we hypothesize will have lower thresholds than the other measurements.

2. We will use whole-cell patch clamp recordings to compare hair cell receptor potentials in the striolar and extra-striolar zones of the rat saccule. Based on morphological and physiological data, we hypothesize that the striolar zone of the saccular epithelium is specialized to respond to high-frequency stimuli. Finally, based on the saccular origin of the VEMP, we hypothesize that the in vivo and in vitro frequency tuning will share overlapping characteristics.

This work is the first step in linking saccular tuning to the frequency tuning of sound-evoked vestibular reflexes. Not only will this help us answer basic science questions pertaining to specializations of the saccule, it may allow us to improve clinical measures of saccular function (the VEMP) and may lead to new clinical measures, such as the VOR in response to long-duration tones, which can be used both inflight and during re-adaptation to assess vestibular function.