Function of bidirectional sensitivity in the otolith organs established by transcription factor Emx2.

Otolith organs of the inner ear are innervated by two parallel afferent projections to the brainstem and cerebellum. These innervations were proposed to segregate across the line of polarity reversal (LPR) within each otolith organ, which divides the organ into two regions of hair cells (HC) with opposite stereociliary orientation. The relationship and functional significance of these anatomical features are not known. Here, we show regional expression of Emx2 in otolith organs, which establishes LPR, mediates the neuronal segregation across LPR and constitutes the bidirectional sensitivity function. Conditional knockout (cKO) of Emx2 in HCs lacks LPR. Tmie cKO, in which mechanotransduction was abolished selectively in HCs within the Emx2 expression domain also lacks bidirectional sensitivity. Analyses of both mutants indicate that LPR is specifically required for mice to swim comfortably and to traverse a balance beam efficiently, but LPR is not required for mice to stay on a rotating rod.

Repair of surviving hair cells in the damaged mouse utricle.

Sensory hair cells (HCs) in the utricle are mechanoreceptors required to detect linear acceleration. After damage, the mammalian utricle partially restores the HC population and organ function, although regenerated HCs are primarily type II and immature. Whether native, surviving HCs can repair and contribute to this recovery is unclear. Here, we generated the Pou4f3DTR/+; Atoh1CreERTM/+; Rosa26RtdTomato/+ mouse to fate map HCs prior to ablation. After HC ablation, vestibular evoked potentials were abolished in all animals, with ∼57% later recovering responses. Relative to nonrecovery mice, recovery animals harbored more Atoh1-tdTomato+ surviving HCs. In both groups, surviving HCs displayed markers of both type I and type II subtypes and afferent synapses, despite distorted lamination and morphology. Surviving type II HCs remained innervated in both groups, whereas surviving type I HCs first lacked and later regained calyces in the recovery, but not the nonrecovery, group. Finally, surviving HCs initially displayed immature and subsequently mature-appearing bundles in the recovery group. These results demonstrate that surviving HCs are capable of self-repair and may contribute to the recovery of vestibular function.

Canonical Wnt Signaling Pathway on Polarity Formation of Utricle Hair Cells.

As part of the inner ear, the vestibular system is responsible for sense of balance, which consists of three semicircular canals, the utricle, and the saccule. Increasing evidence has indicated that the noncanonical Wnt/PCP signaling pathway plays a significant role in the development of the polarity of the inner ear. However, the role of canonical Wnt signaling in the polarity of the vestibule is still not completely clear. In this study, we found that canonical Wnt pathway-related genes are expressed in the early stage of development of the utricle and change dynamically. We conditionally knocked out ß-catenin, a canonical Wnt signaling core protein, and found that the cilia orientation of hair cells was disordered with reduced number of hair cells in the utricle. Moreover, regulating the canonical Wnt pathway (Licl and IWP2) in vitro also affected hair cell polarity and indicated that Axin2 may be important in this process. In conclusion, our results not only confirm that the regulation of canonical Wnt signaling affects the number of hair cells in the utricle but also provide evidence for its role in polarity development.

Utricular Sensitivity during Hydrodynamic Displacements of the Macula.

To explore the effects of cochlear hair cell displacement, researchers have previously monitored functional and mechanical responses during low-frequency (LF) acoustic stimulation of the cochlea. The induced changes are believed to result from modulation of the conductance of mechano-electrical transduction (MET) channels on cochlear hair cells, along with receptor potential modulation. It is less clear how, or if, vestibular hair cell displacement affects vestibular function. Here, we have used LF (<20 Hz) hydrodynamic modulation of the utricular macula position, whilst recording functional and mechanical responses, to investigate the effects of utricular macula displacement. Measured responses included the Utricular Microphonic (UM), the vestibular short-latency evoked potential (VsEP), and laser Doppler vibrometry recordings of macular position. Over 1 cycle of the LF bias, the UM amplitude and waveform were cyclically modulated, with Boltzmann analysis suggesting a cyclic modulation of the vestibular MET gating. The VsEP amplitude was cyclically modulated throughout the LF bias, demonstrating a relative increase (~20-50 %; re baseline) and decrease (~10-20 %; re baseline), which is believed to be related to the MET conductance and vestibular hair cell sensitivity. The relationship between macular displacement and changes in UM and VsEP responses was consistent within and across animals. These results suggest that the sensory structures underlying the VsEP, often thought to be a cranial jerk-sensitive response, are at least partially sensitive to LF (and possibly static) pressures or motion. Furthermore, these results highlight the possibility that some of the vestibular dysfunction related to endolymphatic hydrops may be due to altered vestibular transduction following mechanical (or morphological) changes in the labyrinth.

Evaluation of the utricular function with the virtual-subject visual vertical system: comparison with ocular vestibular-evoked myogenic potentials.

Introduction: The subjective visual vertical (SVV) is the most frequently assessed modality of verticality perception and has been measured in a variety of clinical situations, including peripheral vestibular lesions.Aim: The main objectives are (1) to collect normative data of Virtual SVV™ from healthy subjects, and (2) to study the correlation between Virtual SVV™ and ocular vestibular-evoked myogenic potentials (o-VEMP) on healthy subjects.Materials and methods: Forty-three healthy subjects were recruited. Air conduction (AC)-elicited oVEMPs and bone conduction (BC)-elicited oVEMPs were measured. BC stimuli were produced with a RadioEar B-81 High Output Bone Transducer. Virtual SVV™ were also measured.Results: Virtual SVV™ data from our healthy subjects were consistent with previously published normative SVV data. Normal Virtual SVV™ data did not correlate with normal AC-elicited and BC-elicited oVEMPs.Conclusions: Virtual SVV™ data from our healthy subjects were consistent with previously published normative SVV data. Virtual SVV™ data from our 43 health subjects only had weak correlation with c-VEMP, AC-elicited and BC-elicited oVEMPs. These data serve as a baseline for a future study of patients with unilateral utricular dysfunction.Significance: The Virtual SVV™ can be an attractive substitute for traditional SVV in clinical settings.

Stabilization of Gaze during Early Xenopus Development by Swimming-Related Utricular Signals.

Locomotor maturation requires concurrent gaze stabilization improvement for maintaining visual acuity [1, 2]. The capacity to stabilize gaze, in particular in small aquatic vertebrates where coordinated locomotor activity appears very early, is determined by assembly and functional maturation of inner ear structures and associated sensory-motor circuitries [3-7]. Whereas utriculo-ocular reflexes become functional immediately after hatching [8, 9], semicircular canal-dependent vestibulo-ocular reflexes (VORs) appear later [10]. Thus, small semicircular canals are unable to detect swimming-related head oscillations, despite the fact that corresponding acceleration components are well-suited to trigger an angular VOR [11]. This leaves the utricle as the sole vestibular origin for swimming-related compensatory eye movements [12, 13]. We report a remarkable ontogenetic plasticity of swimming-related head kinematics and vestibular end organ recruitment in Xenopus tadpoles with beneficial consequences for gaze-stabilization. Swimming of older larvae generates sinusoidal head undulations with small, similar curvature angles on the left and right side that optimally activate horizontal semicircular canals. Young larvae swimming causes left-right head undulations with narrow curvatures and strong, bilaterally dissimilar centripetal acceleration components well suited to activate utricular hair cells and to substitute the absent semicircular canal function at this stage. The capacity of utricular signals to supplant semicircular canal function was confirmed by recordings of eye movements and extraocular motoneurons during off-center rotations in control and semicircular canal-deficient tadpoles. Strong alternating curvature angles and thus linear acceleration profiles during swimming in young larvae therefore represents a technically elegant solution to compensate for the incapacity of small semicircular canals to detect angular acceleration components.

The Superiority of the Otolith System.

BACKGROUND: The peripheral vestibular end organ is considered to consist of semi-circular canals (SCC) for detection of angular accelerations and the otoliths for detection of linear accelerations. However, otoliths being phylogenetically the oldest part of the vestibular sensory organs are involved in detection of all motions. SUMMARY: This study elaborates on this property of the otolith organ, as this concept can be of importance for the currently designed vestibular implant devices. Key Message: The analysis of the evolution of the inner ear and examination of clinical examples shows the robustness of the otolith system and inhibition capacity of the SCC. The otolith system must be considered superior to the SCC system as illustrated by evolution, clinical evidence, and physical principles.

Cervical vestibular evoked myogenic potential testing for assessment of vestibular functions in cochlear implant patients.

This study aims to investigate the vestibular function status of cochlear implant patients using cervical vestibular evoked myogenic potential (cVEMP) testing and estimate the effects of cochlear implants on vestibular function. The cVEMPs of 50 cochlear implant patients were measured preoperatively, and at one and six months postoperatively. Then, implanted ears and non-implanted ears were compared in terms of p13/n23 wave response rates, latency, amplitude and threshold. Preoperatively, the binaural cVEMP response rate was 92%, while the cVEMP response rates of implanted ears vs. non-implanted ears at postoperative one and six months were 24% vs. 80% and 52% vs. 82%, respectively. No significant difference between implanted and non-implanted ears was found preoperatively, in terms of latent period, amplitude, or threshold. However, significant changes were found in amplitude and threshold for implanted ears after the operation, but not in latency. No significant postoperative change was found in amplitude, latent period, or threshold for non-implanted ears. Significant differences between implanted and non-implanted ears were found in both amplitude and threshold. Cochlear implants affect vestibular function, especially saccular function, and reduce the cVEMP amplitude and threshold of implanted ears.

Binocular 3D otolith-ocular reflexes: responses of chinchillas to prosthetic electrical stimulation targeting the utricle and saccule.

From animal experiments by Cohen and Suzuki et al. in the 1960s to the first-in-human clinical trials now in progress, prosthetic electrical stimulation targeting semicircular canal branches of the vestibular nerve has proven effective at driving directionally appropriate vestibulo-ocular reflex eye movements, postural responses, and perception. That work was considerably facilitated by the fact that all hair cells and primary afferent neurons in each canal have the same directional sensitivity to head rotation, the three canals' ampullary nerves are geometrically distinct from one another, and electrically evoked three-dimensional (3D) canal-ocular reflex responses approximate a simple vector sum of linearly independent components representing relative excitation of each of the three canals. In contrast, selective prosthetic stimulation of the utricle and saccule has been difficult to achieve, because hair cells and afferents with many different directional sensitivities are densely packed in those endorgans and the relationship between 3D otolith-ocular reflex responses and the natural and/or prosthetic stimuli that elicit them is more complex. As a result, controversy exists regarding whether selective, controllable stimulation of electrically evoked otolith-ocular reflexes (eeOOR) is possible. Using micromachined, planar arrays of electrodes implanted in the labyrinth, we quantified 3D, binocular eeOOR responses to prosthetic electrical stimulation targeting the utricle, saccule, and semicircular canals of alert chinchillas. Stimuli delivered via near-bipolar electrode pairs near the maculae elicited sustained ocular countertilt responses that grew reliably with pulse rate and pulse amplitude, varied in direction according to which stimulating electrode was employed, and exhibited temporal dynamics consistent with responses expected for isolated macular stimulation.NEW & NOTEWORTHY As the second in a pair of papers on Binocular 3D Otolith-Ocular Reflexes, this paper describes new planar electrode arrays and vestibular prosthesis architecture designed to target the three semicircular canals and the utricle and saccule. With this technological advancement, electrically evoked otolith-ocular reflexes due to stimulation via utricle- and saccule-targeted electrodes were recorded in chinchillas. Results demonstrate advances toward achieving selective stimulation of the utricle and saccule.

Binocular 3D otolith-ocular reflexes: responses of normal chinchillas to tilt and translation.

Head rotation, translation, and tilt with respect to a gravitational field elicit reflexive eye movements that partially stabilize images of Earth-fixed objects on the retinas of humans and other vertebrates. Compared with the angular vestibulo-ocular reflex, responses to translation and tilt, collectively called the otolith-ocular reflex (OOR), are less completely characterized, typically smaller, generally disconjugate (different for the 2 eyes) and more complicated in their relationship to the natural stimuli that elicit them. We measured binocular 3-dimensional OOR responses of 6 alert normal chinchillas in darkness during whole body tilts around 16 Earth-horizontal axes and translations along 21 axes in horizontal, coronal, and sagittal planes. Ocular countertilt responses to 40-s whole body tilts about Earth-horizontal axes grew linearly with head tilt amplitude, but responses were disconjugate, with each eye's response greatest for whole body tilts about axes near the other eye's resting line of sight. OOR response magnitude during 1-Hz sinusoidal whole body translations along Earth-horizontal axes also grew with stimulus amplitude. Translational OOR responses were similarly disconjugate, with each eye's response greatest for whole body translations along its resting line of sight. Responses to Earth-horizontal translation were similar to those that would be expected for tilts that would cause a similar peak deviation of the gravitoinertial acceleration (GIA) vector with respect to the head, consistent with the "perceived tilt" model of the OOR. However, that model poorly fit responses to translations along non-Earth-horizontal axes and was insufficient to explain why responses are larger for the eye toward which the GIA vector deviates.NEW & NOTEWORTHY As the first in a pair of papers on Binocular 3D Otolith-Ocular Reflexes, this paper characterizes binocular 3D eye movements in normal chinchillas during tilts and translations. The eye movement responses were used to create a data set to fully define the normal otolith-ocular reflexes in chinchillas. This data set provides the foundation to use otolith-ocular reflexes to back-project direction and magnitude of eye movement to predict tilt axis as discussed in the companion paper.

The Growing Evidence for the Importance of the Otoliths in Spatial Memory.

Many studies have demonstrated that vestibular sensory input is important for spatial learning and memory. However, it has been unclear what contributions the different parts of the vestibular system - the semi-circular canals and otoliths - make to these processes. The advent of mutant otolith-deficient mice has made it possible to isolate the relative contributions of the otoliths, the utricle and saccule. A number of studies have now indicated that the loss of otolithic function impairs normal spatial memory and also impairs the normal function of head direction cells in the thalamus and place cells in the hippocampus. Epidemiological studies have also provided evidence that spatial memory impairment with aging, may be linked to saccular function. The otoliths may be important in spatial cognition because of their evolutionary age as a sensory detector of orientation and the fact that velocity storage is important to the way that the brain encodes its place in space.

Saccular function is associated with both angular and distance errors on the triangle completion test.

OBJECTIVE: The present study was designed to determine whether healthy older adults with age-related vestibular loss have deficits in spatial navigation. METHODS: 154 adults participating in the Baltimore Longitudinal Study of Aging were tested for semicircular canal, saccular, and utricular function and spatial navigation ability using the blindfolded Triangle Completion Test (TCT). Multiple linear regression was used to investigate the relationships between each measure of vestibular function and performance on the TCT (angular error, end point error, and distance walked) while controlling for age and sex. RESULTS: Individuals with abnormal saccular function made larger angular errors (ß = 4.2°, p < 0.05) and larger end point errors (ß = 13.6 cm, p < 0.05). Independent of vestibular function, older age was associated with larger angular (ß's = 2.2-2.8°, p's < 0.005) and end point errors (ß's = 7.5-9.0 cm, p's < 0.005) for each decade increment in age. CONCLUSIONS: Saccular function appears to play a prominent role in accurate spatial navigation during a blindfolded navigation task. SIGNIFICANCE: We hypothesize that gravitational cues detected by the saccule may be integrated into estimation of place as well as heading direction.

Reproductive-state dependent changes in saccular hair cell density of the vocal male plainfin midshipman fish.

The plainfin midshipman fish (Porichthys notatus) is a nocturnal, seasonally breeding, intertidal-nesting teleost fish that produces social acoustic signals for intraspecific communication. Type I or "nesting" males produce agonistic and reproductive-related acoustic signals including a multiharmonic advertisement call during the summer breeding season. Previous work showed that type I male auditory sensitivity of the saccule, the primary midshipman auditory end organ, changes seasonally with reproductive state such that reproductive males become more sensitive and better suited than nonreproductive males to detect the dominant frequencies contained within type I vocalizations. Here, we examine whether reproductive type I males also exhibit reproductive-state dependent changes in hair cell (HC) density in the three putative auditory end organs (saccule, lagena, and utricle). We show that saccular HC density was greater in reproductive type I males compared to nonreproductive type I males, and that the increase in HC density occurs throughout the saccular epithelium in both the central and marginal epithelia regions. We also show as saccular HC density increases there is a concurrent decrease in saccular support cell (SC) density in reproductive type I males with no overall change in total cell density (i.e., HC + SC). In contrast, we did not observe any seasonal changes in HC density in the utricle or lagena between nonreproductive and reproductive type I males. In addition, we compare the saccular HC densities in reproductive type I males with that of reproductive females and show that females have greater saccular HC densities, which suggest a sexually dimorphic difference in HC receptor density between the two sexual phenotypes, at least during the summer breeding season.

Why and when to refer patients for vestibular evoked myogenic potentials: A critical review.

Cervical and ocular vestibular evoked myogenic potentials (cVEMPs and oVEMPs respectively) are now used by an increasing number of laboratories to evaluate otolith inner ear function and their pathways through the central nervous system. However, the literature is incomplete or unclear as to what information both c- and oVEMPs can add beyond what a good clinical examination can provide, and what other paramedical tests can provide also, and the present review aims to clarify what is known so far. The following review will describe what is known with regards to both c- and oVEMPs and their use. MEDLINE (accessed by PubMed, years 1994-2018) was searched with the following string: ("vestibular evoked myogenic potentials" [all fields]). Only articles published in English were evaluated. Both c- and oVEMPs are useful not only for confirming the presence of superior semicircular canal dehiscence (SSCD), but also for confirming the presence of acoustic neuromas when MRI is not available, bilateral vestibulopathies, inferior vestibular neuritis and vestibular dysfunction in inherited neuropathies. Further work is required, especially with respect to oVEMPs. The usefulness of both c- and oVEMPs goes beyond the confirmation of SSCDs, and is useful in many clinical cases.

Uncoordinated maturation of developing and regenerating postnatal mammalian vestibular hair cells.

Sensory hair cells are mechanoreceptors required for hearing and balance functions. From embryonic development, hair cells acquire apical stereociliary bundles for mechanosensation, basolateral ion channels that shape receptor potential, and synaptic contacts for conveying information centrally. These key maturation steps are sequential and presumed coupled; however, whether hair cells emerging postnatally mature similarly is unknown. Here, we show that in vivo postnatally generated and regenerated hair cells in the utricle, a vestibular organ detecting linear acceleration, acquired some mature somatic features but hair bundles appeared nonfunctional and short. The utricle consists of two hair cell subtypes with distinct morphological, electrophysiological and synaptic features. In both the undamaged and damaged utricle, fate-mapping and electrophysiology experiments showed that Plp1+ supporting cells took on type II hair cell properties based on molecular markers, basolateral conductances and synaptic properties yet stereociliary bundles were absent, or small and nonfunctional. By contrast, Lgr5+ supporting cells regenerated hair cells with type I and II properties, representing a distinct hair cell precursor subtype. Lastly, direct physiological measurements showed that utricular function abolished by damage was partially regained during regeneration. Together, our data reveal a previously unrecognized aberrant maturation program for hair cells generated and regenerated postnatally and may have broad implications for inner ear regenerative therapies.

Auditory evoked potentials of the plainfin midshipman fish (Porichthys notatus): implications for directional hearing.

The plainfin midshipman (Porichthys notatus) is an acoustically communicative teleost fish. Here, we evaluated auditory evoked potentials (AEPs) in reproductive female midshipman exposed to tones at or near dominant frequencies of the male midshipman advertisement call. An initial series of experiments characterized AEPs at behaviorally relevant suprathreshold sound levels (130-140 dB SPL re. 1 µPa). AEPs decreased in magnitude with increasing stimulus frequency and featured a stereotyped component at twice the stimulus frequency. Recording electrode position was varied systematically and found to affect AEP magnitude and phase characteristics. Later experiments employed stimuli of a single frequency to evaluate contributions of the saccule to the AEP, with particular attention to the effects of sound source azimuth on AEP amplitude. Unilateral excision of saccular otoliths (sagittae) decreased AEP amplitude; unexpectedly, decreases differed for right versus left otolith excision. A final set of experiments manipulated the sound pressure-responsive swim bladder. Swim bladder excision further reduced the magnitude of AEP responses, effectively eliminating responses at the standard test intensity (130 dB SPL) in some animals. Higher-intensity stimulation yielded response minima at forward azimuths ipsilateral to the excised sagitta, but average cross-azimuth modulation generally remained slight. Collectively, the data underscore that electrode position is an essential variable to control in fish AEP studies and suggest that in female midshipman: (1) the saccule contributes to the AEP, but its directionality as indexed by the AEP is limited, (2) a left-right auditory asymmetry may exist and (3) the swim bladder provides gain in auditory sensitivity that may be important for advertisement call detection and phonotaxis.

Objective methods to measure vestibular evoked myogenic potential response saccular tuning curves.

Objective: To detect cervical vestibular evoked myogenic potential (cVEMP) responses using objective statistical approaches and to apply this approach to estimate saccular frequency-tuning curves in volunteers and Ménière's disease (MD) patients. Design: Estimates of cVEMP threshold were carried out by 3 expert raters at 500 Hz and compared to objective threshold estimates (using Hotelling's T2 [HT2] and Fsp). Saccular tuning curves were objectively estimated. Study sample: Objective and subjective estimates of cVEMP response thresholds were compared for 13 normal hearing adults. Objective measurement of saccular tuning curves was explored in 20 healthy adults and 15 patients with MD. Results: Significant variability was seen between subjective estimates of cVEMP thresholds. Objective analysis with the HT2 test was more sensitive than 2 of 3 experts in detecting responses. The measurement time of cVEMP was considerably reduced with the HT2 test. Objective saccular tuning curves in volunteers showed strongest responses at 500 Hz. A flatter tuning curve was seen for MD patients. Conclusions: There is significant variability in subjective estimations of cVEMP thresholds. Objective analysis methods are more sensitive than subjective analysis, can detect responses rapidly and have potential to reduce variability in threshold estimates, hence they appear well suited to measure cVEMP tuning curves.

Multiscale modeling of mechanotransduction in the utricle.

We review recent progress in using numerical models to relate utricular hair bundle and otoconial membrane (OM) structure to the functional requirements imposed by natural behavior in turtles. The head movements section reviews the evolution of experimental attempts to understand vestibular system function with emphasis on turtles, including data showing that accelerations occurring during natural head movements achieve higher magnitudes and frequencies than previously assumed. The structure section reviews quantitative anatomical data documenting topographical variation in the structures underlying macromechanical and micromechanical responses of the turtle utricle to head movement: hair bundles, OM, and bundle-OM coupling. The macromechanics section reviews macromechanical models that incorporate realistic anatomical and mechanical parameters and reveal that the system is significantly underdamped, contrary to previous assumptions. The micromechanics: hair bundle motion and met currents section reviews work based on micromechanical models, which demonstrates that topographical variation in the structure of hair bundles and OM, and their mode of coupling, result in regional specializations for signaling of low frequency (or static) head position and high frequency head accelerations. We conclude that computational models based on empirical data are especially promising for investigating mechanotransduction in this challenging sensorimotor system.

No evidence for a magnetite-based magnetoreceptor in the lagena of pigeons.

It is well established that an array of avian species sense the Earth's magnetic field and use this information for orientation and navigation. While the existence of a magnetic sense can no longer be disputed, the underlying cellular and biophysical basis remains unknown. It has been proposed that pigeons exploit a magnetoreceptor based on magnetite crystals (Fe3O4) that are located within the lagena [1], a sensory epithelium of the inner ear. It has been hypothesised that these magnetic crystals form a bed of otoconia that stimulate hair cells transducing magnetic information into a neuronal impulse. We performed a systematic high-sensitivity screen for iron in the pigeon lagena using synchrotron X-ray fluorescence microscopy coupled with the analysis of serial sections by transmission electron microscopy. We find no evidence for extracellular magnetic otoconia or intracellular magnetite crystals, suggesting that if an inner ear magnetic sensor does exist it relies on a different biophysical mechanism.