Sustained Loss of Bdnf Affects Peripheral but Not Central Vestibular Targets

The vestibular system is vital for proper balance perception, and its dysfunction contributes significantly to fall-related injuries, especially in the elderly. Vestibular ganglion neurons innervate vestibular hair cells at the periphery and vestibular nuclei and the uvula and nodule of the cerebellum centrally. During aging, these vestibular ganglion neurons degenerate, impairing vestibular function. A complete understanding of the molecular mechanisms involved in neurosensory cell survival in the vestibular system is unknown. Brain-derived neurotrophic factor (BDNF) is specifically required for the survival of vestibular ganglion neurons, as its loss leads to early neuronal death. Bdnf null mice die within 3 weeks of birth, preventing the study of the long-term effects on target cells. We use Pax2-cre to conditionally knock out Bdnf, allowing mice survival to approximately 6 months of age. We show that a long-term loss of Bdnf leads to a significant reduction in the number of vestibular ganglion neurons and a reduction in the number of vestibular hair cells. There was no significant decrease in the central targets lateral vestibular nucleus (LVN) or the cerebellum at 6 months. This suggests that the connectivity between central target cells and other neurons suffices to prevent their loss despite vestibular hair cell and ganglion neuron loss. Whether the central neurons would undergo eventual degeneration in the absence of Bdnf remains to be determined.

Current Response in CaV1.3–/– Mouse Vestibular and Cochlear Hair Cells

Signal transmission by sensory auditory and vestibular hair cells relies upon Ca2+-dependent exocytosis of glutamate. The Ca2+ current in mammalian inner ear hair cells is predominantly carried through CaV1.3 voltage-gated Ca2+ channels. Despite this, CaV1.3 deficient mice (CaV1.3–/–) are deaf but do not show any obvious vestibular phenotype. Here, we compared the Ca2+ current (ICa) in auditory and vestibular hair cells from wild-type and CaV1.3–/– mice, to assess whether differences in the size of the residual ICa could explain, at least in part, the two phenotypes. Using 5 mM extracellular Ca2+ and near-body temperature conditions, we investigated the cochlear primary sensory receptors inner hair cells (IHCs) and both type I and type II hair cells of the semicircular canals. We found that the residual ICa in both auditory and vestibular hair cells from CaV1.3–/– mice was less than 20% (12–19%, depending on the hair cell type and age investigated) compared to controls, indicating a comparable expression of CaV1.3 Ca2+ channels in both sensory organs. We also showed that, different from IHCs, type I and type II hair cells from CaV1.3–/– mice were able to acquire the adult-like K+ current profile in their basolateral membrane. Intercellular K+ accumulation was still present in CaV1.3–/– mice during IK,L activation, suggesting that the K+-based, non-exocytotic, afferent transmission is still functional in these mice. This non-vesicular mechanism might contribute to the apparent normal vestibular functions in CaV1.3–/– mice.

Cy3-ATP labeling of unfixed, permeabilized mouse hair cells

ATP-utilizing enzymes play key roles in hair bundles, the mechanically sensitive organelles of sensory hair cells in the inner ear. We used a fluorescent ATP analog, EDA-ATP-Cy3 (Cy3-ATP), to label ATP-binding proteins in two different preparations of unfixed hair-cell stereocilia of the mouse. In the first preparation, we lightly permeabilized dissected cochleas, then labeled them with Cy3-ATP. Hair cells and their stereocilia remained intact, and stereocilia tips in rows 1 and 2 were labeled particularly strongly with Cy3-ATP. In many cases, vanadate (Vi) traps nucleotides at the active site of myosin isoforms and presents nucleotide dissociation. Co-application with Vi enhanced the tip labeling, which is consistent with myosin isoforms being responsible. By contrast, the actin polymerization inhibitors latrunculin A and cytochalasin D had no effect, suggesting that actin turnover at stereocilia tips was not involved. Cy3-ATP labeling was substantially reduced—but did not disappear altogether—in mutant cochleas lacking MYO15A; by contrast, labeling remained robust in cochleas lacking MYO7A. In the second preparation, used to quantify Cy3-ATP labeling, we labeled vestibular stereocilia that had been adsorbed to glass, which demonstrated that tip labeling was higher in longer stereocilia. We found that tip signal was reduced by ~ 50% in Myo15ash2/sh2 stereocilia as compared to Myo15ash2/+stereocilia. These results suggest that MYO15A accounts for a substantial fraction of the Cy3-ATP tip labeling in vestibular hair cells, and so this novel preparation could be utilized to examine the control of MYO15A ATPase activity in situ.

A Biophysical Model of Nonquantal Transmission at the Vestibular Hair Cell-Calyx Synapse: KLV currents Modulate Fast Electrical and Slow K+ potentials in the Synaptic Cleft

Vestibular hair cells transmit information about head position and motion across synapses to primary afferent neurons. At some of these synapses, the afferent neuron envelopes the hair cell, forming an enlarged synaptic terminal referred to as a calyx. The vestibular hair cell-calyx synapse supports nonquantal transmission (NQT), a neurotransmitter-independent mechanism that is exceptionally fast. The underlying biophysical mechanisms that give rise to NQT are not fully understood. Here we present a computational model of NQT that integrates morphological and electrophysiological data. The model predicts that NQT involves two processes: changes in cleft K+ concentration, as previously recognized, and very fast changes in cleft electrical potential. A significant finding is that changes in cleft electrical potential are faster than changes in [K+] or quantal transmission. The electrical potential mechanism thus provides a basis for the exceptional speed of neurotransmission between type I hair cells and primary neurons and explains experimental observations of fast postsynaptic currents. The [K+] mechanism increases the gain of NQT. Both processes are mediated by current flow through low-voltage-activated K+ (KLV) channels located in both pre-synaptic (hair cell) and post-synaptic (calyx inner face) membranes. The model further demonstrates that the calyx morphology is necessary for NQT; as calyx height is increased, NQT increases in size, speed and efficacy at depolarizing the afferent neuron. We propose that the calyx evolved to enhance NQT and speed up signals that drive vestibular reflexes essential for stabilizing the eyes and neck and maintaining balance during rapid and complex head motions.

Peripheral Vestibular Dysfunction Is a Common Occurrence in Children With Non-syndromic and Syndromic Genetic Hearing Loss

Hearing loss (HL) is the most common sensory deficit in humans and is frequently accompanied by peripheral vestibular loss (PVL). While often overlooked, PVL is an important sensory dysfunction that may impair development of motor milestones in children and can have a significant negative impact on quality of life. In addition, many animal and in vitro models of deafness use vestibular hair cells as a proxy to study cochlear hair cells. The extent of vestibular end organ dysfunction associated with genetic pediatric hearing loss is not well-understood. We studied children with a known genetic cause of hearing loss who underwent routine preoperative vestibular testing prior to cochlear implantation between June 2014 and July 2020. Vestibular testing included videonystagmography, rotary chair, video head impulse testing, and/or vestibular evoked myogenic potentials. Etiology of HL was determined through history, physical examination, imaging, laboratory testing, and/or genetic testing. Forty-four children (21 female/23 male) met inclusion criteria; 24 had genetic non-syndromic and 20 had genetic syndromic forms of HL. Mean age at the time of testing was 2.8 ± 3.8 years (range 7 months−17 years). The most common cause of non-syndromic HL was due to mutations in GJB2 (n = 13) followed by MYO15A (3), MYO6 (2), POU3F4 (2), TMPRSS3 (1), CDH23 (1), TMC1 (1), and ESRRB (1). The most common forms of syndromic HL were Usher syndrome (4) and Waardenburg (4), followed by SCID/reticular dysgenesis (3), CHARGE (2), CAPOS (1), Coffin-Siris (1), Jervell and Lange-Nielsen (1), Noonan (1), peroxisome biogenesis disorder (1), Perrault (1), and Trisomy 21 (1). Overall, 23 patients (52%) had PVL. A larger proportion of children with syndromic forms of HL had PVL (12/20, 60%) compared with children with genetic non-syndromic HL (11/24, 46%), though without statistical significant (p = 0.3). The occurrence of PVL varied by affected gene. In conclusion, PVL is a common finding in children with syndromic and non-syndromic genetic HL undergoing vestibular evaluation prior to cochlear implantation. Improved understanding of the molecular physiology of vestibular hair cell dysfunction is important for clinical care as well as research involving vestibular hair cells in model organisms and in vitro models.

Conserved and Divergent Principles of Planar Polarity Revealed by Hair Cell Development and Function

Planar polarity describes the organization and orientation of polarized cells or cellular structures within the plane of an epithelium. The sensory receptor hair cells of the vertebrate inner ear have been recognized as a preeminent vertebrate model system for studying planar polarity and its development. This is principally because planar polarity in the inner ear is structurally and molecularly apparent and therefore easy to visualize. Inner ear planar polarity is also functionally significant because hair cells are mechanosensors stimulated by sound or motion and planar polarity underlies the mechanosensory mechanism, thereby facilitating the auditory and vestibular functions of the ear. Structurally, hair cell planar polarity is evident in the organization of a polarized bundle of actin-based protrusions from the apical surface called stereocilia that is necessary for mechanosensation and when stereociliary bundle is disrupted auditory and vestibular behavioral deficits emerge. Hair cells are distributed between six sensory epithelia within the inner ear that have evolved unique patterns of planar polarity that facilitate auditory or vestibular function. Thus, specialized adaptations of planar polarity have occurred that distinguish auditory and vestibular hair cells and will be described throughout this review. There are also three levels of planar polarity organization that can be visualized within the vertebrate inner ear. These are the intrinsic polarity of individual hair cells, the planar cell polarity or coordinated orientation of cells within the epithelia, and planar bipolarity; an organization unique to a subset of vestibular hair cells in which the stereociliary bundles are oriented in opposite directions but remain aligned along a common polarity axis. The inner ear with its complement of auditory and vestibular sensory epithelia allows these levels, and the inter-relationships between them, to be studied using a single model organism. The purpose of this review is to introduce the functional significance of planar polarity in the auditory and vestibular systems and our contemporary understanding of the developmental mechanisms associated with organizing planar polarity at these three cellular levels.

Fast adaptation of cooperative channels engenders Hopf bifurcations in auditory hair cells

Since the pioneering work of Thomas Gold published in 1948, it has been known that we owe our sensitive sense of hearing to a process in the inner ear that can amplify incident sounds on a cycle-by-cycle basis. Termed the active process, it uses energy to counteract the viscous dissipation associated with sound-evoked vibrations of the ear's mechanotransduction apparatus. Despite its importance, the mechanism of the active process and the proximate source of energy that powers it have remained elusive—especially at the high frequencies characteristic of mammalian hearing. This is partly due to our insufficient understanding of the mechanotransduction process in hair cells, the sensory receptors and amplifiers of the inner ear. It has previously been proposed that a cyclical binding of Ca2+ ions to individual mechanotransduction channels could power the active process. That model, however, relied on tailored reaction rates that structurally forced the direction of the cycle. Here, we ground our study on our previous model of hair-cell mechanotransduction, which relied on the cooperative gating of pairs of channels, and incorporate into it the cyclical binding of Ca2+ ions. With a single binding site per channel and reaction rates drawn from thermodynamic principles, our model shows that hair cells behave as nonlinear oscillators that exhibit Hopf bifurcations, dynamical instabilities long understood to be signatures of the active process. Using realistic parameter values, we find bifurcations at frequencies in the kilohertz range with physiological Ca2+ concentrations. In contrast to the myosin-based mechanism, responsible for low-frequency relaxation oscillations in the vestibular hair cells of amphibians, the current model relies on the electrochemical gradient of Ca2+ as the only energy source for the active process and on the relative motion of cooperative channels within the stereociliary membrane as the single mechanical driver. Equipped with these two mechanisms, a hair bundle proves capable of operating at frequencies in the kilohertz range, characteristic of mammalian hearing.

Cy3-ATP Labeling of Unfixed, Permeabilized Hair Cells

ATP-utilizing enzymes play key roles in hair bundles, the mechanically sensitive organelles of sensory hair cells in the inner ear. We used a fluorescent ATP analog, EDA-ATP-Cy3 (Cy3-ATP), to label ATP-binding proteins in two different preparations of unfixed hair-cell stereocilia. In the first preparation, we lightly permeabilized dissected cochleas, then labeled them with Cy3-ATP. Hair cells and their stereocilia remained intact, and stereocilia tips in rows 1 and 2 were labeled particularly strongly with Cy3-ATP. Co-application with vanadate (VO43-) enhanced the tip labeling, which is consistent with myosin isoforms being responsible; by contrast, the actin polymerization inhibitors latrunculin A and cytochalasin D had no effect, suggesting that actin turnover at stereocilia tips was not involved. Cy3-ATP labeling was substantially reduced—but did not disappear altogether—in mutant cochleas lacking MYO15A; by contrast, labeling remained robust in cochleas lacking MYO7A. In the second preparation, used to quantify Cy3-ATP labeling, we labeled vestibular stereocilia that had been adsorbed to glass, which demonstrated that tip labeling was higher in longer stereocilia. We found that tip signal was reduced by ~50% in Myo15ash2/sh2 stereocilia as compared to Myo15ash2/+ stereocilia of the same length range. These results suggest that MYO15A accounts for a substantial fraction of the Cy3-ATP tip labeling in vestibular hair cells, and so this novel preparation could be utilized to examine the control of MYO15A ATPase activity in situ.

Modulation of Vestibular Microphonics; a Historical Note

Modulation of microphonics has recently been used to investigate the sensitivity of the utricle in the vestibular organ of the guinea pig. The same technique was used more than 30 years ago to obtain information on the processing of rotational stimuli in the horizontal semicircular canals of the pigeon. Data from that time were reanalysed to give a relation that describes the mechano-electrical transduction (MET) process in vestibular hair cells.