Contributions of the individual domains of αIIbß3 integrin to its extension: Insights from multiscale modeling.

The platelet integrin αIIbß3 undergoes long-range conformational transitions between bent and extended conformations to regulate platelet aggregation during hemostasis and thrombosis. However, how exactly αIIbß3 transitions between conformations remains largely elusive. Here, we studied how transitions across bent and extended-closed conformations of αIIbß3 integrin are regulated by effective interactions between its functional domains. We first carried out µs-long equilibrium molecular dynamics (MD) simulations of full-length αIIbß3 integrins in bent and intermediate conformations, the latter characterized by an extended headpiece and closed legs. Then, we built heterogeneous elastic network models, perturbed inter-domain interactions, and evaluated their relative contributions to the energy barriers between conformations. Results showed that integrin extension emerges from: (i) changes in interfaces between functional domains; (ii) allosteric coupling of the head and upper leg domains with flexible lower leg domains. Collectively, these results provide new insights into integrin conformational activation based on short- and long-range interactions between its functional domains and highlight the importance of the lower legs in the regulation of integrin allostery.

Evidence That Ultrafast Nonquantal Transmission Underlies Synchronized Vestibular Action Potential Generation.

Amniotes evolved a unique postsynaptic terminal in the inner ear vestibular organs called the calyx that receives both quantal and nonquantal (NQ) synaptic inputs from Type I sensory hair cells. The nonquantal synaptic current includes an ultrafast component that has been hypothesized to underlie the exceptionally high synchronization index (vector strength) of vestibular afferent neurons in response to sound and vibration. Here, we present three lines of evidence supporting the hypothesis that nonquantal transmission is responsible for synchronized vestibular action potentials of short latency in the guinea pig utricle of either sex. First, synchronized vestibular nerve responses are unchanged after administration of the AMPA receptor antagonist CNQX, while auditory nerve responses are completely abolished. Second, stimulus evoked vestibular nerve compound action potentials (vCAP) are shown to occur without measurable synaptic delay and three times shorter than the latency of auditory nerve compound action potentials (cCAP), relative to the generation of extracellular receptor potentials. Third, paired-pulse stimuli designed to deplete the readily releasable pool (RRP) of synaptic vesicles in hair cells reveal forward masking in guinea pig auditory cCAPs, but a complete lack of forward masking in vestibular vCAPs. Results support the conclusion that the fast component of nonquantal transmission at calyceal synapses is indefatigable and responsible for ultrafast responses of vestibular organs evoked by transient stimuli.SIGNIFICANCE STATEMENT The mammalian vestibular system drives some of the fastest reflex pathways in the nervous system, ensuring stable gaze and postural control for locomotion on land. To achieve this, terrestrial amniotes evolved a large, unique calyx afferent terminal which completely envelopes one or more presynaptic vestibular hair cells, which transmits mechanosensory signals mediated by quantal and nonquantal (NQ) synaptic transmission. We present several lines of evidence in the guinea pig which reveals the most sensitive vestibular afferents are remarkably fast, much faster than their auditory nerve counterparts. Here, we present neurophysiological and pharmacological evidence that demonstrates this vestibular speed advantage arises from ultrafast NQ electrical synaptic transmission from Type I hair cells to their calyx partners.

Stem cell-derived brain organoids for controlled studies of transcranial neuromodulation.

Transcranial neuromodulation methods have the potential to diagnose and treat brain disorders at their neural source in a personalized manner. However, it has been difficult to investigate the direct effects of transcranial neuromodulation on neurons in human brain tissue. Here, we show that human brain organoids provide a detailed and artifact-free window into neuromodulation-evoked electrophysiological effects. We derived human cortical organoids from induced pluripotent stem cells and implanted 32-channel electrode arrays. Each organoid was positioned in the center of the human skull and subjected to low-intensity transcranial focused ultrasound. We found that ultrasonic stimuli modulated network activity in the gamma and delta ranges of the frequency spectrum. The effects on the neural networks were a function of the ultrasound stimulation frequency. High gamma activity remained elevated for at least 20 minutes following stimulation offset. This approach is expected to provide controlled studies of the effects of ultrasound and other transcranial neuromodulation modalities on human brain tissue.

Using macular velocity measurements to relate parameters of bone conduction to vestibular compound action potential responses.

To examine mechanisms responsible for vestibular afferent sensitivity to transient bone conducted vibration, we performed simultaneous measurements of stimulus-evoked vestibular compound action potentials (vCAPs), utricular macula velocity, and vestibular microphonics (VMs) in anaesthetized guinea pigs. Results provide new insights into the kinematic variables of transient motion responsible for triggering mammalian vCAPs, revealing synchronized vestibular afferent responses are not universally sensitive to linear jerk as previously thought. For short duration stimuli (< 1 ms), the vCAP increases magnitude in close proportion to macular velocity and temporal bone (linear) acceleration, rather than other kinematic elements. For longer duration stimuli, the vCAP magnitude switches from temporal bone acceleration sensitive to linear jerk sensitive while maintaining macular velocity sensitivity. Frequency tuning curves evoked by tone-burst stimuli show vCAPs increase in proportion to onset macular velocity, while VMs increase in proportion to macular displacement across the entire frequency bandwidth tested between 0.1 and 2 kHz. The subset of vestibular afferent neurons responsible for synchronized firing and vCAPs have been shown previously to make calyceal synaptic contacts with type I hair cells in the striolar region of the epithelium and have irregularly spaced inter-spike intervals at rest. Present results provide new insight into mechanical and neural mechanisms underlying synchronized action potentials in these sensitive afferents, with clinical relevance for understanding the activation and tuning of neurons responsible for driving rapid compensatory reflex responses.

A mathematical model for mechanical activation and compound action potential generation by the utricle in response to sound and vibration.

Introduction: Calyx bearing vestibular afferent neurons innervating type I hair cells in the striolar region of the utricle are exquisitely sensitive to auditory-frequency air conducted sound (ACS) and bone conducted vibration (BCV). Here, we present experimental data and a mathematical model of utricular mechanics and vestibular compound action potential generation (vCAP) in response to clinically relevant levels of ACS and BCV. Vibration of the otoconial layer relative to the sensory epithelium was simulated using a Newtonian two-degree-of-freedom spring-mass-damper system, action potential timing was simulated using an empirical model, and vCAPs were simulated by convolving responses of the population of sensitive neurons with an empirical extracellular voltage kernel. The model was validated by comparison to macular vibration and vCAPs recorded in the guinea pig, in vivo. Results: Transient stimuli evoked short-latency vCAPs that scaled in magnitude and timing with hair bundle mechanical shear rate for both ACS and BCV. For pulse BCV stimuli with durations <0.8 ms, the vCAP magnitude increased in proportion to temporal bone acceleration, but for pulse durations >0.9 ms the magnitude increased in proportion to temporal bone jerk. Once validated using ACS and BCV data, the model was applied to predict blast-induced hair bundle shear, with results predicting acute mechanical damage to bundles immediately upon exposure. Discussion: Results demonstrate the switch from linear acceleration to linear jerk as the adequate stimulus arises entirely from mechanical factors controlling the dynamics of sensory hair bundle deflection. The model describes the switch in terms of the mechanical natural frequencies of vibration, which vary between species based on morphology and mechanical factors.

A parametric blueprint for optimum cochlear outer hair cell design.

The present work examines the hypothesis that cochlear outer hair cell (OHC) properties vary in precise proportions along the tonotopic map to optimize electromechanical power conversion. We tested this hypothesis using a very simple model of a single isolated OHC driving a mechanical load. Results identify three non-dimensional ratios that are predicted to optimize power conversion: the ratio of the resistive-capacitive (RC) corner to the characteristic frequency (CF), the ratio of nonlinear to linear capacitance and the ratio of OHC stiffness to cochlear load stiffness. Optimum efficiency requires all three ratios to be universal constants, independent of CF and species. The same ratios are cardinal control parameters that maximize power output by positioning the OHC operating point on the edge of a dynamic instability. Results support the hypothesis that OHC properties evolved to optimize electro-mechanical power conversion. Identification of the RC corner frequency as a control parameter reveals a powerful mechanism used by medial olivocochlear efferent system to control OHC power output. Results indicate the upper-frequency limit of OHC power output is not constrained by the speed of the motor itself but instead is probably limited by the size of the nucleus and membrane surface area available for ion-channel expression.

Otolith Membrane Herniation, not Semicircular Canal Duct Dilation, Is Associated with Decreased Caloric Responses in Ménière's Disease.

Ménière's disease (MD) is a debilitating disorder with unclear pathophysiology whose diagnosis often relies on clinical judgment rather than objective testing. To complicate matters further, a dissociation has emerged between two vestibular function tests commonly used in patients with MD to examine the same end-organ (the semicircular canals): the caloric test and video head impulse testing (vHIT). Caloric responses are often abnormal, while vHIT results remain normal. Explaining this dissociation could reveal novel insights into MD pathophysiology. Here, we conduct a histopathological study using temporal bone specimens (N = 58, 21 MD-affected ears and 37 age-matched controls) and their clinical testing data to examine current hypotheses aimed at this dissociation. We find otolith membrane herniation into the horizontal semicircular canal in 69% of MD ears, with 90% of these ears demonstrating a diminished caloric response. No ears with a normal response had this herniation. Moreover, we evaluated the semicircular canals for endolymphatic hydrops, which had been hypothesized to contribute to the dissociation, and found no evidence of duct dilation/hydrops. We did, however, note a potentially novel morphologic finding-smaller bony labyrinth cross-sectional diameters/areas in some MD ear canals compared to controls, suggesting relative size of the membranous duct to the bony canal rather than absolute size may be of importance. Taken together, this study refines hypotheses on the vestibular test dissociation in MD, holding diagnostic implications and expanding our understanding of the mechanisms underlying this enigmatic disease.

Analysis of outer hair cell electromechanics reveals power delivery at the upper-frequency limits of hearing.

Outer hair cells are the cellular motors in the mammalian inner ear responsible for sensitive high-frequency hearing. Motor function over the frequency range of human hearing requires expression of the protein prestin in the OHC lateral membrane, which imparts piezoelectric properties to the cell membrane. In the present report, electrical power consumption and mechanical power output of the OHC membrane-motor complex are determined using previously published voltage-clamp data from isolated OHCs and membrane patches. Results reveal that power output peaks at a best frequency much higher than implied by the low-pass character of nonlinear capacitance, and much higher than the whole-cell resistive-capacitive corner frequency. High frequency power output is enabled by a -90° shift in the phase of electrical charge displacement in the membrane, manifested electrically as emergence of imaginary-valued nonlinear capacitance.

Differential Activation of Canal and Otolith Afferents by Acoustic Tone Bursts in Rats.

Vestibular evoked myogenic potentials (VEMPs) are routinely used to test otolith function, but which specific vestibular afferent neurons and central circuits are activated by auditory frequency VEMP stimuli remains unclear. To examine this question, we analyzed the sensitivity of individual vestibular afferents in adult Sprague-Dawley rats to tone bursts delivered at 9 frequencies (125-4000 Hz) and 3 intensity levels (60, 70, 80 dB SL re: acoustic brainstem response (ABR) threshold). Afferent neuron tone sensitivity was quantified by the cumulative probability of evoking a spike (CPE). Based on a threshold CPE of 0.1, acoustic stimuli in the present study evoked responses in 78.2 % (390/499) of otolith afferent neurons vs. 48.4 % (431/891) of canal afferent neurons. Organ-specific vestibular inputs to the central nervous system in response to tone bursts differ based on intensity and frequency content of the stimulus. At frequencies below 500 Hz, tone bursts primarily activated both otolith afferents, even at the highest intensity tested (80 dB SL re ABR threshold). At 1500 Hz, however, tone bursts activated the canal and otolith afferents at the moderate and high intensities tested (70, 80 dB SL), but activated only otolith afferents at the low intensity tested (60 dB SL). Within an end organ, diversity of sensitivity between individual afferent neurons correlated with spontaneous discharge rate and regularity. Examination of inner ear fluid mechanics in silico suggests that the frequency response and preferential activation of the otolith organs likely arise from inner ear fluid motion trapped near the oval and round windows. These results provide insight into understanding the mechanisms of sound activation of the vestibular system and developing novel discriminative VEMP testing protocols and interpretative guidelines in humans.

ATP and ACh Evoked Calcium Transients in the Neonatal Mouse Cochlear and Vestibular Sensory Epithelia.

Hair cells in the mammalian inner ear sensory epithelia are surrounded by supporting cells which are essential for function of cochlear and vestibular systems. In mice, support cells exhibit spontaneous intracellular Ca2+ transients in both auditory and vestibular organs during the first postnatal week before the onset of hearing. We recorded long lasting (>200 ms) Ca2+ transients in cochlear and vestibular support cells in neonatal mice using the genetic calcium indicator GCaMP5. Both cochlear and vestibular support cells exhibited spontaneous intracellular Ca2+ transients (GCaMP5 ΔF/F), in some cases propagating as waves from the apical (endolymph facing) to the basolateral surface with a speed of ∼25 µm per second, consistent with inositol trisphosphate dependent calcium induced calcium release (CICR). Acetylcholine evoked Ca2+ transients were observed in both inner border cells in the cochlea and vestibular support cells, with a larger change in GCaMP5 fluorescence in the vestibular support cells. Adenosine triphosphate evoked robust Ca2+ transients predominantly in the cochlear support cells that included Hensen's cells, Deiters' cells, inner hair cells, inner phalangeal cells and inner border cells. A Ca2+ event initiated in one inner border cells propagated in some instances longitudinally to neighboring inner border cells with an intercellular speed of ∼2 µm per second, and decayed after propagating along ∼3 cells. Similar intercellular propagation was not observed in the radial direction from inner border cell to inner sulcus cells, and was not observed between adjacent vestibular support cells.

Biomechanics of Third Window Syndrome.

Third window syndrome describes a set of vestibular and auditory symptoms that arise when a pathological third mobile window is present in the bony labyrinth of the inner ear. The pathological mobile window (or windows) adds to the oval and round windows, disrupting normal auditory and vestibular function by altering biomechanics of the inner ear. The most commonly occurring third window syndrome arises from superior semicircular canal dehiscence (SSCD), where a section of bone overlying the superior semicircular canal is absent or thinned (near-dehiscence). The presentation of SSCD syndrome is well characterized by clinical audiological and vestibular tests. In this review, we describe how the third compliant window introduced by a SSCD alters the biomechanics of the inner ear and thereby leads to vestibular and auditory symptoms. Understanding the biomechanical origins of SSCD further provides insight into other third window syndromes and the potential of restoring function or reducing symptoms through surgical repair.

The cochlear outer hair cell speed paradox.

Cochlear outer hair cells (OHCs) are among the fastest known biological motors and are essential for high-frequency hearing in mammals. It is commonly hypothesized that OHCs amplify vibrations in the cochlea through cycle-by-cycle changes in length, but recent data suggest OHCs are low-pass filtered and unable to follow high-frequency signals. The fact that OHCs are required for high-frequency hearing but appear to be throttled by slow electromotility is the "OHC speed paradox." The present report resolves this paradox and reveals origins of ultrafast OHC function and power output in the context of the cochlear load. Results demonstrate that the speed of electromotility reflects how fast the cell can extend against the load, and does not reflect the intrinsic speed of the motor element itself or the nearly instantaneous speed at which the coulomb force is transmitted. OHC power output at auditory frequencies is revealed by emergence of an imaginary nonlinear capacitance reflecting the phase of electrical charge displacement required for the motor to overcome the viscous cochlear load.

Developmental GAD2 Expression Reveals Progenitor-like Cells with Calcium Waves in Mammalian Crista Ampullaris.

Sense of motion, spatial orientation, and balance in vertebrates relies on sensory hair cells in the inner ear vestibular system. Vestibular supporting cells can regenerate hair cells that are lost from aging, ototoxicity, and trauma, although not all factors or specific cell types are known. Here we report a population of GAD2-positive cells in the mouse crista ampullaris and trace GAD2 progenitor-like cells that express pluripotent transcription factors SOX2, PROX1, and CTBP2. GAD2 progenitor-like cells organize into rosettes around a central branched structure in the eminentia cruciatum (EC) herein named the EC plexus. GCaMP5G calcium indicator shows spontaneous and acetylcholine-evoked whole-cell calcium waves in neonatal and adult mice. We present a hypothetical model that outlines the lineage and potential regenerative capacity of GAD2 cells in the mammalian vestibular neuroepithelium.

Cooperativity of Kv7.4 channels confers ultrafast electromechanical sensitivity and emergent properties in cochlear outer hair cells.

The mammalian cochlea relies on active electromotility of outer hair cells (OHCs) to resolve sound frequencies. OHCs use ionic channels and somatic electromotility to achieve the process. It is unclear, though, how the kinetics of voltage-gated ionic channels operate to overcome extrinsic viscous drag on OHCs at high frequency. Here, we report ultrafast electromechanical gating of clustered Kv7.4 in OHCs. Increases in kinetics and sensitivity resulting from cooperativity among clustered-Kv7.4 were revealed, using optogenetics strategies. Upon clustering, the half-activation voltage shifted negative, and the speed of activation increased relative to solitary channels. Clustering also rendered Kv7.4 channels mechanically sensitive, confirmed in consolidated Kv7.4 channels at the base of OHCs. Kv7.4 clusters provide OHCs with ultrafast electromechanical channel gating, varying in magnitude and speed along the cochlea axis. Ultrafast Kv7.4 gating provides OHCs with a feedback mechanism that enables the cochlea to overcome viscous drag and resolve sounds at auditory frequencies.

Spontaneous and Acetylcholine Evoked Calcium Transients in the Developing Mouse Utricle.

Spontaneous calcium transients are present during early postnatal development in the mouse retina and cochlea, and play an important role in maturation of the sensory organs and neural circuits in the central nervous system (CNS). It is not known whether similar calcium transients occur during postnatal development in the vestibular sensory organs. Here we demonstrate spontaneous intracellular calcium transients in sensory hair cells (HCs) and supporting cells (SCs) in the murine utricular macula during the first two postnatal weeks. Calcium transients were monitored using a genetically encoded calcium indicator, GCaMP5G (G5), at 100 ms-frame-1 in excised utricle sensory epithelia, including HCs, SCs, and neurons. The reporter line expressed G5 and tdTomato (tdT) in a Gad2-Cre dependent manner within a subset of utricular HCs, SCs and neurons. Kinetics of the G5 reporter limited temporal resolution to calcium events lasting longer than 200 ms. Spontaneous calcium transients lasting 1-2 s were observed in the expressing population of HCs at birth and slower spontaneous transients lasting 10-30 s appeared in SCs by P3. Beginning at P5, calcium transients could be modulated by application of the efferent neurotransmitter acetylcholine (ACh). In mature mice, calcium transients in the utricular macula occurred spontaneously, had a duration 1-2 s, and could be modulated by the exogenous application of acetylcholine (ACh) or muscarine. Long-lasting calcium transients evoked by ACh in mature mice were blocked by atropine, consistent with previous reports describing the role of muscarinic receptors expressed in calyx bearing afferents in efferent control of vestibular sensation. Large spontaneous and ACh evoked transients were reversibly blocked by the inositol trisphosphate receptor (IP3R) antagonist aminoethoxydiphenyl borate (2-APB). Results demonstrate long-lasting calcium transients are present in the utricular macula during the first postnatal week, and that responses to ACh mature over this same time period.

Endothiodon cf. bathystoma (Synapsida: Dicynodontia) bony labyrinth anatomy, variation and body mass estimates.

The semicircular canal (SC) system of the inner ear detects head angular accelerations and is essential for navigation and spatial awareness in vertebrates. Because the bony labyrinth encloses the membranous labyrinth SCs, it can be used as a proxy for animal behavior. The bony labyrinth of dicynodonts, a clade of herbivorous non-mammalian synapsids, has only been described in a handful of individuals and remains particularly obscure. Here we describe the bony labyrinth anatomy of three Endothiodon cf. bathystoma specimens from Mozambique based on digital reconstructions from propagation phase-contrast synchrotron micro-computed tomography. We compare these findings with the bony labyrinth anatomy of their close relative Niassodon. The bony labyrinths of Endothiodon and Niassodon are relatively similar and show only differences in the shape of the horizontal SCs and the orientation of the vertical SCs. When compared to extant mammals, Endothiodon and Niassodon have highly eccentric SCs. In addition, the Endothiodon SCs are nearly orthogonal. An eccentric and orthogonal SC morphology is consistent with a specialization in rapid head movements, which are typical of foraging or feeding behaviors. Furthermore, we estimate the body mass of these Endothiodon specimens at ~116 to 182 kg, based on the average SC radii calculated using a linear regression model optimized by the Amemiya Prediction Criterion. Our findings provide novel insights into the paleobiology of Endothiodon which are consistent with the peculiar feeding mechanism among dicynodonts presumed from their multiple postcanine toothrows.

ACh-induced hyperpolarization and decreased resistance in mammalian type II vestibular hair cells.

In the mammalian vestibular periphery, electrical activation of the efferent vestibular system (EVS) has two effects on afferent activity: 1) it increases background afferent discharge and 2) decreases afferent sensitivity to rotational stimuli. Although the cellular mechanisms underlying these two contrasting afferent responses remain obscure, we postulated that the reduction in afferent sensitivity was attributed, in part, to the activation of α9- containing nicotinic acetylcholine (ACh) receptors (α9*nAChRs) and small-conductance potassium channels (SK) in vestibular type II hair cells, as demonstrated in the peripheral vestibular system of other vertebrates. To test this hypothesis, we examined the effects of the predominant EVS neurotransmitter ACh on vestibular type II hair cells from wild-type (wt) and α9-subunit nAChR knockout (α9-/-) mice. Immunostaining for choline acetyltransferase revealed there were no obvious gross morphological differences in the peripheral EVS innervation among any of these strains. ACh application onto wt type II hair cells, at resting potentials, produced a fast inward current followed by a slower outward current, resulting in membrane hyperpolarization and decreased membrane resistance. Hyperpolarization and decreased resistance were due to gating of SK channels. Consistent with activation of α9*nAChRs and SK channels, these ACh-sensitive currents were antagonized by the α9*nAChR blocker strychnine and SK blockers apamin and tamapin. Type II hair cells from α9-/- mice, however, failed to respond to ACh at all. These results confirm the critical importance of α9nAChRs in efferent modulation of mammalian type II vestibular hair cells. Application of exogenous ACh reduces electrical impedance, thereby decreasing type II hair cell sensitivity. NEW & NOTEWORTHY Expression of α9 nicotinic subunit was crucial for fast cholinergic modulation of mammalian vestibular type II hair cells. These findings show a multifaceted efferent mechanism for altering hair cell membrane potential and decreasing membrane resistance that should reduce sensitivity to hair bundle displacements.

Wave Mechanics of the Vestibular Semicircular Canals.

The semicircular canals are biomechanical sensors responsible for detecting and encoding angular motion of the head in 3D space. Canal afferent neurons provide essential inputs to neural circuits responsible for representation of self-position/orientation in space, and to compensatory circuits including the vestibulo-ocular and vestibulo-collic reflex arcs. In this work we derive, to our knowledge, a new 1D mathematical model quantifying canal biomechanics based on the morphology, dynamics of the inner ear fluids, and membranous labyrinth deformability. The model takes the form of a dispersive wave equation and predicts canal responses to angular motion, sound, and mechanical stimulation. Numerical simulations were carried out for the morphology of the human lateral canal using known physical properties of the endolymph and perilymph in three diverse conditions: surgical plugging, rotation, and mechanical indentation. The model reproduces frequency-dependent attenuation and phase shift in cases of canal plugging. During rotation, duct deformability extends the frequency bandwidth and enhances the high frequency gain. Mechanical indentation of the membranous duct at high frequencies evokes traveling waves that move away from the location of indentation and at low frequencies compels endolymph displacement along the canal. These results demonstrate the importance of the conformal perilymph-filled bony labyrinth to pressure changes and to high frequency sound and vibration.