Vestibular Compound Action Potentials and Macular Velocity Evoked by Sound and Vibration in the Guinea Pig1

Vestibular Compound Action Potentials and Macular Velocity Evoked by Sound and Vibration in the Guinea Pig1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Vestibular Compound Action Potentials and Macular Velocity Evoked by Sound and Vibration in the Guinea Pig 1 Christopher J. Pastras, Ian S. Curthoys, Richard D. Rabbitt, Daniel J. Brown This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2323465/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Jun, 2023 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract To examine mechanisms responsible for vestibular afferent sensitivity to transient air conducted sounds (ACS) and inter-aural bone conducted vibration (BCV), we performed simultaneous measurements of stimulus-evoked vestibular compound action potentials (vCAPs), utricular macula or stapes velocity, and vestibular microphonics (VMs) in the anaesthetized guinea pig. For short duration punctate stimuli (< 1ms), the vCAP increases magnitude in close proportion to macular velocity and temporal bone (ear-bar) acceleration, rather than other kinematic variables. For longer duration stimuli, the vCAP magnitude switches from temporal bone acceleration sensitive to linear jerk sensitive while maintaining macular velocity sensitivity. vCAP input-output (IO) functions suggest primary afferent response generation has the same origins for both BCV and ACS, with similar macular velocity thresholds and IO functions for both stimuli. Frequency tuning curves evoked by tone-burst stimuli also show the vCAP increases magnitude in proportion to macular velocity, while in contrast, the VM increases magnitude in proportion to macular displacement across the entire frequency bandwidth tested. 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. Biological sciences/Neuroscience Biological sciences/Physiology Biological sciences/Systems biology Health sciences/Medical research inner ear vestibular compound action potential macula vibration sound chirp Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Significant Statement Calyx-bearing afferents in the utricle have the remarkable ability to fire an action potential at a precise time following the onset of a transient stimulus and provide temporal information required for compensatory vestibular reflex circuits, but specifically how transient high-frequency stimuli lead to mechanical activation of hair cells and neural responses is poorly understood. Here, we dissect the relative contributions of mechanics, hair cell transduction, and action potential generation on short-latency responses to transient stimuli. Results provide a framework for the interpretation of synchronized vestibular afferent responses, with relevance to understanding origins of myogenic reflex responses commonly used in the clinic to assay vestibular function, and vestibular short latency potentials commonly used for vestibular phenotyping in rodents. Introduction Vestibular otolith organs are phylogenetically ancient inertial sensors that evolved hundreds of millions of years ago in primitive fish 1 , and successfully endowed extant land-dwelling vertebrates with the sensory neural inputs necessary for locomotion and navigation in a complex terrestrial environment 2 – 5 . In amniotes, some otolith afferent neurons preferentially respond to low-frequency gravito-inertial acceleration 6 – 11 , while others preferentially respond to high-frequency air conducted sound (ACS) or bone conducted vibration (BCV) 12 – 19 . The full population of otolith sensory neurons provide the central nervous system with broad-band detection of linear acceleration and head orientation in three-dimensional (3D) space, providing critical inputs to the autonomic nervous system to modulate heart rate and respiration during movements 20 , 21 , and to motor circuits responsible for the vestibular-ocular, -spinal, and -colic reflexes 9 , 22 . The compensatory nature of vestibular circuits makes disorders of the otolith organs particularly debilitating, often leading to sensory conflict and symptoms of dizziness, nausea, blurred vision, anxiety, and disorientation. Otolith function is commonly tested in the clinic using transient ACS or BCV to evoke reflexive cervical or ocular myogenic potentials (VEMPs), but precisely how high-frequency transient stimuli lead to mechano-transduction and neural responses in otolith organs is not well understood. The broad dynamic range of otolith sensitivity from DC to several kilohertz 23 arises from diverse properties of hair cells, synapses, and vestibular afferent spike generators 24 , 25 . Amniote neuroepithelia have two major hair cell types (I and II) and two major synaptic terminal types (bouton, calyx, or their combination; dimorphic) 26 – 28 which combine with spike generation properties to provide the broad frequency bandwidth and diversity in action potential generation between different afferent neurons. The larger diameter calyx bearing afferents, which evolved in land-dwelling amniotes 2 , 3 , 29 , make synaptic contacts with type-I hair cells in the striolar region of the macula 30 – 32 , and are characterized by their irregular action potential discharge rate, phasic responses to maintained stimuli, and sensitive short-latency responses to linear acceleration 33 . Calyx synaptic terminals completely envelop the lateral and basal surface of one or more type-I hair cells and are exquisitely sensitive to transient stimuli 18 , 34 . Three modes of excitatory synaptic transmission occur at calyx terminals: quantal glutamatergic vesicular release (QT) 35 , 36 , ultrafast nonquantal ephaptic coupling (NQf) 24 , 37 , and slow nonquantal accumulation of K + within the synaptic cleft (NQs) 24 , 38 , 39 . Direct ephaptic electrical coupling (NQf) is the component responsible for ultrashort latency and high sensitivity of calyx bearing vestibular afferents to transient inputs 37 . Sensitivity of calyx bearing otolith afferent neurons to transient ACS and BCV is routinely exploited in the clinic and the laboratory to test otolith function. In the clinic, reflexive cervical and ocular vestibular evoked myogenic potentials (cVEMP and oVEMP) are used to test saccular and utricular function 40 , and in the laboratory short latency vestibular stimulus evoked potentials (VsEP) are used to screen otolith function in mice and other rodents 41 . VsEPs are compound action potentials arising from transient stimuli that evoke nearly synchronous firing of a large number of calyx-bearing afferent neurons. When the vestibular compound action potential (vCAP) is recorded from localized sites near the vestibular nerve branch such as the facial nerve canal, the signal-to-noise ratio is enhanced providing recordings similar to auditory CAPs recorded from the round window niche 42 , 43 . vCAPs reflect combined responses of the population of sensitive afferent neurons and have been recorded in both acute and chronic animal models of health and disease 44 . Although whole-nerve neural responses to transient ACS and BCV stimuli have been reported for otolith organs, it is currently not known how high frequency transient stimuli lead to mechano-electrical transduction (MET) by sensory hair cells or the generation of synchronized action potentials. The present report quantifies the relationship between mechanical vibration of the macula, gating of hair cell MET channels, and generation of vCAPs in the guinea pig utricle for ACS and BCV stimuli. This was achieved by simultaneous measurement of temporal bone acceleration, macular (and stapes) velocity, vestibular microphonics (VMs), and extracellular vCAPs. Results provide new insight into mechanical and receptor mechanisms underlying synchronized neural responses in phasic vestibular afferents, with clinical relevance for understanding vestibular reflex responses, used to diagnose vestibular health and disease at the bedside. Methods Animal preparation & surgery. Experiments were performed on 28 adult tri-colored guinea pigs ( Cavia porcellus ) weighing between 300-500g of either sex. All experiments performed in this study were approved by the University of Sydney Animal Care and Ethics Committee (Approval number: #2019/1533). All methods were carried out in accordance with the relevant guidelines and regulations, which included the Australian Code for the Care and Use of Animals for Scientific Purposes (8th edition, 2013), and the ARRIVE guidelines 45 . Prior to procedures, animals first received pre-anesthetic intraperitoneal injections of Atropine Sulphate; 0.1mg/kg (0.6mg/ml; Apex Laboratories, NSW, Australia) and Buprenorphine Hydrochloride; 0.05mg/kg (Temgesic; 324µg/ml; Reckitt Benckiser, Auckland, NZ). Thereafter, animals were anesthetized in an induction chamber with Isoflurane (2–4%; Henry Schein, NSW, Australia) saturated in medical O 2 (Coregas, NSW, Australia). Once lacking a foot-withdrawal reflex, guinea pigs were transferred to the surgical table, and received anesthetic via a nose cone, whilst local injections of lignocaine hydrochloride (Lidocaine, Troy Laboratories, NSW, Australia) were delivered to surgery sites. Animals were then tracheotomized and artificially ventilated using Isoflurane (~ 2%) with oxygen, with the aid of a small animal ventilator (Model 683, Harvard Apparatus, MA, USA). Animals were thereafter rigidly mounted in custom-made ear-bar frames, housing a ‘canalphone’ speaker (ATH-IM70, Audio-Technica, Tokyo, Japan) predominately used for broadband masking and high-frequency stimuli. (Note, high-frequency with regards to the vestibular system, being between 1-2kHz, which is considered low-frequency for the mammalian cochlea). Low-frequency air-conducted sound was delivered to the ear-canal via a silastic tube sealed into the ear-bar opening, which was coupled to a modified ‘sub-woofer’ speaker (Beyerdynamic, Heilbronn, Germany) acting as a volume velocity source. For sound pressure level calibration, a low-noise microphone probe (ER10B, Etymotic Inc., IL, USA) was sealed into the ear-bar opening. For the delivery of vibrational stimuli, an electrodynamic mini-shaker (Type-4810, Brüel & Kjær, Denmark) was attached to the ear-bar in the inter-aural plane via a 5cm metal rod (Fig. 1 ). vCAP recording. To record the vestibular Compound Action Potential (vCAP), the dorsolateral bulla was exposed and opened via a postauricular surgical approach, with the guinea pig laying supine, mounted in custom-made ear-bars (modular setup using components from Thorlabs, NJ, USA). A single channel two-electrode differential recording montage was used to measure vCAPs. Here, the non-inverting (active) electrode was a fabricated 200 µm Ag/AgCl electrode that was inserted ~ 3mm into the bony facial nerve canal, near the vestibular branch of cranial nerve VIII (see. Figure 1 A). The inverting (reference) electrode was a custom-made Ag/AgCl electrode that was inserted into nearby neck musculature. All biopotentials were grounded via a low-resistance earth electrode placed in the nape of the neck, covered in saline-soaked gauze. vCAPs were evoked by transient pulses or tone-burst stimuli; neural origins were confirmed with chemical ablation of vCAPs following tetrodotoxin (TTX; 100µM in artificial perilymph; Sigma Aldrich, AUS) (Supplementary Fig. 1, S1). VM recording. To record localized Vestibular Microphonic (VM) potentials from the basal surface of the utricular macula, the cochlea was surgically exposed and ablated using a ventral surgical approach, to provide a full view of the utricular macular epithelium under the observation of the operating microscope (see. Figure 1 , 3 , & Pastras et al., 2021). The VM was measured using a two-electrode single-ended recording montage. The active electrode was an Ag/AgCl electrode placed into a pulled Borosilicate pipette with a tip diameter of ~ 10µm and backfilled with 250mM of NaCl. The pipette was positioned in the vestibule using a manual 3-axis micromanipulator fixed to an isolation table. The pipette electrode was guided down to the surface of the macula until touching the thin layer of perilymph above the epithelium (see. Pastras et al., 2017). Laser Doppler Vibrometry measurements. A single-point LDV (type 8338 - Brüel & Kjær, Denmark) was used to measure the dynamic response of the utricular macula and stapes during transient vibration and sound stimulation. LDV output was calibrated against a triaxial accelerometer prior to each experiment (Supplementary Fig. 2, S2). To increase the LDV signal strength, reflective microbeads (20µm diam., > 1.93 Refractive Index, Cospheric, CA, USA) were positioned on the macula and stapes under guidance of a surgical microscope. The LDV laser beam was then directed onto the microbead targets via an adjustable optical mirror in 3D (Thorlabs, NJ, USA) (Fig. 1 B). Perilymph build-up over the bead was controlled by the placement of tissue wicks into the vestibule, which minimised artifacts in the LDV recordings due to fluid surface motion effects. When recording vCAP responses, attempts were made to position the bead at the dark band at the centre of the macula, which corresponds approximately to the striolar region (Fig. 1 & Supplementary Fig. 3, S3). However, measures of macular vibration at the lateral striolar region revealed minimal to no differences to that of the central ‘striolar’ zone for pulsatile vibration (Supplementary Fig. 3, S3). This suggested that discrepancies in bead placement across animals did not alter mechanical results based on spatial tuning of the macula. Ear-bar acceleration and jerk. A triaxial piezoelectric accelerometer (Model 832M1-0200, TE connectivity, NSW, Australia) with a frequency response of 2-6000Hz and range of ± 25g, was mounted to the ear-bar frame using a screw thread adapter, in the same plane as the bone-conductor (inter-aural axis). Ear-bar jerk was calculated by taking the first derivative of ear-bar acceleration. Stimuli and recordings. Stimuli and responses were generated and recorded using custom-developed LabVIEW programs (National Instruments, TX, USA). BCV and ACS were generated using a high-resolution external soundcard, USB DAC (SoundblasterX7; Creative Inc., Singapore). Analogue responses were amplified by 80dB (x10,000), with a 0.1Hz to 10kHz band-pass filter (IsoDAM8, WPI, Florida, USA) before being digitized at a rate of 40,000Hz. All responses were averaged using 100 stimulus presentations. Data Availability The datasets used during the current study, as well as the code for data acquisition and analysis are available from the corresponding author on reasonable request. Results vCAP sensitivity with changes in rise-time. Primary striolar afferents and their myogenic counterpart, the VEMP, have been shown to be sensitive to the very onset of the stimulus envelope and are attenuated with increases in the stimulus rise-fall time 46 . However, the associated mechanical activation during vestibular afferent response generation under these conditions is unknown. To examine the stimulation sensitivity of the vestibular striolar afferents, vCAPs were monitored with simultaneous measures of macular epithelial vibration during changes in input drive duration (or rise-time) across several paradigms: Iso-drive, iso-macular velocity, iso-ear-bar acceleration, and iso-ear-bar jerk. The general approach was to examine the stimulation induced changes in the vCAP and associated mechanics in relation to the changes in various stimulus parameters. Iso-drive. Command voltages (drive) supplied to the Bruel & Kjaer minishaker as a 4ms square wave pulse were kept constant, whilst varying the stimulus rise-time between 0-2ms (Fig. 2 A). vCAPs, macular vibration, ear-bar acceleration, and its derivative, ear-bar jerk, were simultaneously measured. All responses declined as a function of drive rise-time, albeit at different rates (Fig. 2 B-E). Normalizing data by the shortest rise-time result revealed changes in vCAP sensitivity (Fig. 5 , red) were closely correlated with the changes in macular velocity (Fig. 3 , blue circles). Both the vCAP amplitude and macular velocity declined approximately linearly with increases in drive rise-time for all stimulus intensities tested (Fig. 3 A & B). By contrast, ear-bar acceleration and ear-bar jerk declined nonlinearly with increased rise-time, with ear-bar jerk displaying a greater rate of decline than acceleration, especially for brief rise-times < 1ms (Fig. 3 A & B). Response amplitudes normalized to the vCAP (normalized by the vCAP magnitude in A-B) reveal a close correlation between vCAP and macular velocity magnitudes (Fig. 3 C & D). Of the two ear-bar kinematic variables, the vCAP (and macular velocity) scaled most closely with ear-bar acceleration (magenta) compared to ear-bar jerk (grey), for all stimuli used during the iso-input drive paradigm (Fig. 3 C & D). Doubling the BCV input command voltage drive (0.03V vs 0.06V) resulted in a doubling of the mechanical response sensitivity, which included macular velocity, ear-bar acceleration, and ear-bar jerk (Fig. 3 E). By comparison, the same two-fold increase in BCV drive resulted in a compressive scaling of the vCAP (~ 1.2-1.5x increase), suggesting vestibular neural output is nonlinear, whereas macular mechanics is linear and passive. Iso-macular velocity. Macular response sensitivity was further characterized by monitoring vCAP response amplitudes during an iso-macular velocity paradigm with associated changes in input drive rise-time. Here, the voltage drive to the minishaker was varied to produce a fixed first negative macular velocity peak (N1) associated with changes in stimulus rise-time between 0 and 2ms (Fig. 4 A). Macular displacement and ear-bar velocity increased linearly as a function of increased BCV drive rise-time associated with constant macular velocity (Fig. 4 B & E). vCAP peak-peak amplitude scaled closely with onset macular velocity and remained consistent between rise-times of 0 to 1ms. However, vCAP sensitivity began to decline between 1 and 2ms, associated with longer BCV rise times (Fig. 4 C). Ear-bar acceleration declined linearly between 0 and 2ms (Fig. 4 D), and ear-bar jerk, declined exponentially (Fig. 4 F) over a 2ms change in stimulus drive rise-time. Iso-ear-bar acceleration and jerk. To further probe the kinematic sensitivity of the vCAP with regards to the ear-bar (and cranium), vCAPs were recorded while keeping ear-bar acceleration (Fig. 5 ), or ear-bar jerk (Fig. 6 ), constant as rise-time was varied. For a fixed ear-bar acceleration (1mG, Fig. 5 A), ear-bar jerk declined exponentially (Fig. 5 C), while ear-bar velocity and macular displacement increased sigmoidally, beginning to saturate at long duration drive rise-times (Fig. 5 B & F). vCAPs increased in amplitude with brief drive rise-times between 0 and 0.5ms, but began to decline with longer rise-times, between 0.5 and 2ms (Fig. 5 D). This is consistent with a switch in vCAP sensitivity from temporal bone acceleration to jerk for longer BCV durations. Macular velocity increased as a function of input rise-time, up until 1.5ms, where epithelial vibration began to decline (Fig. 5 E), consistent with the saturation of macular displacement at long rise times (Fig. 5 F). For a fixed ear-bar jerk (6mG/ms, Fig. 6 A), vCAPs scaled approximately with ear-bar acceleration and macular velocity for brief rise-times (0-0.5ms) (Fig. 6 B, C & D). However, for longer duration rise-times (> 0.5ms), vCAP amplitudes saturated and scaled with ear-bar jerk (Fig. 6 A-B). A fixed ear-bar jerk resulted in a proportional scaling of ear-bar velocity with macular displacement (Fig. 6 D & F), and a similar scaling relationship of ear-bar acceleration to macular velocity (Fig. 6 C & E). Waveform and magnitude data from Figs. 4 – 6 were normalized for each paradigm (Fig. 7 ), showing the relative scaling of each response as a function of input drive rise-time. For iso-macular velocity, iso-ear-bar acceleration, and iso-ear-bar jerk (Fig. 7 A-C), the vCAP scales almost in proportion to macular velocity and ear-bar acceleration for short duration rise-times (< 1ms). However, for longer duration rise-times, the scaling of the vCAP becomes divergent with macular velocity (and ear-bar acceleration) and begins to approximate the scaling of ear-bar jerk. BCV chirps were used to assess the relationship between mechanical activation of the macula and vCAP generation for more complex vibrational stimuli. A 10ms backward chirp (0ms rise-time) with broadband spectra (Fig. 8 A,B) generates a highly synchronous vCAP with robust macular vibration, and relatively broadband ear-bar vibration (Fig. 8 C-F). Increasing the chirp stimulus rise-time from 0ms to 5ms completely abolishes the vCAP response, leaving behind a contralateral Auditory Brainstem Response (ABR), which disappears following contralateral cochlear ablation (data not shown). The ABR response scales closely, in timing and amplitude, with the mid-latency (high frequency) components of ear-bar acceleration and jerk (Fig. 8 F), whereas the vCAP scales closely with onset macular velocity and onset ear-bar acceleration. These results reveal that transient onset stimuli are needed to produce sufficient macular vibration for the synchronization of otolithic afferent responses and the generation of sensory vCAPs. To further probe the relevant stimulus characteristics for evoking transient vestibular responses using broadband input, both backward and forward chirps were used to evoke vCAPs, with corresponding measures of skull vibration (Fig. 9 A-D). Data reveal that the latency and generation of the vCAP closely follows the timing of the low frequency component of the broadband stimulus, with most of its spectral power falling below 1kHz (Fig. 9 A). At its simplest, this follows from the undamped low-pass biomechanics of the otoliths, with a natural frequency around 500Hz. BCV vs ACS vCAP IO functions. To characterize the sensitivity of irregular striolar afferents to increasing levels of mechanical stimulation, pulsatile BCV and ACS stimuli (0.5ms duration, 0.25ms rise-fall) were used to evoke vCAPs, with simultaneous measurements of macular epithelial vibration using LDV. Representative waveforms from one example animal show typical BCV (Fig. 10 A-B) and ACS (Fig. 10 D-E) evoked vCAP responses, along with macular velocity measurements to a range of stimulus intensities. The corresponding IO function (vCAP amplitude vs macular velocity) of these example plots is shown below (Black lines; Fig. 10 C & F), along with the IO function from 18 (BCV) and 11 (ACS) other animals. It should be noted that in this intra-animal comparison, vCAP responses are approximately 5x larger for BCV than ACS stimuli, for a similar level of macular velocity. Furthermore, when comparing vCAP IOs across animals and stimuli (Inter-animal; Fig. 10 C, F & Inset), the overall sensitivity (slope) of vCAP IOs was greater for BCV than ACS stimuli (Fig. 10 C & F), which is represented in the Fig. 10 inset, with ACS & BCV overlaid. Given, ACS relies on impedance matching between the atmosphere and the viscous environment of the inner ear, we wanted to investigate whether sensitivity differences between BCV and ACS vCAPs where due to reduced fluid coupling between the stapes footplate and the macula, providing inadequate mechanical drive to the vestibular hair cells. To investigate these effects, fluid was removed from within the vestibule for both BCV and ACS responses (Fig. 11 A & B) and was compared to recordings made when perilymph was fully covering the macular surface (Fig. 11 C), in the same animal. With fluid in the vestibule, direct LDV measures of macular vibration were infeasible. Instead, as a proxy, we measured stapes footplate vibration with LDV (Fig. 11 C). Without fluid coupling, the vCAP amplitude and IO slope were much smaller for ACS than for the BCV stimulation (Fig. 11 D). When fluid coupling was improved, the ACS vCAP amplitude and IO slope increased to the level of the BCV vCAP IO (Fig. 11 D & E) revealing the differences in the ACS vCAP were likely due to altered fluid coupling and changes impedance matching. This reveals the sensitivity of the utricular nerve in generating synchronized action potentials of short latency is equivalent for punctate ACS and BCV stimuli in the guinea pig. These recordings also provide good estimates for the level of mechanical vibration of the macula and stapes at the threshold of action potential generation in the anaesthetized guinea pig. Results reveal that the magnitude of macular velocity for vCAP threshold is ~ 0.3µm/s for both BCV and ACS (Fig. 11 D). vCAP sensitivity with changes in frequency. BCV and ACS tone bursts are routinely used in the neuro-otology clinic to evoke vestibular reflex responses, such as the VEMP, as a part of a standard assay of otolith function. Although there are mixed data on VEMP tuning curves, likely due to differences across recording setups, the optimal VEMP frequency is generally reported to be around 500Hz. However, the basis for this tuning is unclear relative to mechanical input and the generation of MET currents. To characterize the vCAP frequency response as a proxy from utricular afferent sensitivity across frequency, BCV tone bursts between 100-2000Hz of varying intensity levels were used to evoke a fixed amplitude vCAP, with simultaneous measures of epithelial vibration (iso-vCAP frequency tuning curve; Fig. 12 A). Associated macular velocity, macular displacement, ear-bar acceleration, and ear-bar jerk were also plotted against the frequency of the BCV stimulus (Fig. 12 B-D). Results reveal that for an iso-onset vCAP response (Fig. 12 A), the associated onset macular velocity (taken as the initial N1 transient bump) remains relatively flat across frequency (Fig. 12 A & B), suggesting that the vCAP scales with macular velocity for transient stimuli such as onset tone-bursts and pulses. By comparison, macular displacement declined exponentially with stimulus frequency, with displacement being largest at low frequencies. Ear-bar acceleration approximated a parabolic function over frequency (Fig. 12 C), whereas ear-bar jerk increased exponentially (Fig. 12 D). At low frequencies (< 450Hz), ear-bar jerk was relatively flat and had comparable scaling to the onset vCAP, consistent with the finding that vestibular afferents scale with jerk for spectral power below the natural frequency of the otoliths. Iso-VM Frequency tuning curves. To test the extent vCAP tuning curve was related to pre-synaptic hair cell responses, Iso-macular velocity tuning curves were recorded in the same animal. Voltage drives to the mini shaker were programmatically altered to produce a constant macular velocity across frequency from 100-2000Hz (Fig. 13 A), whilst simultaneously recording the VM, macular displacement, ear-bar acceleration, and total harmonic distortion of the recording system (Fig. 13 B-D). Results reveal that for a fixed macular velocity across BCV frequency (Fig. 13 A), VM amplitude and sensitivity is closely correlated with macular displacement (Fig. 13 B), and this tuning is independent of temporal bone acceleration and distortion in the recording setup (Fig. 13 C-D). Discussion Punctate linear vibration stimuli such as brief hammer or finger taps 47 , transient BCV or ACS stimuli, and tone-bursts delivered by speakers or audiometric bone transducers are routinely used in the clinic or laboratory to evoke robust VEMP and vCAP responses. However, the mechanisms underlying these neurophysiological responses are not well understood. In the present work, we directly measured mechanical vibration of the macula, VMs and vCAPs in guinea pigs to determine how clinically relevant BCV and ACS stimuli evoke synchronized action potentials in the utricular nerve. We first examined the relationship between the BCV stimulus and the vibration of the macula by comparing the peak macular velocity to the peak linear ear-bar acceleration (G) and jerk (G/s) for a series of stimulus strengths. Results in Figs. 4 and 5 demonstrate the peak macular velocity increases roughly in proportion to the acceleration stimulus, consistent with the prediction of simple one degree-of-freedom (1-DOF) models of the utricle for stimuli at or below the corner frequency 48 , 49 . Mechanical simulations using a 2-DOF model of utricular mechanics 50 reproduce the LDV velocities reported here, further confirming that the utricle behaves as a simple inertial sensor that responds to acceleration. The present LDV measurements are consistent with a slightly underdamped mechanical response, exhibiting low-pass sensitivity to sinusoidal inter-aural vibration with a corner frequency near 500 Hz 50 , 51 . In terms of the applied BCV stimulus, present results reveal the vCAP magnitude scales most closely with acceleration for short drive rise-times ( 1ms). These results were reproduced across three experimental paradigms, which included iso-macular velocity (Fig. 4 ), iso-ear-bar acceleration (Fig. 5 ), and iso-ear-bar jerk (Fig. 6 ). For short rise-times, the vCAP magnitude scaled most closely with macular velocity, and ear-bar acceleration, rather than other kinematic components such as macular displacement or ear-bar jerk (or macular acceleration; not shown, or ear-bar displacement; also, not shown). Hence, for very brief BCV stimuli, linear acceleration was the adequate stimulus to generate synchronized vCAPs in the present guinea pig experiments. However, at longer stimulus pulse widths, vCAP scaling approximated the time-derivative of ear-bar acceleration, which is consistent with previous VsEP experiments in rodents where linear jerk was clearly identified as the adequate stimulus to generate evoked responses 41 , 52 . Despite this, there are key differences between the present report and previous VsEP studies that likely underlie the difference in sensitivity including: 1) Animal model: use of the guinea pig ( Cavia porcellus ) in the present report vs. mice (C57BL/6J) or rats (Sprague Dawley); 2) Stimulus: ~3mG inter-aural acceleration at ~ 20mG/ms in the present report vs. ~2000mG nasal-occipital acceleration at ~ 1000mG/s jerk in a supine position; 3) vCAP recording: non-inverting (active) electrode inserted in the facial nerve canal in the present report vs. scalp; 4) Surgical Approach: ablation of the cochlea in the present report vs. keeping the cochlea intact; 5) Anesthetics and medications: isoflurane vs. ketamine/xylazine, and the use of pre-anesthetics medications in the present report, such as opioids, i.e., buprenorphine, and mAChR antagonists, such as atropine, which may alter primary afferent or even efferent neuron sensitivity. Among all of these differences, a theoretical model of mechanical activation of the utricle by BCV 50 suggests the primary determinant of acceleration vs. jerk sensitivity is the frequency content of the stimulus relative to the major corner frequency of the otolith organ in the direction stimulated. Stimuli below the corner are predicted to show jerk sensitivity, while stimuli near the corner are expected to show acceleration sensitivity. Therefore, differences between species in size of the utricle and differences between stimuli likely explain jerk vs. acceleration scaling of the vCAP. A broad-band stimulus would be expected to evoke more complex vCAPs that do not clearly scale with jerk or acceleration. For this reason, we use the term vCAP for compound action potentials evoked by any vestibular stimulus and reserve VsEP for vCAPs that scale with linear jerk. Macular velocity was not recorded in previous VsEP experiments but based on the present results we would expect the relationship between vCAP and macular velocity to hold even for stimuli where the VsEP scales with linear jerk. Chirps are used to evoke cochlear responses in animal models and the clinic, such as the chirp-evoked ABR 53 . Special stimuli have been created to overcome travelling wave delays associated with cochlear mechanics 54 . Recent studies have extended these stimuli to the vestibular system to generate VEMPs 55 , 56 , however, it is unclear how these relatively complex stimuli evoke synchronous neural responses at the end-organ level. Moreover, many of the stimuli which have translated from the cochlea to the vestibular system have been designed to suit unique features of auditory transduction 57 . Hence, it is not apparent if chirps are well suited for otolithic receptor activation. Present results reveal that chirps produce robust macular vibration and sensory vCAPs, providing support for their use as part of the neuro-otology test battery. However, it is important to consider the relevant stimulus characteristics for generating transient vestibular responses. Data shows vCAPs respond to the initial onset or offset of the stimulus waveform, with relevant spectral power below 1kHz (Figs. 8 & 9 ). When the transient onset (or offset) is smoothed by increasing the rise-time, the response drops off abruptly (Fig. 8 ). These results provide a neurophysiological framework for earlier findings, which reported robust VEMPs in humans evoked by band limited chirps (250-1000Hz), chosen because of the ideal sensitivity range of the otoliths 55 , 58 . Hence, a band limited chirp accompanied with a short rise-time should be considered when designing specific parameters to generate VsEPs, vCAPs, or VEMPs in the laboratory or clinic. Moreover, as the relevant power spectrum approximates the natural frequency of the guinea pig utricle (~ 500Hz), these vCAPs scale with temporal bone acceleration, rather than jerk, as predicted by the modelling. This relation will of course change with different stimulus parameters and end-organ properties, as mentioned above. Vibration and sound are excellent stimuli for activating vestibular reflex responses in the clinic, driven by sensitivity of irregular primary afferent neurons 14 . However, precisely how BCV and ACS generate vestibular functional responses at the level of the macula are not well understood. Previous results have demonstrated that there are likely differences in macromechanical activation modes of macular receptors for sinusoidal BCV and ACS across frequencies. That is, the relative phase of the VM and macular velocity evoked by sinusoidal stimuli has been shown to be different for BCV, than for ACS, especially at frequencies beyond 300Hz up to kilohertz 59 . At low frequencies (< 300Hz), both BCV and ACS VMs and macular vibration responses are approximately ‘in-phase’. Beyond this frequency, BCV microphonics undergo a complex phase shift (lag) relative to macular vibration, up to 2–3 cycles at 1kHz. By contrast, the relative phase of the ACS microphonic and macular velocity remains relatively flat up to high frequencies. Despite differences in timing and activation for sinusoidal BCV and ACS hair cell and macular responses, it is not clear if differences exist in neural responses evoked by transient BCV and ACS. This is relevant for understanding the generation of the BCV and ACS VEMP to punctate stimuli at the bedside for diagnostic purposes. To examine differences in BCV and ACS neural response generation, simultaneous measures of the vCAP and macular velocity were recorded by pulsatile stimulation across animals. Without significant perilymph in the vestibule, results reveal larger vCAP input-output (vCAP IO) amplitudes and slopes for BCV compared to ACS (Fig. 10 ). However, further investigation identified this difference as a conductive loss from inadequate ACS stimulus coupling and reduced mechanical sensitivity. Here, discrepancies in vCAP IO slope fell away when recording ACS responses with significant perilymph overlying the macula (Fig. 11 D & E). Hence, pulsatile sound and vibration vCAP responses share equivalent IO functions and macular vibration thresholds to brief BCV and ACS stimuli, suggesting analogous mechanical activation modes of their sensory receptors across stimuli. Simultaneous recordings of macular velocity and vCAP responses provide the ability to quantify the level of epithelial vibration at threshold, and suprathreshold levels such as response saturation. Results indicate that for both vibration and sound stimulation the level of macular vibration for vCAP threshold is < 0.3µm/s across animals. Moreover, the level of macular vibration for vCAP IO response saturation is < 1µm/s. By comparison, for ACS, the level of stapes vibration for vCAP threshold is over an order of magnitude larger than that of the macula, at ~ 25µm/s. To determine how macular vibration is related to MET currents entering sensory hair cells, we compared the VM to the macular velocity and macular displacement for sinusoidal tone bursts. The VM is the voltage modulation in the endolymph relative to reference ground measured adjacent to epithelium and reflects changes in the net MET current entering hair cells caused by hair bundle deflection. Results in Fig. 13 show the VM, and therefore the net MET current, is closely aligned with macular displacement over the entire bandwidth tested. Results are consistent with the hypothesis that hair bundles are deflected primarily by otoconial layer displacement, not velocity, and that hair bundle shear is directly related to the macular displacement measured here using LDV 60 . While the magnitude of VMs measuring the net MET currents scaled with macular displacement, the magnitude of vCAPs measuring the action potential synchronization scaled with macular velocity (Fig. 12 ). This difference highlights rate-sensitive signal processing occurring after the MET current 61 manifests primarily as a time derivative in sensitive calyx bearing afferents that synchronize to transient stimuli. Conclusion This work sought to examine the relationship between macular macromechanics and action potential generation from irregular striolar afferents to improve our understanding of their stimulation sensitivity and tuning to clinical stimulation modes. Unlike previous studies, which characterized the operation of vestibular primary afferents relative to intense cranial acceleration, this work goes one step further and characterizes synchronous vestibular afferent responses (vCAPs) relative to macular epithelial vibration, VMs and their input drives. Results demonstrate that vCAPs increase in proportion to macular velocity for both ACS and BCV, suggesting the mechanical mechanism of MET activation is the same for both stimuli. In contrast to vCAPs, VMs increased in proportion to macular displacement, indicating that the net MET current entering all hair cells was gated primarily by displacement, not velocity. The difference between VM and vCAP dynamics reflects adaptation signal processing interposed between the MET current and action potential generation in sensitive vestibular afferents 25 , 61 , and is the same process responsible for phase-locking of utricular afferent action potentials to audio frequency stimuli 34 . For brief BCV pulses (< 1ms) used in the present study, macular velocities and vCAPs both increased in proportion to temporal bone acceleration. At longer duration BCV pulses, vCAPs increased in proportion to temporal bone jerk, which aligns with previous VsEPs measurements in rodents at lower stimulus frequencies and higher stimulus strengths relative to the present study 52 . Declarations Acknowledgments: This work was supported by a Macquarie University Research Fellowship, MQRF0001126 (CJP), and a NIH DC 006685 (RDR). Competing interests: The authors declare no competing interests. References Straka, H. & Baker, R. Vestibular blueprint in early vertebrates. Front Neural Circuits 7 , 182 (2013). https://doi.org:10.3389/fncir.2013.00182 Eatock, R. A. Specializations for fast signaling in the amniote vestibular inner ear. Integrative and comparative biology 58 , 341–350 (2018). Fuchs, P. A. Vestibular calyx, potassium: Kalium in calyx regnat. 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A mathematical model for mechanical activation and compound action potential generation by the utricle in response to sound and vibration. In review (2022). Dunlap, M. D. & Grant, J. W. Experimental measurement of utricle system dynamic response to inertial stimulus. J Assoc Res Otolaryngol 15 , 511–528 (2014). https://doi.org:10.1007/s10162-014-0456-x Jones, T. A. et al. The adequate stimulus for mammalian linear vestibular evoked potentials (VsEPs). Hear Res 280 , 133–140 (2011). https://doi.org:10.1016/j.heares.2011.05.005 Wegner, O. & Dau, T. Frequency specificity of chirp-evoked auditory brainstem responses. The Journal of the Acoustical Society of America 111 , 1318–1329 (2002). Elberling, C., Don, M., Cebulla, M. & Stürzebecher, E. Auditory steady-state responses to chirp stimuli based on cochlear traveling wave delay. The Journal of the Acoustical Society of America 122 , 2772–2785 (2007). Walther, L. E. & Cebulla, M. Band limited chirp stimulation in vestibular evoked myogenic potentials. European Archives of Oto-Rhino-Laryngology 273 , 2983–2991 (2016). Özgür, A. et al. Comparison of tone burst, click and chirp stimulation in vestibular evoked myogenic potential testing in healthy people. (2015). Elberling, C. & Don, M. A direct approach for the design of chirp stimuli used for the recording of auditory brainstem responses. The Journal of the Acoustical Society of America 128 , 2955–2964 (2010). Cebulla, M. & Walther, L. E. Cervical vestibular evoked myogenic potentials via air conduction delivered by either sequentially or quasi-simultaneously presented narrow-band chirp stimuli. International Journal of Audiology 58 , 174–179 (2019). Pastras, C. J., Curthoys, I. S. & Brown, D. J. Dynamic response to sound and vibration of the guinea pig utricular macula, measured in vivo using Laser Doppler Vibrometry. Hearing Research 370 , 232–237 (2018). https://doi.org:https://doi.org/10.1016/j.heares.2018.08.005 Pastras, C. J., Stefani, S. P., Camp, A. J., Curthoys, I. S. & Brown, D. J. Summating potentials from the utricular macula of anaesthetized guinea pigs. Hear Res 406 , 108259 (2021). https://doi.org:10.1016/j.heares.2021.108259 Songer, J. E. & Eatock, R. A. Tuning and timing in mammalian type I hair cells and calyceal synapses. J Neurosci 33 , 3706–3724 (2013). https://doi.org:10.1523/JNEUROSCI.4067-12.2013 Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 23 Jun, 2023 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 13 Feb, 2023 Reviews received at journal 02 Feb, 2023 Reviewers agreed at journal 21 Jan, 2023 Reviewers agreed at journal 05 Jan, 2023 Reviewers invited by journal 30 Dec, 2022 Editor assigned by journal 21 Dec, 2022 Editor invited by journal 07 Dec, 2022 Submission checks completed at journal 07 Dec, 2022 First submitted to journal 29 Nov, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2323465","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":158128200,"identity":"01452f89-cd1d-4db6-bb55-b602772188fc","order_by":0,"name":"Christopher J. Pastras","email":"data:image/png;base64,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","orcid":"","institution":"Macquarie University","correspondingAuthor":true,"prefix":"","firstName":"Christopher","middleName":"J.","lastName":"Pastras","suffix":""},{"id":158128201,"identity":"a485d3a4-0b43-4c91-8eee-658c415cc919","order_by":1,"name":"Ian S. Curthoys","email":"","orcid":"","institution":"The University of Sydney","correspondingAuthor":false,"prefix":"","firstName":"Ian","middleName":"S.","lastName":"Curthoys","suffix":""},{"id":158128203,"identity":"f6ea7f83-1af6-4f06-8ddb-dc4eb0ae2371","order_by":2,"name":"Richard D. Rabbitt","email":"","orcid":"","institution":"University of Utah","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"D.","lastName":"Rabbitt","suffix":""},{"id":158128205,"identity":"6b012028-f533-4131-bc42-9f4881879c52","order_by":3,"name":"Daniel J. Brown","email":"","orcid":"","institution":"Curtin University","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"J.","lastName":"Brown","suffix":""}],"badges":[],"createdAt":"2022-11-29 05:14:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2323465/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2323465/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-023-37102-3","type":"published","date":"2023-06-23T21:17:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":30229908,"identity":"f7eea240-8a56-4dda-802b-484ee6bb9e25","added_by":"auto","created_at":"2022-12-12 21:56:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":172008,"visible":true,"origin":"","legend":"\u003cp\u003eThe experimental approach to record vestibular afferent and vibration responses. A. Transient BCV and ACS (magenta) were used to evoke B. synchronized vCAPs (blue) recorded from the facial nerve canal in anesthetized guinea pigs. C. Simultaneous measurements of utricular macular vibration (green) were measured via Laser Doppler Vibrometry (LDV).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2323465/v1/7f4e72509ed5f2a6fa352b6e.png"},{"id":30229720,"identity":"0dc57d1f-d726-4274-9eac-fe7eecbe19e2","added_by":"auto","created_at":"2022-12-12 21:48:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":108905,"visible":true,"origin":"","legend":"\u003cp\u003eMacular responses during BCV iso-drive at different stimulus intensities, between 0.032V-0.063V. A. The BCV voltage drive to the mini shaker was kept constant (iso-drive) whilst the stimulus rise fall-time was varied (0-2ms; 0-50%) for a 4ms BCV pulse. Simultaneously measured B. vestibular compound action potentials (red), C. macular velocity (blue), D. ear-bar acceleration (magenta), and its derivative, E. ear-bar jerk (grey). Responses in the left and middle panel correspond to the lowest (0.03V) and highest (0.06V) stimulus intensity, respectively.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2323465/v1/c8f5337b7f41448d7c321ea6.png"},{"id":30229906,"identity":"17fb5408-8df9-4be7-98d4-292e93aa7712","added_by":"auto","created_at":"2022-12-12 21:56:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":93911,"visible":true,"origin":"","legend":"\u003cp\u003eNormalized data from Fig. 2 showing macular response scaling and stimulation sensitivity. A, B. Responses during iso-drive stimulation of the macula normalized to maximal amplitude for 0.032V and 0.063V BCV stimulus intensities, respectively. C, D. Response amplitudes normalized to vCAP amplitude, corresponding to data in A, B, respectively. E. Response scaling with changes in stimulus rise-time associated with a x2 BCV drive increase.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2323465/v1/b19adb5d0c3309d0578838bf.png"},{"id":30229727,"identity":"26ba4cf3-11e7-432f-8f0f-4327c369df8f","added_by":"auto","created_at":"2022-12-12 21:48:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":140918,"visible":true,"origin":"","legend":"\u003cp\u003eIso-macular velocity and associated response waveforms and magnitudes. A. The magnitude of macular velocity (blue) was kept constant (\u003cem\u003eiso-macular velocity\u003c/em\u003e) with changes in drive rise-time (0-50%; 0-2ms) associated with a 4ms BCV pulse. B. Its integral, macular displacement (grey) was quantified, along with C. synchronized vCAPs (red) recorded from the facial nerve canal, D. ear-bar acceleration (magneta), E. its integral, ear-bar velocity (cyan), and F. ear-bar jerk (green).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2323465/v1/06efc72e39677b7ce732a904.png"},{"id":30229722,"identity":"d44ea8cf-bd1d-4c00-8443-1b26c31a08b9","added_by":"auto","created_at":"2022-12-12 21:48:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":107051,"visible":true,"origin":"","legend":"\u003cp\u003eIso-ear-bar acceleration and associated response waveforms and magnitudes. A. The magnitude of ear-bar acceleration (magenta) was kept constant (\u003cem\u003eiso-ear-bar acceleration\u003c/em\u003e) with changes in input drive rise fall time (0-50%; 0-2ms) associated with a 4ms BCV pulse. B. Its integral, ear-bar velocity (cyan), and C. its derivative, ear-bar jerk (green) was quantified, along with D. vCAPs (red), E. macular velocity (blue), and F. its integral, macular displacement (grey).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2323465/v1/4160adae20f2f535878276ac.png"},{"id":30230117,"identity":"aac4b418-df9d-475a-ba19-748e9c5d9212","added_by":"auto","created_at":"2022-12-12 22:04:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":92915,"visible":true,"origin":"","legend":"\u003cp\u003eIso-ear-bar jerk and associated response waveforms and magnitudes. A. The magnitude of ear-bar jerk (green) was kept constant (iso-ear-bar jerk) with changes in input drive rise fall time (0-2ms) associated with a 4ms BCV pulse. B. vCAPs were measured from the facial nerve canal, (red), alongside C. ear-bar acceleration (magenta), D. ear-bar velocity (cyan), E. macular velocity (blue), and F. its integral, macular displacement (grey).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2323465/v1/54d71c0394b3dcf0d85da4ee.png"},{"id":30229731,"identity":"7f9a1700-a675-4b7f-af49-2ce27a868439","added_by":"auto","created_at":"2022-12-12 21:48:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":28065,"visible":true,"origin":"","legend":"\u003cp\u003eNormalized amplitudes associated with changes in stimulus paradigm. Macular velocity (blue), vCAP (red), ear-bar acceleration (magenta), and ear-bar jerk (green) for A. iso-macular velocity, B. iso-ear-bar acceleration, and C. iso-ear-bar jerk. Normalized amplitude plots in A-C correspond to waveforms in Figs 4-6, respectively.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2323465/v1/df4e5f9ba50623e9c15b195d.png"},{"id":30230212,"identity":"a2320596-f62d-45d9-8e8e-7d6598850cb0","added_by":"auto","created_at":"2022-12-12 22:12:36","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":106598,"visible":true,"origin":"","legend":"\u003cp\u003evCAP sensitivity to broadband chirps. A. 10ms BCV chirps with varying rise-times, B. Chirp power spectra, C. measured vCAPs, D. macular velocity using LDV, E. ear-bar acceleration, and F. jerk.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-2323465/v1/b9663dbf2f1ca41867c943f8.png"},{"id":30230213,"identity":"47a2156a-9271-4910-92be-b81ec8ecce57","added_by":"auto","created_at":"2022-12-12 22:12:36","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":94330,"visible":true,"origin":"","legend":"\u003cp\u003eChirp direction and vCAP generation. A. Backward and forward BCV chirps generated B. vCAPs which followed the low-frequency component of the broadband stimulus. C. Simultaneously measured skull jerk, and D. acceleration. Inset: Chirp stimulus power spectrum (hanning window).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-2323465/v1/b0b2836c7d1323dd95b20ec9.png"},{"id":30230119,"identity":"6cefdb41-c1fc-4094-a549-7e2c3fbef337","added_by":"auto","created_at":"2022-12-12 22:04:36","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":153265,"visible":true,"origin":"","legend":"\u003cp\u003eInter-animal comparisons of vCAP Input-Output (IO) functions for BCV and ACS. A. Representative BCV macular velocity and B. vCAP waveforms in 1 animal, with the associated IO curve displayed as the black trace below. C. BCV IO functions across 18 animals. D. Representative ACS macular velocity and E. vCAP waveforms in the same animal, with its corresponding IO curve displayed as the black trace below. F. ACS IO functions across 11 animals. Inset: Overlay comparison of BCV and ACS IO functions. Although, IO thresholds are similar, IO slopes are larger for BCV data.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-2323465/v1/c789dc9672267083f2bdfe6d.png"},{"id":30230122,"identity":"749b3674-3c4f-4126-9a05-19962aef9ca2","added_by":"auto","created_at":"2022-12-12 22:04:36","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":88212,"visible":true,"origin":"","legend":"\u003cp\u003eA. Intra-animal comparisons of vCAP Input-Output (IO) functions for BCV and ACS for Minimal and Maximal Fluid Coupling between the stapes and macula. A, B. Ear-bar acceleration, macular vibration and vCAP waveforms associated with BCV and ACS, respectively, during Minimal Fluid Coupling. C. Ear-bar acceleration, macular vibration and vCAP waveforms during ACS for Maximal Fluid Coupling. D. Corresponding vCAP IO functions associated with waveforms in A, B, \u0026amp; C, above. E. Normalized vCAP IO function data.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-2323465/v1/b82a7078c8192b0dfc29be35.png"},{"id":30229914,"identity":"67d3ca70-7125-4fa3-a55f-eb24bfc5f139","added_by":"auto","created_at":"2022-12-12 21:56:36","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":125465,"visible":true,"origin":"","legend":"\u003cp\u003eIso-vCAP frequency response tuning curve. A. Onset vCAPs were kept constant during a 30ms BCV tone burst (0ms rf) across frequency (up to 1.5kHz), with simultaneous measurements of B. macular velocity, C. ear-bar acceleration, and D. its kinematic derivative, ear-bar jerk. E. Representative waveform comparisons for the onset vCAP, macular velocity, ear-bar acceleration, and ear-bar jerk associated with a 500Hz (black) and 800Hz (coloured) tone-burst, respectively (10ms window). Inset: Entire 50ms time-domain window of the tone-burst response.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-2323465/v1/3dad2e58e8f3284284ecd724.png"},{"id":30230443,"identity":"799c3e1e-397f-40a6-806f-a7572b422d8b","added_by":"auto","created_at":"2022-12-12 22:20:36","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":64920,"visible":true,"origin":"","legend":"\u003cp\u003eVM frequency tuning curve. A. Macular velocity (blue) was kept constant over the full bandwidth (iso-macular velocity), with simultaneous measurements of LDV total harmonic distortion (light blue), B. vestibular microphonics, associated macular displacement, C. VM THD, D. ear-bar acceleration and associated THD.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-2323465/v1/da814c0eedf89e0c3df775bd.png"},{"id":44732424,"identity":"aad1958b-d386-4cea-a7a8-d74a162370b4","added_by":"auto","created_at":"2023-10-16 21:55:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1458842,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2323465/v1/51ca1c9b-0a00-44f2-834e-5bfe35d88308.pdf"},{"id":30229912,"identity":"205c26d8-6c1e-4a8e-9c91-1a986226dd04","added_by":"auto","created_at":"2022-12-12 21:56:36","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2077967,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-2323465/v1/d9d14e1a83d4db7c41ef9b82.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eVestibular Compound Action Potentials and Macular Velocity Evoked by Sound and Vibration in the Guinea Pig\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e","fulltext":[{"header":"Significant Statement","content":"\u003cp\u003eCalyx-bearing afferents in the utricle have the remarkable ability to fire an action potential at a precise time following the onset of a transient stimulus and provide temporal information required for compensatory vestibular reflex circuits, but specifically how transient high-frequency stimuli lead to mechanical activation of hair cells and neural responses is poorly understood. Here, we dissect the relative contributions of mechanics, hair cell transduction, and action potential generation on short-latency responses to transient stimuli. Results provide a framework for the interpretation of synchronized vestibular afferent responses, with relevance to understanding origins of myogenic reflex responses commonly used in the clinic to assay vestibular function, and vestibular short latency potentials commonly used for vestibular phenotyping in rodents.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eVestibular otolith organs are phylogenetically ancient inertial sensors that evolved hundreds of millions of years ago in primitive fish \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and successfully endowed extant land-dwelling vertebrates with the sensory neural inputs necessary for locomotion and navigation in a complex terrestrial environment \u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In amniotes, some otolith afferent neurons preferentially respond to low-frequency gravito-inertial acceleration \u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, while others preferentially respond to high-frequency air conducted sound (ACS) or bone conducted vibration (BCV) \u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The full population of otolith sensory neurons provide the central nervous system with broad-band detection of linear acceleration and head orientation in three-dimensional (3D) space, providing critical inputs to the autonomic nervous system to modulate heart rate and respiration during movements \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and to motor circuits responsible for the vestibular-ocular, -spinal, and -colic reflexes \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The compensatory nature of vestibular circuits makes disorders of the otolith organs particularly debilitating, often leading to sensory conflict and symptoms of dizziness, nausea, blurred vision, anxiety, and disorientation. Otolith function is commonly tested in the clinic using transient ACS or BCV to evoke reflexive cervical or ocular myogenic potentials (VEMPs), but precisely how high-frequency transient stimuli lead to mechano-transduction and neural responses in otolith organs is not well understood.\u003c/p\u003e \u003cp\u003eThe broad dynamic range of otolith sensitivity from DC to several kilohertz \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e arises from diverse properties of hair cells, synapses, and vestibular afferent spike generators \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Amniote neuroepithelia have two major hair cell types (I and II) and two major synaptic terminal types (bouton, calyx, or their combination; dimorphic)\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e which combine with spike generation properties to provide the broad frequency bandwidth and diversity in action potential generation between different afferent neurons. The larger diameter calyx bearing afferents, which evolved in land-dwelling amniotes \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, make synaptic contacts with type-I hair cells in the striolar region of the macula \u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, and are characterized by their irregular action potential discharge rate, phasic responses to maintained stimuli, and sensitive short-latency responses to linear acceleration\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Calyx synaptic terminals completely envelop the lateral and basal surface of one or more type-I hair cells and are exquisitely sensitive to transient stimuli \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Three modes of excitatory synaptic transmission occur at calyx terminals: quantal glutamatergic vesicular release (QT) \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, ultrafast nonquantal ephaptic coupling (NQf) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, and slow nonquantal accumulation of K\u003csup\u003e+\u003c/sup\u003e within the synaptic cleft (NQs) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Direct ephaptic electrical coupling (NQf) is the component responsible for ultrashort latency and high sensitivity of calyx bearing vestibular afferents to transient inputs \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSensitivity of calyx bearing otolith afferent neurons to transient ACS and BCV is routinely exploited in the clinic and the laboratory to test otolith function. In the clinic, reflexive cervical and ocular vestibular evoked myogenic potentials (cVEMP and oVEMP) are used to test saccular and utricular function \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, and in the laboratory short latency vestibular stimulus evoked potentials (VsEP) are used to screen otolith function in mice and other rodents \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. VsEPs are compound action potentials arising from transient stimuli that evoke nearly synchronous firing of a large number of calyx-bearing afferent neurons. When the vestibular compound action potential (vCAP) is recorded from localized sites near the vestibular nerve branch such as the facial nerve canal, the signal-to-noise ratio is enhanced providing recordings similar to auditory CAPs recorded from the round window niche \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. vCAPs reflect combined responses of the population of sensitive afferent neurons and have been recorded in both acute and chronic animal models of health and disease\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Although whole-nerve neural responses to transient ACS and BCV stimuli have been reported for otolith organs, it is currently not known how high frequency transient stimuli lead to mechano-electrical transduction (MET) by sensory hair cells or the generation of synchronized action potentials.\u003c/p\u003e \u003cp\u003eThe present report quantifies the relationship between mechanical vibration of the macula, gating of hair cell MET channels, and generation of vCAPs in the guinea pig utricle for ACS and BCV stimuli. This was achieved by simultaneous measurement of temporal bone acceleration, macular (and stapes) velocity, vestibular microphonics (VMs), and extracellular vCAPs. Results provide new insight into mechanical and receptor mechanisms underlying synchronized neural responses in phasic vestibular afferents, with clinical relevance for understanding vestibular reflex responses, used to diagnose vestibular health and disease at the bedside.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eAnimal preparation \u0026amp; surgery.\u003c/span\u003e Experiments were performed on 28 adult tri-colored guinea pigs (\u003cem\u003eCavia porcellus\u003c/em\u003e) weighing between 300-500g of either sex. All experiments performed in this study were approved by the University of Sydney Animal Care and Ethics Committee (Approval number: #2019/1533). All methods were carried out in accordance with the relevant guidelines and regulations, which included the Australian Code for the Care and Use of Animals for Scientific Purposes (8th edition, 2013), and the ARRIVE guidelines\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Prior to procedures, animals first received pre-anesthetic intraperitoneal injections of Atropine Sulphate; 0.1mg/kg (0.6mg/ml; Apex Laboratories, NSW, Australia) and Buprenorphine Hydrochloride; 0.05mg/kg (Temgesic; 324\u0026micro;g/ml; Reckitt Benckiser, Auckland, NZ). Thereafter, animals were anesthetized in an induction chamber with Isoflurane (2\u0026ndash;4%; Henry Schein, NSW, Australia) saturated in medical O\u003csub\u003e2\u003c/sub\u003e (Coregas, NSW, Australia). Once lacking a foot-withdrawal reflex, guinea pigs were transferred to the surgical table, and received anesthetic via a nose cone, whilst local injections of lignocaine hydrochloride (Lidocaine, Troy Laboratories, NSW, Australia) were delivered to surgery sites. Animals were then tracheotomized and artificially ventilated using Isoflurane (~\u0026thinsp;2%) with oxygen, with the aid of a small animal ventilator (Model 683, Harvard Apparatus, MA, USA). Animals were thereafter rigidly mounted in custom-made ear-bar frames, housing a \u0026lsquo;canalphone\u0026rsquo; speaker (ATH-IM70, Audio-Technica, Tokyo, Japan) predominately used for broadband masking and high-frequency stimuli. (Note, high-frequency with regards to the vestibular system, being between 1-2kHz, which is considered low-frequency for the mammalian cochlea). Low-frequency air-conducted sound was delivered to the ear-canal via a silastic tube sealed into the ear-bar opening, which was coupled to a modified \u0026lsquo;sub-woofer\u0026rsquo; speaker (Beyerdynamic, Heilbronn, Germany) acting as a volume velocity source. For sound pressure level calibration, a low-noise microphone probe (ER10B, Etymotic Inc., IL, USA) was sealed into the ear-bar opening. For the delivery of vibrational stimuli, an electrodynamic mini-shaker (Type-4810, Br\u0026uuml;el \u0026amp; Kj\u0026aelig;r, Denmark) was attached to the ear-bar in the inter-aural plane via a 5cm metal rod (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003evCAP recording.\u003c/span\u003e To record the vestibular Compound Action Potential (vCAP), the dorsolateral bulla was exposed and opened via a postauricular surgical approach, with the guinea pig laying supine, mounted in custom-made ear-bars (modular setup using components from Thorlabs, NJ, USA). A single channel two-electrode differential recording montage was used to measure vCAPs. Here, the non-inverting (active) electrode was a fabricated 200 \u0026micro;m Ag/AgCl electrode that was inserted\u0026thinsp;~\u0026thinsp;3mm into the bony facial nerve canal, near the vestibular branch of cranial nerve VIII (see. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The inverting (reference) electrode was a custom-made Ag/AgCl electrode that was inserted into nearby neck musculature. All biopotentials were grounded via a low-resistance earth electrode placed in the nape of the neck, covered in saline-soaked gauze. vCAPs were evoked by transient pulses or tone-burst stimuli; neural origins were confirmed with chemical ablation of vCAPs following tetrodotoxin (TTX; 100\u0026micro;M in artificial perilymph; Sigma Aldrich, AUS) (Supplementary Fig.\u0026nbsp;1, S1).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eVM recording.\u003c/span\u003e To record localized Vestibular Microphonic (VM) potentials from the basal surface of the utricular macula, the cochlea was surgically exposed and ablated using a ventral surgical approach, to provide a full view of the utricular macular epithelium under the observation of the operating microscope (see. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u0026amp; Pastras et al., 2021). The VM was measured using a two-electrode single-ended recording montage. The active electrode was an Ag/AgCl electrode placed into a pulled Borosilicate pipette with a tip diameter of ~\u0026thinsp;10\u0026micro;m and backfilled with 250mM of NaCl. The pipette was positioned in the vestibule using a manual 3-axis micromanipulator fixed to an isolation table. The pipette electrode was guided down to the surface of the macula until touching the thin layer of perilymph above the epithelium (see. Pastras et al., 2017).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eLaser Doppler Vibrometry measurements.\u003c/span\u003e A single-point LDV (type 8338 - Br\u0026uuml;el \u0026amp; Kj\u0026aelig;r, Denmark) was used to measure the dynamic response of the utricular macula and stapes during transient vibration and sound stimulation. LDV output was calibrated against a triaxial accelerometer prior to each experiment (Supplementary Fig.\u0026nbsp;2, S2). To increase the LDV signal strength, reflective microbeads (20\u0026micro;m diam., \u0026gt;\u0026thinsp;1.93 Refractive Index, Cospheric, CA, USA) were positioned on the macula and stapes under guidance of a surgical microscope. The LDV laser beam was then directed onto the microbead targets via an adjustable optical mirror in 3D (Thorlabs, NJ, USA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Perilymph build-up over the bead was controlled by the placement of tissue wicks into the vestibule, which minimised artifacts in the LDV recordings due to fluid surface motion effects. When recording vCAP responses, attempts were made to position the bead at the dark band at the centre of the macula, which corresponds approximately to the striolar region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026amp; Supplementary Fig.\u0026nbsp;3, S3). However, measures of macular vibration at the lateral striolar region revealed minimal to no differences to that of the central \u0026lsquo;striolar\u0026rsquo; zone for pulsatile vibration (Supplementary Fig.\u0026nbsp;3, S3). This suggested that discrepancies in bead placement across animals did not alter mechanical results based on spatial tuning of the macula.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eEar-bar acceleration and jerk.\u003c/span\u003e A triaxial piezoelectric accelerometer (Model 832M1-0200, TE connectivity, NSW, Australia) with a frequency response of 2-6000Hz and range of \u0026plusmn;\u0026thinsp;25g, was mounted to the ear-bar frame using a screw thread adapter, in the same plane as the bone-conductor (inter-aural axis). Ear-bar jerk was calculated by taking the first derivative of ear-bar acceleration.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eStimuli and recordings.\u003c/span\u003e Stimuli and responses were generated and recorded using custom-developed LabVIEW programs (National Instruments, TX, USA). BCV and ACS were generated using a high-resolution external soundcard, USB DAC (SoundblasterX7; Creative Inc., Singapore). Analogue responses were amplified by 80dB (x10,000), with a 0.1Hz to 10kHz band-pass filter (IsoDAM8, WPI, Florida, USA) before being digitized at a rate of 40,000Hz. All responses were averaged using 100 stimulus presentations.\u003c/p\u003e\n\u003ch3\u003eData Availability\u003c/h3\u003e\n\u003cp\u003eThe datasets used during the current study, as well as the code for data acquisition and analysis are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003evCAP sensitivity with changes in rise-time.\u003c/b\u003e Primary striolar afferents and their myogenic counterpart, the VEMP, have been shown to be sensitive to the very onset of the stimulus envelope and are attenuated with increases in the stimulus rise-fall time \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. However, the associated mechanical activation during vestibular afferent response generation under these conditions is unknown. To examine the stimulation sensitivity of the vestibular striolar afferents, vCAPs were monitored with simultaneous measures of macular epithelial vibration during changes in input drive duration (or rise-time) across several paradigms: Iso-drive, iso-macular velocity, iso-ear-bar acceleration, and iso-ear-bar jerk. The general approach was to examine the stimulation induced changes in the vCAP and associated mechanics in relation to the changes in various stimulus parameters.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIso-drive.\u003c/b\u003e Command voltages (drive) supplied to the Bruel \u0026amp; Kjaer minishaker as a 4ms square wave pulse were kept constant, whilst varying the stimulus rise-time between 0-2ms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). vCAPs, macular vibration, ear-bar acceleration, and its derivative, ear-bar jerk, were simultaneously measured. All responses declined as a function of drive rise-time, albeit at different rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-E). Normalizing data by the shortest rise-time result revealed changes in vCAP sensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e, red) were closely correlated with the changes in macular velocity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e, blue circles). Both the vCAP amplitude and macular velocity declined approximately linearly with increases in drive rise-time for all stimulus intensities tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u0026amp; B). By contrast, ear-bar acceleration and ear-bar jerk declined nonlinearly with increased rise-time, with ear-bar jerk displaying a greater rate of decline than acceleration, especially for brief rise-times\u0026thinsp;\u0026lt;\u0026thinsp;1ms (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u0026amp; B). Response amplitudes normalized to the vCAP (normalized by the vCAP magnitude in A-B) reveal a close correlation between vCAP and macular velocity magnitudes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC \u0026amp; D). Of the two ear-bar kinematic variables, the vCAP (and macular velocity) scaled most closely with ear-bar acceleration (magenta) compared to ear-bar jerk (grey), for all stimuli used during the iso-input drive paradigm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC \u0026amp; D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDoubling the BCV input command voltage drive (0.03V vs 0.06V) resulted in a doubling of the mechanical response sensitivity, which included macular velocity, ear-bar acceleration, and ear-bar jerk (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). By comparison, the same two-fold increase in BCV drive resulted in a compressive scaling of the vCAP (~\u0026thinsp;1.2-1.5x increase), suggesting vestibular neural output is nonlinear, whereas macular mechanics is linear and passive.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIso-macular velocity.\u003c/b\u003e Macular response sensitivity was further characterized by monitoring vCAP response amplitudes during an iso-macular velocity paradigm with associated changes in input drive rise-time. Here, the voltage drive to the minishaker was varied to produce a fixed first negative macular velocity peak (N1) associated with changes in stimulus rise-time between 0 and 2ms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Macular displacement and ear-bar velocity increased linearly as a function of increased BCV drive rise-time associated with constant macular velocity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB \u0026amp; E). vCAP peak-peak amplitude scaled closely with onset macular velocity and remained consistent between rise-times of 0 to 1ms. However, vCAP sensitivity began to decline between 1 and 2ms, associated with longer BCV rise times (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Ear-bar acceleration declined linearly between 0 and 2ms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), and ear-bar jerk, declined exponentially (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) over a 2ms change in stimulus drive rise-time.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIso-ear-bar acceleration and jerk.\u003c/b\u003e To further probe the kinematic sensitivity of the vCAP with regards to the ear-bar (and cranium), vCAPs were recorded while keeping ear-bar acceleration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e), or ear-bar jerk (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), constant as rise-time was varied. For a fixed ear-bar acceleration (1mG, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), ear-bar jerk declined exponentially (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), while ear-bar velocity and macular displacement increased sigmoidally, beginning to saturate at long duration drive rise-times (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB \u0026amp; F). vCAPs increased in amplitude with brief drive rise-times between 0 and 0.5ms, but began to decline with longer rise-times, between 0.5 and 2ms (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). This is consistent with a switch in vCAP sensitivity from temporal bone acceleration to jerk for longer BCV durations. Macular velocity increased as a function of input rise-time, up until 1.5ms, where epithelial vibration began to decline (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), consistent with the saturation of macular displacement at long rise times (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). For a fixed ear-bar jerk (6mG/ms, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), vCAPs scaled approximately with ear-bar acceleration and macular velocity for brief rise-times (0-0.5ms) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C \u0026amp; D). However, for longer duration rise-times (\u0026gt;\u0026thinsp;0.5ms), vCAP amplitudes saturated and scaled with ear-bar jerk (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). A fixed ear-bar jerk resulted in a proportional scaling of ear-bar velocity with macular displacement (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD \u0026amp; F), and a similar scaling relationship of ear-bar acceleration to macular velocity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC \u0026amp; E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWaveform and magnitude data from Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e were normalized for each paradigm (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), showing the relative scaling of each response as a function of input drive rise-time. For iso-macular velocity, iso-ear-bar acceleration, and iso-ear-bar jerk (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C), the vCAP scales almost in proportion to macular velocity and ear-bar acceleration for short duration rise-times (\u0026lt;\u0026thinsp;1ms). However, for longer duration rise-times, the scaling of the vCAP becomes divergent with macular velocity (and ear-bar acceleration) and begins to approximate the scaling of ear-bar jerk.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBCV chirps were used to assess the relationship between mechanical activation of the macula and vCAP generation for more complex vibrational stimuli. A 10ms backward chirp (0ms rise-time) with broadband spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA,B) generates a highly synchronous vCAP with robust macular vibration, and relatively broadband ear-bar vibration (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-F). Increasing the chirp stimulus rise-time from 0ms to 5ms completely abolishes the vCAP response, leaving behind a contralateral Auditory Brainstem Response (ABR), which disappears following contralateral cochlear ablation (data not shown). The ABR response scales closely, in timing and amplitude, with the mid-latency (high frequency) components of ear-bar acceleration and jerk (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF), whereas the vCAP scales closely with onset macular velocity and onset ear-bar acceleration. These results reveal that transient onset stimuli are needed to produce sufficient macular vibration for the synchronization of otolithic afferent responses and the generation of sensory vCAPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further probe the relevant stimulus characteristics for evoking transient vestibular responses using broadband input, both backward and forward chirps were used to evoke vCAPs, with corresponding measures of skull vibration (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-D). Data reveal that the latency and generation of the vCAP closely follows the timing of the low frequency component of the broadband stimulus, with most of its spectral power falling below 1kHz (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). At its simplest, this follows from the undamped low-pass biomechanics of the otoliths, with a natural frequency around 500Hz.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBCV vs ACS vCAP IO functions.\u003c/b\u003e To characterize the sensitivity of irregular striolar afferents to increasing levels of mechanical stimulation, pulsatile BCV and ACS stimuli (0.5ms duration, 0.25ms rise-fall) were used to evoke vCAPs, with simultaneous measurements of macular epithelial vibration using LDV. Representative waveforms from one example animal show typical BCV (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA-B) and ACS (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eD-E) evoked vCAP responses, along with macular velocity measurements to a range of stimulus intensities. The corresponding IO function (vCAP amplitude vs macular velocity) of these example plots is shown below (Black lines; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC \u0026amp; F), along with the IO function from 18 (BCV) and 11 (ACS) other animals. It should be noted that in this intra-animal comparison, vCAP responses are approximately 5x larger for BCV than ACS stimuli, for a similar level of macular velocity. Furthermore, when comparing vCAP IOs across animals and stimuli (Inter-animal; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC, F \u0026amp; Inset), the overall sensitivity (slope) of vCAP IOs was greater for BCV than ACS stimuli (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC \u0026amp; F), which is represented in the Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e inset, with ACS \u0026amp; BCV overlaid.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven, ACS relies on impedance matching between the atmosphere and the viscous environment of the inner ear, we wanted to investigate whether sensitivity differences between BCV and ACS vCAPs where due to reduced fluid coupling between the stapes footplate and the macula, providing inadequate mechanical drive to the vestibular hair cells.\u003c/p\u003e \u003cp\u003eTo investigate these effects, fluid was removed from within the vestibule for both BCV and ACS responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA \u0026amp; B) and was compared to recordings made when perilymph was fully covering the macular surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eC), in the same animal. With fluid in the vestibule, direct LDV measures of macular vibration were infeasible. Instead, as a proxy, we measured stapes footplate vibration with LDV (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eC). Without fluid coupling, the vCAP amplitude and IO slope were much smaller for ACS than for the BCV stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eD). When fluid coupling was improved, the ACS vCAP amplitude and IO slope increased to the level of the BCV vCAP IO (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eD \u0026amp; E) revealing the differences in the ACS vCAP were likely due to altered fluid coupling and changes impedance matching. This reveals the sensitivity of the utricular nerve in generating synchronized action potentials of short latency is equivalent for punctate ACS and BCV stimuli in the guinea pig. These recordings also provide good estimates for the level of mechanical vibration of the macula and stapes at the threshold of action potential generation in the anaesthetized guinea pig. Results reveal that the magnitude of macular velocity for vCAP threshold is ~\u0026thinsp;0.3\u0026micro;m/s for both BCV and ACS (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003evCAP sensitivity with changes in frequency.\u003c/b\u003e BCV and ACS tone bursts are routinely used in the neuro-otology clinic to evoke vestibular reflex responses, such as the VEMP, as a part of a standard assay of otolith function. Although there are mixed data on VEMP tuning curves, likely due to differences across recording setups, the optimal VEMP frequency is generally reported to be around 500Hz. However, the basis for this tuning is unclear relative to mechanical input and the generation of MET currents. To characterize the vCAP frequency response as a proxy from utricular afferent sensitivity across frequency, BCV tone bursts between 100-2000Hz of varying intensity levels were used to evoke a fixed amplitude vCAP, with simultaneous measures of epithelial vibration (iso-vCAP frequency tuning curve; Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAssociated macular velocity, macular displacement, ear-bar acceleration, and ear-bar jerk were also plotted against the frequency of the BCV stimulus (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eB-D). Results reveal that for an iso-onset vCAP response (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eA), the associated onset macular velocity (taken as the initial N1 transient bump) remains relatively flat across frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eA \u0026amp; B), suggesting that the vCAP scales with macular velocity for transient stimuli such as onset tone-bursts and pulses. By comparison, macular displacement declined exponentially with stimulus frequency, with displacement being largest at low frequencies. Ear-bar acceleration approximated a parabolic function over frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eC), whereas ear-bar jerk increased exponentially (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eD). At low frequencies (\u0026lt;\u0026thinsp;450Hz), ear-bar jerk was relatively flat and had comparable scaling to the onset vCAP, consistent with the finding that vestibular afferents scale with jerk for spectral power below the natural frequency of the otoliths.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIso-VM Frequency tuning curves.\u003c/b\u003e To test the extent vCAP tuning curve was related to pre-synaptic hair cell responses, Iso-macular velocity tuning curves were recorded in the same animal. Voltage drives to the mini shaker were programmatically altered to produce a constant macular velocity across frequency from 100-2000Hz (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eA), whilst simultaneously recording the VM, macular displacement, ear-bar acceleration, and total harmonic distortion of the recording system (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eB-D). Results reveal that for a fixed macular velocity across BCV frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eA), VM amplitude and sensitivity is closely correlated with macular displacement (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eB), and this tuning is independent of temporal bone acceleration and distortion in the recording setup (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePunctate linear vibration stimuli such as brief hammer or finger taps\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, transient BCV or ACS stimuli, and tone-bursts delivered by speakers or audiometric bone transducers are routinely used in the clinic or laboratory to evoke robust VEMP and vCAP responses. However, the mechanisms underlying these neurophysiological responses are not well understood. In the present work, we directly measured mechanical vibration of the macula, VMs and vCAPs in guinea pigs to determine how clinically relevant BCV and ACS stimuli evoke synchronized action potentials in the utricular nerve.\u003c/p\u003e \u003cp\u003eWe first examined the relationship between the BCV stimulus and the vibration of the macula by comparing the peak macular velocity to the peak linear ear-bar acceleration (G) and jerk (G/s) for a series of stimulus strengths. Results in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e demonstrate the peak macular velocity increases roughly in proportion to the acceleration stimulus, consistent with the prediction of simple one degree-of-freedom (1-DOF) models of the utricle for stimuli at or below the corner frequency \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Mechanical simulations using a 2-DOF model of utricular mechanics\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e reproduce the LDV velocities reported here, further confirming that the utricle behaves as a simple inertial sensor that responds to acceleration. The present LDV measurements are consistent with a slightly underdamped mechanical response, exhibiting low-pass sensitivity to sinusoidal inter-aural vibration with a corner frequency near 500 Hz \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn terms of the applied BCV stimulus, present results reveal the vCAP magnitude scales most closely with acceleration for short drive rise-times (\u0026lt;\u0026thinsp;1ms), and switches to linear jerk for longer duration rise-times (\u0026gt;\u0026thinsp;1ms). These results were reproduced across three experimental paradigms, which included iso-macular velocity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e), iso-ear-bar acceleration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e), and iso-ear-bar jerk (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). For short rise-times, the vCAP magnitude scaled most closely with macular velocity, and ear-bar acceleration, rather than other kinematic components such as macular displacement or ear-bar jerk (or macular acceleration; not shown, or ear-bar displacement; also, not shown). Hence, for very brief BCV stimuli, linear acceleration was the \u003cem\u003eadequate stimulus\u003c/em\u003e to generate synchronized vCAPs in the present guinea pig experiments. However, at longer stimulus pulse widths, vCAP scaling approximated the time-derivative of ear-bar acceleration, which is consistent with previous VsEP experiments in rodents where linear jerk was clearly identified as the adequate stimulus to generate evoked responses \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Despite this, there are key differences between the present report and previous VsEP studies that likely underlie the difference in sensitivity including: 1) Animal model: use of the guinea pig (\u003cem\u003eCavia porcellus\u003c/em\u003e) in the present report vs. mice (C57BL/6J) or rats (Sprague Dawley); 2) Stimulus: ~3mG inter-aural acceleration at ~\u0026thinsp;20mG/ms in the present report vs. ~2000mG nasal-occipital acceleration at ~\u0026thinsp;1000mG/s jerk in a supine position; 3) vCAP recording: non-inverting (active) electrode inserted in the facial nerve canal in the present report vs. scalp; 4) Surgical Approach: ablation of the cochlea in the present report vs. keeping the cochlea intact; 5) Anesthetics and medications: isoflurane vs. ketamine/xylazine, and the use of pre-anesthetics medications in the present report, such as opioids, i.e., buprenorphine, and mAChR antagonists, such as atropine, which may alter primary afferent or even efferent neuron sensitivity. Among all of these differences, a theoretical model of mechanical activation of the utricle by BCV \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e suggests the primary determinant of acceleration vs. jerk sensitivity is the frequency content of the stimulus relative to the major corner frequency of the otolith organ in the direction stimulated. Stimuli below the corner are predicted to show jerk sensitivity, while stimuli near the corner are expected to show acceleration sensitivity. Therefore, differences between species in size of the utricle and differences between stimuli likely explain jerk vs. acceleration scaling of the vCAP. A broad-band stimulus would be expected to evoke more complex vCAPs that do not clearly scale with jerk or acceleration. For this reason, we use the term vCAP for compound action potentials evoked by any vestibular stimulus and reserve VsEP for vCAPs that scale with linear jerk. Macular velocity was not recorded in previous VsEP experiments but based on the present results we would expect the relationship between vCAP and macular velocity to hold even for stimuli where the VsEP scales with linear jerk.\u003c/p\u003e \u003cp\u003eChirps are used to evoke cochlear responses in animal models and the clinic, such as the chirp-evoked ABR\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Special stimuli have been created to overcome travelling wave delays associated with cochlear mechanics\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Recent studies have extended these stimuli to the vestibular system to generate VEMPs\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, however, it is unclear how these relatively complex stimuli evoke synchronous neural responses at the end-organ level. Moreover, many of the stimuli which have translated from the cochlea to the vestibular system have been designed to suit unique features of auditory transduction\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Hence, it is not apparent if chirps are well suited for otolithic receptor activation. Present results reveal that chirps produce robust macular vibration and sensory vCAPs, providing support for their use as part of the neuro-otology test battery. However, it is important to consider the relevant stimulus characteristics for generating transient vestibular responses. Data shows vCAPs respond to the initial onset or offset of the stimulus waveform, with relevant spectral power below 1kHz (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). When the transient onset (or offset) is smoothed by increasing the rise-time, the response drops off abruptly (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These results provide a neurophysiological framework for earlier findings, which reported robust VEMPs in humans evoked by band limited chirps (250-1000Hz), chosen because of the ideal sensitivity range of the otoliths\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Hence, a band limited chirp accompanied with a short rise-time should be considered when designing specific parameters to generate VsEPs, vCAPs, or VEMPs in the laboratory or clinic. Moreover, as the relevant power spectrum approximates the natural frequency of the guinea pig utricle (~\u0026thinsp;500Hz), these vCAPs scale with temporal bone acceleration, rather than jerk, as predicted by the modelling. This relation will of course change with different stimulus parameters and end-organ properties, as mentioned above.\u003c/p\u003e \u003cp\u003eVibration and sound are excellent stimuli for activating vestibular reflex responses in the clinic, driven by sensitivity of irregular primary afferent neurons\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, precisely how BCV and ACS generate vestibular functional responses at the level of the macula are not well understood. Previous results have demonstrated that there are likely differences in macromechanical activation modes of macular receptors for sinusoidal BCV and ACS across frequencies. That is, the relative phase of the VM and macular velocity evoked by sinusoidal stimuli has been shown to be different for BCV, than for ACS, especially at frequencies beyond 300Hz up to kilohertz\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. At low frequencies (\u0026lt;\u0026thinsp;300Hz), both BCV and ACS VMs and macular vibration responses are approximately \u0026lsquo;in-phase\u0026rsquo;. Beyond this frequency, BCV microphonics undergo a complex phase shift (lag) relative to macular vibration, up to 2\u0026ndash;3 cycles at 1kHz. By contrast, the relative phase of the ACS microphonic and macular velocity remains relatively flat up to high frequencies. Despite differences in timing and activation for sinusoidal BCV and ACS hair cell and macular responses, it is not clear if differences exist in neural responses evoked by transient BCV and ACS. This is relevant for understanding the generation of the BCV and ACS VEMP to punctate stimuli at the bedside for diagnostic purposes. To examine differences in BCV and ACS neural response generation, simultaneous measures of the vCAP and macular velocity were recorded by pulsatile stimulation across animals. Without significant perilymph in the vestibule, results reveal larger vCAP input-output (vCAP IO) amplitudes and slopes for BCV compared to ACS (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). However, further investigation identified this difference as a conductive loss from inadequate ACS stimulus coupling and reduced mechanical sensitivity. Here, discrepancies in vCAP IO slope fell away when recording ACS responses with significant perilymph overlying the macula (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eD \u0026amp; E). Hence, pulsatile sound and vibration vCAP responses share equivalent IO functions and macular vibration thresholds to brief BCV and ACS stimuli, suggesting analogous mechanical activation modes of their sensory receptors across stimuli.\u003c/p\u003e \u003cp\u003eSimultaneous recordings of macular velocity and vCAP responses provide the ability to quantify the level of epithelial vibration at threshold, and suprathreshold levels such as response saturation. Results indicate that for both vibration and sound stimulation the level of macular vibration for vCAP threshold is \u0026lt;\u0026thinsp;0.3\u0026micro;m/s across animals. Moreover, the level of macular vibration for vCAP IO response saturation is \u0026lt;\u0026thinsp;1\u0026micro;m/s. By comparison, for ACS, the level of stapes vibration for vCAP threshold is over an order of magnitude larger than that of the macula, at ~\u0026thinsp;25\u0026micro;m/s.\u003c/p\u003e \u003cp\u003eTo determine how macular vibration is related to MET currents entering sensory hair cells, we compared the VM to the macular velocity and macular displacement for sinusoidal tone bursts. The VM is the voltage modulation in the endolymph relative to reference ground measured adjacent to epithelium and reflects changes in the net MET current entering hair cells caused by hair bundle deflection. Results in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e show the VM, and therefore the net MET current, is closely aligned with macular displacement over the entire bandwidth tested. Results are consistent with the hypothesis that hair bundles are deflected primarily by otoconial layer displacement, not velocity, and that hair bundle shear is directly related to the macular displacement measured here using LDV \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile the magnitude of VMs measuring the net MET currents scaled with macular displacement, the magnitude of vCAPs measuring the action potential synchronization scaled with macular velocity (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). This difference highlights rate-sensitive signal processing occurring after the MET current \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e manifests primarily as a time derivative in sensitive calyx bearing afferents that synchronize to transient stimuli.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis work sought to examine the relationship between macular macromechanics and action potential generation from irregular striolar afferents to improve our understanding of their stimulation sensitivity and tuning to clinical stimulation modes. Unlike previous studies, which characterized the operation of vestibular primary afferents relative to intense cranial acceleration, this work goes one step further and characterizes synchronous vestibular afferent responses (vCAPs) relative to macular epithelial vibration, VMs and their input drives. Results demonstrate that vCAPs increase in proportion to macular velocity for both ACS and BCV, suggesting the mechanical mechanism of MET activation is the same for both stimuli. In contrast to vCAPs, VMs increased in proportion to macular displacement, indicating that the net MET current entering all hair cells was gated primarily by displacement, not velocity. The difference between VM and vCAP dynamics reflects adaptation signal processing interposed between the MET current and action potential generation in sensitive vestibular afferents \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, and is the same process responsible for phase-locking of utricular afferent action potentials to audio frequency stimuli \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. For brief BCV pulses (\u0026lt;\u0026thinsp;1ms) used in the present study, macular velocities and vCAPs both increased in proportion to temporal bone acceleration. At longer duration BCV pulses, vCAPs increased in proportion to temporal bone jerk, which aligns with previous VsEPs measurements in rodents at lower stimulus frequencies and higher stimulus strengths relative to the present study \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments:\u003c/h2\u003e \u003cp\u003e\u003cem\u003eThis work was supported by a Macquarie University Research Fellowship, MQRF0001126 (CJP), and a NIH DC 006685 (RDR).\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStraka, H. \u0026amp; Baker, R. 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Hearing Research \u003cb\u003e370\u003c/b\u003e, 232\u0026ndash;237 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:https://doi.org/10.1016/j.heares.2018.08.005\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.heares.2018.08.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePastras, C. J., Stefani, S. P., Camp, A. J., Curthoys, I. S. \u0026amp; Brown, D. J. Summating potentials from the utricular macula of anaesthetized guinea pigs. Hear Res \u003cb\u003e406\u003c/b\u003e, 108259 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.heares.2021.108259\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.heares.2021.108259\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSonger, J. E. \u0026amp; Eatock, R. A. Tuning and timing in mammalian type I hair cells and calyceal synapses. J Neurosci \u003cb\u003e33\u003c/b\u003e, 3706\u0026ndash;3724 (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1523/JNEUROSCI.4067-12.2013\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1523/JNEUROSCI.4067-12.2013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"inner ear, vestibular, compound action potential, macula, vibration, sound, chirp","lastPublishedDoi":"10.21203/rs.3.rs-2323465/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2323465/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo examine mechanisms responsible for vestibular afferent sensitivity to transient air conducted sounds (ACS) and inter-aural bone conducted vibration (BCV), we performed simultaneous measurements of stimulus-evoked vestibular compound action potentials (vCAPs), utricular macula or stapes velocity, and vestibular microphonics (VMs) in the anaesthetized guinea pig. For short duration punctate stimuli (\u0026lt; 1ms), the vCAP increases magnitude in close proportion to macular velocity and temporal bone (ear-bar) acceleration, rather than other kinematic variables. For longer duration stimuli, the vCAP magnitude switches from temporal bone acceleration sensitive to linear jerk sensitive while maintaining macular velocity sensitivity. vCAP input-output (IO) functions suggest primary afferent response generation has the same origins for both BCV and ACS, with similar macular velocity thresholds and IO functions for both stimuli. Frequency tuning curves evoked by tone-burst stimuli also show the vCAP increases magnitude in proportion to macular velocity, while in contrast, the VM increases magnitude in proportion to macular displacement across the entire frequency bandwidth tested. 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.\u003c/p\u003e","manuscriptTitle":"Vestibular Compound Action Potentials and Macular Velocity Evoked by Sound and Vibration in the Guinea Pig1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-12 21:48:31","doi":"10.21203/rs.3.rs-2323465/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-02-13T06:15:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-02-03T00:56:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"05c300b0-19aa-4b51-9326-e08e1308aeb1","date":"2023-01-22T04:55:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0b0718ca-1c1a-4585-94ba-26f8ace5dc5f","date":"2023-01-05T13:09:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-12-30T10:02:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-12-21T10:03:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-12-07T09:30:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-12-07T09:22:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-11-29T05:04:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e2cc7bbf-bd85-4e4a-b0b9-1396a921c4a0","owner":[],"postedDate":"December 12th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":17603417,"name":"Biological sciences/Neuroscience"},{"id":17603418,"name":"Biological sciences/Physiology"},{"id":17603419,"name":"Biological sciences/Systems biology"},{"id":17603420,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2023-10-16T21:38:35+00:00","versionOfRecord":{"articleIdentity":"rs-2323465","link":"https://doi.org/10.1038/s41598-023-37102-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2023-06-23 21:17:56","publishedOnDateReadable":"June 23rd, 2023"},"versionCreatedAt":"2022-12-12 21:48:31","video":"","vorDoi":"10.1038/s41598-023-37102-3","vorDoiUrl":"https://doi.org/10.1038/s41598-023-37102-3","workflowStages":[]},"version":"v1","identity":"rs-2323465","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2323465","identity":"rs-2323465","version":["v1"]},"buildId":"J0_U0BvcaRcwD8yVFaRlm","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}