Much of our knowledge regarding the properties of HCs comes from ex vivo recordings of the VM from bullfrog otolithic HCs (Corey and Hudspeth, 1983; Azimzadeh and Salvi, 2017). Here, the otolithic maccula (most studies have used the sacculus) is extracted and placed between perilymph/endolymph filled baths in an Ussing chamber (Figure 1A; from Corey and Hudspeth, 1983), with a region of the epithelia exposed to both baths. Vibration is directly applied to the macula, or overlying otolithic membrane (OM), via a stiff probe (Figures 1A,B). Recording the bath potential provides a global measure of the VM generated from the HCs exposed to both baths (i.e., a summed response of all HCs), or alternatively intracellular potentials can be recorded with glass microelectrodes. VM recordings have been made with either the OM intact (Figure 1C), partially removed so as to only stimulate HCs with stereocilia of a particular orientation (Figure 1D), or totally removed. Removing the OM uncouples hair bundle motions from neighboring HCs, and has substantial effects on their excitability and sensitivity (Benser et al., 1993; Dierkes et al., 2008; Fredrickson-Hemsing et al., 2012; Ó Maoiléidigh et al., 2012). With the otolith membrane intact and all HCs are stimulated, the global VM will exhibit a response with twice the frequency of the vibration stimulus (Figures 1C,E). This is because HCs of both polarities are stimulated (Flock, 1965; Corey and Hudspeth, 1983). When only HCs on one side of the line of polarity reversal (Li et al., 2008) are stimulated the VM is cyclic, following the vibration stimulus (Figures 1D,E), although it will saturate at high stimulus levels (Hudspeth and Corey, 1977; Corey and Hudspeth, 1983).

Several other studies have examined the microphonic from the SCC HCs using an ex vivo preparation (De Vries and Bleeker, 1949; Van Eyck, 1951a,b,c; Masetto et al., 1995; Botta et al., 1998; Rabbitt et al., 2005). Here, the polarity of mechanical sensitivity is the same for all hair bundle stereocilia, such that mechanical displacements of the cupula either increases the conductance of all SCC HCs, or decreases it. This results in an asymmetrically distorted microphonic, which can be recorded some distance from the cristae in the vestibular fluids (Botta et al., 1998).

Few studies over the last 50 years have recorded the VM in vivo. This is arguably because evoking the VM requires low-frequency (10–1000 Hz) stimulation, which induces hair bundle displacements (Huizinga and Van Der Meulen, 1951; Trincker and Partsch, 1959; Bleeker et al., 1980; Wit et al., 1981, 1990), yet this will evoke a CM that will dominate the inner ear fluid potentials. That is, compared to VM responses, the CM is large (1–2 millivolts in the perilymph, and several times larger in endolymph; Honrubia et al., 1973) because there is a large electrochemical driving potential for the receptor current through cochlear HCs of +150mV (involving a +90 mV electrogenic potential on the apical surface, and a transmembrane potential of −60 mV; Davis, 1965), whereas the driving potential for the receptor current through HCs in the SCCs, utricle or saccule is most likely to be closer to +65 mV due to a much lower endolymphatic potential (Schmidt, 1963; Ono and Tachibana, 1990; He et al., 1997). Additionally, the CM is large because the polarization of HCs stereocilia sensitivity, within a given region of the cochlea, are aligned in the same direction (Russell, 1983), and cochlea scalae are separated by an epithelium with an electrical impedance of 40–50 kOhm (Johnstone et al., 1966). Conversely, the otolith HCs microphonic will cancel in the fluids due to opposite polarity of HCs either side of the line of polarity reversal, which generates microphonic potentials in the fluids which are 180° out of phase (Corey and Hudspeth, 1983). Furthermore, vestibular HCs are either supported by bone-anchored epithelia, or in the case of the utricle, suspended on a membrane which most likely has an electrical impedance close to 13 kOhm, and therefore the circuit potential related to vestibular HC stimulation will be comparatively low.

Most in vivo studies of the VM have necessarily abolished cochlear function prior to monitoring the VM, and have measured the VM within the inner ear fluids (Adrian et al., 1938; Wever and Vernon, 1956; Trincker and Partsch, 1959; Wit et al., 1981, 1986, 1990). Only a few studies, mostly using fish, have recorded the VM without destroying the cochlea (Zotterman, 1943; Furukawa and Ishii, 1967; Fay and Popper, 1974; Rabbitt et al., 2005; Sisneros, 2007; Yao et al., 2016). VM recordings in fish, particularly zebrafish, are emerging as a powerful tool for studying inner ear developmental biology (Trapani and Nicolson, 2010; Yao et al., 2016). Here, both the lateral line organ and the inner ear (the otic capsule) will respond to alternating pressures and generate microphonic potentials, and differentiating the source of the VM (i.e., explicitly which HCs generate the VM), will be complex due to the small size of the organ.

De Vries and Bleeker (1949) and Van Eyck (1949) were the first to measure VM in vivo, from the SCCs of pigeons. De Vries and Vrolijk (1953), used sinusoidal tympanic membrane displacements to evoke SCC microphonics in pigeons after the cochlea and otoliths had been destroyed. The otoliths were destroyed because they too were stimulated by displacement of the tympanic membrane, and the otolith responses contaminated the SCC responses. Here, the VM was recorded both in the vestibule, and in the SCC after a small hole had been made in the canal wall, which was shown to induce the Tullio effect and enhance SCC responses. Ultimately, the VM from the SCCs demonstrated phase relationships which supported Ewald’s laws, demonstrating highly nonlinear microphonic potentials, where each SCC was maximally stimulated for fluid motion in a given direction. Later Wit et al. (1986) used ACS stimuli, with a SCC fenestration and cochlear extirpation, to evoke VM responses in pigeons (Figure 2). Increasing the level of the stimulus resulted in the response frequency doubling, similar to that obtained with ex vivo experiments where the whole otolith was stimulated (Figure 1C), suggesting that additional vestibular HCs were being recruited with high level ACS, which had a response phase difference of 180°. No attempt was made to separate the response components.

Trincker and Partsch (1959) performed arguably the most extensive in vivo assessment of the VM in mammals, using guinea pigs, and stimulated microphonic potentials from the SCCs, utricle, and saccule, using both BCV and ACS tones, after the cochlea was completely destroyed. Recordings were performed with electrodes within the cochlear fluids, within the SCCs, or within the ampulla. Selective ablation of each end organ was used to confirm the specific origin of the microphonic. VM responses from all vestibular end organs were evoked with sinusoidal stimuli of frequencies between 300 Hz and 120 kHz. Given that CM responses are known to be evoked in mammals by sinusoidal stimuli up to 30 kHz (Cheatham et al., 2011), it seems highly unlikely that either cochlear or vestibular microphonic responses would have been evoked by the ultrasonic stimuli by Trincker and Partsch, and suggests that potentially some of the ultrasonic responses in their study may have included an artifact component.

Ultimately, whilst much research continues to utilize ex vivo measurements of vestibular HCs function, there is a need to substantiate the use of such ex vivo preparations as a reliable measure of the in vivo properties of vestibular HCs. Certainly for cochlear research, the CM remains a mainstay of experimental research measures, and has been used to support and further our understanding of the properties of HCs transduction, derived from intracellular receptor potential measurements (Patuzzi and Sellick, 1983; Patuzzi et al., 1989). For example, the in vivo CM has been used to demonstrate the underlying HCs related cause of many forms of sensorineural hearing loss (Patuzzi et al., 1989), which may have otherwise been attributed to neural dysfunction. Unfortunately, there has been little work done to establish techniques for measuring the VM in vivo, and most in vivo animal studies of the vestibular system are limited to measuring single-unit afferent responses (Fernández and Goldberg, 1976; Curthoys et al., 2006; Curthoys and Vulovic, 2011), single cell receptor potentials (Rabbitt et al., 2005), and VsEP responses (see below). Thus, our understanding of the origin of many forms of vestibular dysfunction may be lacking, as we have not utilized methods that may separate vestibular HCs from neural dysfunction. VM recordings offer an opportunity to perform simple recordings of vestibular HCs sensitivity in vivo, and may demonstrate changes that drive or differ from neural dysfunction.

The majority of VsEP studies have recorded the response with the non-inverting electrode placed subcutaneously at the vertex, or sub-cranially at different locations overlying the cortex. Here, responses typically start with a small (~0.5–1 μV) P1 peak (Figure 3A; which corresponds to the initial peak in facial nerve recordings; (Aran et al., 1980; Jones, 1992; Nazareth and Jones, 1998), and a series of slightly larger positive and negative peaks thereafter (Elidan et al., 1987a; Jones and Pedersen, 1989; Jones and Jones, 1999; Plotnik et al., 1999b; Bremer et al., 2014). This VsEP primarily reflects the response of various vestibular brainstem nuclei and nerves (Nazareth and Jones, 1998), much the same way the ABR reflects central auditory neuron responses (Figure 3B). Importantly, ACS evoked ABR responses are suppressed by acoustic forward-masking noise (Figure 3B), whereas BCV evoked VsEP responses are not (Figure 3A).

VsEP recordings performed with the non-inverting electrode within the cochlea or facial nerve canal will appear similar in waveshape to the cochlear CAP, with an initial negative and positive peak (with amplitudes between 20 μV and 100 μV), termed N1 and P1, with a few smaller peaks thereafter (Böhmer, 1995; Bremer et al., 2012; Chihara et al., 2013); Figure 4A). That said, other studies have suggested that VsEPs recorded within the facial nerve begin with a large positive peak (Oei et al., 2001; Kingma and Wit, 2009), and appear similar to an inverted version of a cochlear CAP. Regardless of the polarity of the first VsEP peak, this activity primarily reflects the compound field potential of the vestibular nerve.

The most widely utilized stimulus for evoking the VsEP involves delivering a rapid, linear-BCV impulse to the skull, in a naso-occipital direction, transduced by a large electrodynamic shaker bolted or clamped to the skull (Figure 5A). This theoretically permits a controlled, rapid push-pull of the animal’s entire head (with <100 μm displacement) in the naso-occipital direction. An extensive examination of the appropriate parameters for evoking the VsEP in mice and rats using this setup has been performed by Jones et al. (Jones and Jones, 1999; Jones et al., 2002; Jones T. A. et al., 2011). Here, it has been suggested that a rapid acceleration of the head, producing a 1 ms to 4 ms pulsed “jerk” (the derivative of acceleration; Figure 5B) is ideal for evoking the VsEP. Indeed, the level of BCV jerk, rather than the level of acceleration, velocity, or displacement, appears to be the main factor determining the amplitude of the VsEP response, and suggests the VsEP is a response of the primary afferents that innervate otolith jerk-sensitive HCs (Jones T. A. et al., 1998; Jones T. A. et al., 2011). Jones T. A. et al. (2011) also suggest that an ideal duration of the linear BCV jerk pulse is approximately 2 ms, which preferentially stimulates the vestibular system, with less cochlear activation. Most studies have demonstrated a reliable VsEP in response to a linear BCV stimulation between 0.5 g and 8 g, or 0.1 g/ms to 6 g/ms.

It should be noted that a 2 ms duration jerk pulse requires an acceleration pulse that increases from zero, peaks at 2 ms, and slowly declines thereafter (Figure 5B). The head velocity change will peak several milliseconds after the onset of the movement, and the peak displacement will occur several milliseconds after that (typically well after the VsEP has occurred). Such a movement of the head can be difficult to produce (particularly for larger heads), but may be necessary to maximally stimulate the jerk-sensitive HCs of the otoliths with minimal cochlear stimulation. Importantly, the head acceleration in this setup is measured on the mechanism attached to the shaker and skull, which arguably may not faithfully represent the acceleration of the vestibular system (Jones et al., 2015). That is, the otolith acceleration may be more complex than that recorded elsewhere in the system, given that the skull can compress and resonate in a complex manner in response to BCV pulses (Durrant and Hyre, 1993), and viscous forces act on the otolith organs (Jones et al., 2015). Moreover, it is not clear how much inter-aural or rostro-caudal movement of the skull is induced by a BCV pulse applied directly to the vertex in a naso-occipital direction.

Other studies have utilized a linear BCV pulse without necessarily controlling for jerk, and most often recording the VsEP from the facial nerve canal (Böhmer, 1995; Kingma and Wit, 2009, 2010; Brown et al., 2013; Chihara et al., 2013). These later studies have all utilized simultaneous acoustic masking to suppress ECochG responses evoked by the BCV click stimulus. Importantly, click-like BCV stimulation can induce a highly synchronized response of the vestibular afferents (Figure 6; Curthoys et al., 2006), where typically only one spike is initiated by the BCV pulse, but the latency of this spike relative to the peak skull acceleration may vary slightly (by 0.2 ms to 0.5 ms) between afferent neurons. This latency variability is most likely related to the indirect nature of measuring skull acceleration as a means of interpreting the displacement of otolith HCs, although it may also demonstrate variability in the response of different HCs to a given vibration of the vestibular end-organ. Regardless of this slight variability, single-unit recordings suggest that the histogram of afferent responses to a BCV-click should be highly synchronized, and therefore the VsEP response should provide a faithful representation of the vestibular nerve field potential. This raises a question—what are the later peaks in the VsEP recorded from the facial nerve canal (Figure 4A)? Are they derived from brainstem activity, or are they a result of a complex resonance of the skull producing multiple successive VsEP responses, or are they the result of different vestibular afferent nerve responses to the BCV stimulus?

Chihara et al. (2013) attempted to determine if the later peaks were the result of a skull-resonance, evoking multiple vestibular nerve responses. Here, we (the experiments were performed in the author’s laboratory) used an audiometric bone conductor rigidly attached to the skull of a guinea pig, with an accelerometer placed nearby on the skull, to deliver a brief linear-BCV stimulus that resulted in an acceleration profile that had minimal later peaks or resonant features (Figure 4B). Acoustic masking was used to suppress cochlear responses. This approach reduced some, but not all of the later components in the VsEP response. Again, it should be realized that skull acceleration responses, particularly at high frequencies, are unlikely to represent the vibration of the end-organ. We have now abandoned this approach, and instead simply deliver brief (0.2–4 ms) monophasic pulses to the bone conductor, which is attached to the ear-bar (Brown et al., 2016). The later peaks in the VsEP responses remain, but we have so far been unable to clarify their origin.

Regardless of the exact vibration of the vestibule, using variants of this setup, several studies have demonstrated that the linear-BCV evoked VsEP is a response of otolith organs. That is, the VsEP remains after cochlear extirpation, or SCC plugging, but is abolished after death (Jones and Jones, 1999; Plotnik et al., 1999b). Moreover, selective otolith destruction abolishes the linear VsEP (Chihara et al., 2013), and otoconia deficient mice have absent or reduced VsEP responses (Jones et al., 1999, 2004). A few studies (Freeman et al., 1999a; Plotnik et al., 1999a) have attempted to stimulate selected vestibular end-organs with pulsed BCV applied in either the naso-occipital, dorso-ventral, or inter-aural directions (along with rotatory pulses), and found similar VsEP response waveforms evoked by all stimuli, but with different response amplitudes. Moreover, Jones et al. (2001) demonstrated in chickens that the initial directional polarity of the linear BCV (relative to the vestibular system), particularly for inter-aural directed stimuli, significantly alters the response waveform. It is not clear if such selective linear BCV stimulation permits a selective activation of the different vestibular end-organs, but this result highlights that that the VsEP is, at least partly, directionally sensitive.

Whilst the linear-BCV evoked VsEP is believed to originate from otolith afferent neurons, several studies have suggested that different stimuli, such as a rapid rotation of the head may generate a SCC afferent VsEP response (Elidan et al., 1982, 1987b; Li et al., 1993; Freeman et al., 1999b; Sohmer et al., 1999). Other studies have used brief low-frequency sinusoidal ACS tones, with fenestration of a given SCC canal, to stimulate a nerve response from the SCC (Wit et al., 1981; Curthoys, 2017). Some studies have suggested that high-intensity ACS can stimulate SCC afferent neurons (Zhu et al., 2014), whereas others have suggested that it does not (Curthoys et al., 2006; Curthoys, 2017). Certainly, it would seem that the otoliths are far more sensitive to transient ACS or BCV than the SCCs. Ultimately, the majority of VsEP studies that have performed additional experimental measures to investigate the origin of the VsEP response, such as selective end-organ ablation, have used a linear-BCV stimulus, and currently more evidence is required to demonstrate that a VsEP can be evoked via a stimulus designed to selectively, or preferentially activate the SCCs afferent neurons.

There are several potential pitfalls that need to be considered when recording EVestG responses. First, most EVestG responses are evoked using BCV stimuli generated by an electrodynamic shaker. This can produce a significant amount of electromagnetic radiation, which should be prevented from radiating to the electrodes using standard techniques such as shielded or twisted cables, and electrical and magnetic shielding of the shaker with grounded MU-metal shielding (Ford et al., 2004). Moreover, BCV of the head can produce significant electrode movement artifact, although electrode stabilization techniques can be of benefit (Comert and Hyttinen, 2015). Using alternating polarity (i.e., reverse direction) BCV stimulation can attenuate much of the artifact in VsEP measurements, but this should only be employed if the VsEP has the same waveshape and latency for either polarity stimuli, otherwise responses may partially cancel. Jones et al. (2002) demonstrated that the VsEP amplitude changed slightly with stimulus polarity, but the latency did not,² and therefore alternating polarity responses could be averaged together to minimize any electrical or movement artifact, with minimal changes to the VsEP waveshape. Both Plotnik et al. (1997) and Jones et al. (2002) demonstrated that the amplitude of the VsEP decreased by up to 15% with increasing stimulus presentation rates, suggesting that an ideal rate should be around 16 per second, which is similar to the ideal repetition rate used for ECochG responses (Eggermont, 1974).

In order to suppress ECochG responses from VsEP recordings, most studies have utilized broad-band acoustic masking noise. This is often necessary because transient BCV stimuli can produce an acoustic click that is transmitted to the cochlea either as an ACS or through direct BCV (Puria and Rosowski, 2012). Acoustic masking noise can either be presented simultaneously with BCV stimulus (Böhmer, 1995; Jones and Jones, 1999; Oei et al., 2001; Chihara et al., 2013), or it can be silenced immediately prior to it (Jones T. A. et al., 2011; King et al., 2017), where forward-masking effects are sufficient to suppress any cochlear responses (Verschooten et al., 2012). It’s not clear if the primary purpose for silencing the masking noise just prior to the BCV stimulus is because the masking noise itself generates CM or electrical artifact, which can contaminate the VsEP response, or if it is believed that the acoustic masking noise may directly interfere with the BCV stimulation of the vestibular system. Several studies have suggested that high levels of noise (>110 dB SPL) can reduce the linear-VsEP amplitude (Böhmer, 1995; Sohmer et al., 1999), particularly if there is a fenestration of the SCC (Wit et al., 1981; Biron et al., 2002). This suggests that the otolith jerk-responsive HCs may be sensitive to high levels of ACS, as is known from single-unit recordings (Curthoys and Vulovic, 2011), and studies have demonstrated that loud noise exposure can produce a permanent reduction in the VsEP (Biron et al., 2002), although this conflicts with previous studies (Sohmer et al., 1999). Nevertheless, moderate continuous or forward-masking acoustic noise most likely provides an adequate suppression of cochlear activity, without overly attenuating otolith responses. Interestingly, Jones and Jones (1999) and Jones et al. (2002) suggest that VsEP responses, recorded with sub-cranial electrodes, are often unaffected by forward masking noise, suggesting that there is little contamination from ABR. This likely reflects the fact that they use a stimulus designed to maximize jerk stimulation of the otoliths, whilst minimizing cochlear stimulation.

Lastly, whilst several studies have demonstrated that the VsEP is a response of peripheral and central vestibular neurones (Nazareth and Jones, 1998; Jones and Jones, 1999; Jones et al., 2002), some studies have suggested that the VsEP measured within the inner ear can contain components that reflect vestibular HCs activity (Wit et al., 1986, 1990). This raises the possibility that there may be an SP-like component of the VsEP when it is measured close to the vestibular HCs. Moreover, it suggests that it may be possible to measure vestibular HCs responses, such as VM, from electrode montages that enable recording of both vestibular nerve and HCs activity.

A concern with interpreting VsEP responses is the uncertainty of which vestibular end-organs contribute to the response. That is, BCV stimuli can induce neural responses from all vestibular end-organs, despite primarily activating otolithic irregular afferent neurons (Curthoys et al., 2006). Whilst researchers have attempted to use the direction of the applied BCV to activate selected vestibular HCs, it is unlikely that this circumvents the complex 3-dimensional vibration of the inner ear and the complex transduction pathways (Stenfelt, 2015, 2016; Chhan et al., 2016). Mechanical engineers are well aware of the complexity of interpreting the vibrational response of a structure via its “impulse response”. An alternative method involves measuring the “steady-state” or continuous vibrational response, where the complexities of the impulse response have dissipated. For the vestibular system, this would involve measuring its response to a continuous sinusoidal linear (or rotatory) BCV stimulus, which should provide a stimulation of the vestibule that is easier to interpret, and would provide a response that could be more readily compared to single-unit recordings obtained during sinusoidal BCV (Curthoys et al., 2006; Curthoys and Vulovic, 2011). Indeed, a few studies have demonstrated that a continuous sinusoidal stimulus can evoke both a sinusoidal VM (Wit et al., 1986) and cyclic neural responses (Wit et al., 1981, 1986 Figure 7). These responses are reminiscent of the auditory nerve neurophonic, used to assess low-frequency sensitivity of the cochlea during a tone (Henry, 1995; Lichtenhan et al., 2014). It may therefore be possible to use sinusoidal ACS or BCV to evoke vestibular neurophonic, and this may provide a means to obtain responses from vestibular neurones which are most sensitive to vibration in a specific direction. Meanwhile, the VsEP obtained using impulse stimuli should assume that the VsEP is “mostly” a response of the afferent neurons synapsing with the jerk-sensitive HCs in the otoliths, with some potential contributions from all vestibular end-organs (see “VsEP Stimulus” Section).

Whilst it may be tempting to use static tilts to probe the origin of the VsEP response, the issue of static head position during VsEP measurements is one which still needs to be resolved. Plotnik et al. (1999a) suggested that, in addtion to changes related to stimulus delivery direction, VsEP responses were altered by the static orientation of the head, suggesting that gravity may alter the sensitivity of the jerk-sensitive HCs. This contrasts with a lack of static head-orientation changes in similar measures otolith function in humans (Kastanioudakis et al., 2016).

Encouragingly, for researchers using the VsEP as a measure of peripheral vestibular function in longitudinal studies, Honaker et al. (2015) demonstrated that the VsEP amplitude and threshold do not change significantly across repeated recordings, which includes repositioning of electrodes (at fixed/standardized positions). Thus, as long as the delivery of the BCV stimulus is consistent between successive recording sessions, the VsEP should provide a sensitive measure of changes in peripheral vestibular sensitivity. It should be noted that response variability will also depend on the signal-to-noise ratio of the response, which greatly depends on the number of averages. For the VsEP measured at the vertex, the response is typically averaged of over 200 times, due to the low signal-to-noise ratio (Jones et al., 2002). To reduce variability in the responses due to the noise-floor of the recording, responses can be band-pass filtered between 300 kHz and 10 kHz (Jones et al., 2002), although these filter settings were obtained for VsEP responses recorded at the vertex, and may differ for VsEP responses measured in the periphery.

An important factor to consider when monitoring VsEP responses during an intervention, is how to assess changes. Previously, many studies have monitored the peak-to-peak amplitude of the response, however because the later peaks in the VsEP reflect central responses, they may be altered without an equivalent change in the 8th nerve’s sensitivity, resulting in changes in the VsEP waveform (Jones et al., 2000; Morley et al., 2017). Therefore, VsEP thresholds should ideally be used to assess changes in the sensitivity of the irregular otolith afferents, although changes in the VsEP waveform, such as changes in inter peak intervals and peak latencies, may provide additional information. That said, the source of the later peaks in VsEP responses recorded from the vertex is not as well defined as the origin in the later peaks in ABR responses (Kaga et al., 1997), although several studies have used electrical source analysis to localize VsEP activity (Todd et al., 2014, 2017).

One final issue to consider is the potential influence of anesthetics on EVestG responses (Gaines and Jones, 2013). Although anesthesia is known to suppress certain cortical activity, there seems to be little difference in the VsEP measured at the vertex, between anesthetized and awake animals, other than a suppression of a late (>7 ms) component, which may potentially reflect cortical vestibular activity (Jones, 1992). Nonetheless, it is possible that different anesthetics may induce changes in the VsEP response, particularly of the later, central components.