The mammalian efferent vestibular system plays a crucial role in vestibulo-ocular reflex compensation after unilateral labyrinthectomy

Loss of the α9-nicotinic acetylcholine receptor (α9-nAChR) subunit utilized by the efferent vestibular system (EVS) has been shown to significantly affect vestibulo-ocular reflex (VOR) adaptation. In our present study we have shown that loss of α9-nAChRs also affects VOR compensation, suggesting that the mammalian EVS plays an important role in vestibular plasticity, in general, and that VOR compensation is a more distributed process than previously thought, relying on both central and peripheral changes.

Abstract

The α9-nicotinic acetylcholine receptor (α9-nAChR) subunit is expressed in the vestibular and auditory periphery, and its loss of function could compromise peripheral input from the predominantly cholinergic efferent vestibular system (EVS). A recent study has shown that α9-nAChRs play an important role in short-term vestibulo-ocular reflex (VOR) adaptation. We hypothesize that α9-nAChRs could also be important for other forms of vestibular plasticity, such as that needed for VOR recovery after vestibular organ injury. We measured the efficacy of VOR compensation in α9 knockout mice. These mice have deletion of most of the gene (chrna9) encoding the nAChR and thereby lack α9-nAChRs. We measured the VOR gain (eye velocity/head velocity) in 20 α9 knockout mice and 16 cba129 controls. We measured the sinusoidal (0.2–10 Hz, 20–100°/s) and transient (1,500–6,000°/s²) VOR in complete darkness before (baseline) unilateral labyrinthectomy (UL) and then 1, 5, and 28 days after UL. On day 1 after UL, cba129 mice retained ~50% of their initial function for contralesional rotations, whereas α9 knockout mice only retained ~20%. After 28 days, α9 knockout mice had ~50% lower gain for both ipsilesional and contralesional rotations compared with cba129 mice. Cba129 mice regained ~75% of their baseline function for ipsilesional and ~90% for contralesional rotations. In contrast, α9 knockout mice only regained ~30% and ~50% function, respectively, leaving the VOR severely impaired for rotations in both directions. Our results show that loss of α9-nAChRs severely affects VOR compensation, suggesting that complimentary central and peripheral EVS-mediated adaptive mechanisms might be affected by this loss.

NEW & NOTEWORTHY Loss of the α9-nicotinic acetylcholine receptor (α9-nAChR) subunit utilized by the efferent vestibular system (EVS) has been shown to significantly affect vestibulo-ocular reflex (VOR) adaptation. In our present study we have shown that loss of α9-nAChRs also affects VOR compensation, suggesting that the mammalian EVS plays an important role in vestibular plasticity, in general, and that VOR compensation is a more distributed process than previously thought, relying on both central and peripheral changes.

in a recent study, we investigated whether the α9-nicotinic acetylcholine receptor (α9-nAChR), important for the mammalian efferent vestibular system (EVS), plays a role in vestibular plasticity mechanisms that drive vestibulo-ocular reflex (VOR) adaptation (Hübner et al. 2015). We compared the VOR response of α9 knockout mice, a mouse strain that lacks α9-nAChRs due to deletion of most of the gene (chrna9) encoding that receptor (Vetter et al. 1999), with data obtained in cba129 (control) mice. In cba129 mice, α9-nAChRs interact with acetylcholine (ACh), the main neurotransmitter of the EVS, to alter the output of the vestibular peripheral organs (Gacek and Lyon 1974; Goldberg and Fernández 1980; Highstein 1991; Marco et al. 1993; Purcell and Perachio 1997). In our previous study in α9 knockout mice, we demonstrated that loss of the α9-nAChR subunit causes a significant reduction (>70%) in the capacity for VOR adaptation compared with controls. Thus the utilization of α9-nAChRs by the mammalian EVS appears to be essential for plasticity mechanisms involved in VOR adaptation. However, it is unclear if VOR compensation, another form of vestibular plasticity involved in the (partial) restoration of static and dynamic deficits after vestibular organ injury, is similarly dependent on the EVS (Curthoys 2000; Dieringer 1995, 2003; Vibert et al. 1997).

Historically, the process of vestibular compensation was thought to be independent from plasticity mechanisms involved in the adaptive modification of the VOR response (e.g., during visual-vestibular mismatch adaptation and vergence-mediated gain changes). Increasing evidence, however, suggests that vestibular compensation is a distributed process involving numerous parallel events at various sites in the brain and vestibular periphery. Some of these processes are closely related to mechanisms that are active during VOR adaptation (Beraneck et al. 2004; Dieringer 1995, 2003; Dutia 2010; Straka et al. 2005; Vibert et al. 1999a, Vibert et al. 1999b). For example, in mice with cerebellar dysfunction (Lurcher mutants) it was shown that long-term cerebellum-dependent motor learning is just as important for VOR compensation after unilateral labyrinthectomy (UL) (Aleisa et al. 2007; Beraneck et al. 2008; Faulstich et al. 2006) as it is for VOR adaptation (Koekkoek et al. 1997; Van Alphen et al. 2002). The restoration of dynamic deficits (e.g., reduced VOR response gains; gain = eye/head velocity) seems to be especially dependent on intact cerebellar innervation of brain stem vestibular nuclei (Beraneck et al. 2004). Furthermore, both forms of vestibular plasticity (VOR adaptation and compensation) were found to rely on an increase in gain of phasic peripheral signals (i.e., irregularly discharging primary vestibular afferents) as well as an increase in the proportion and sensitivity of phasic central neurons in the brain stem vestibular nuclei (i.e., type-B neurons) (Clendaniel et al. 2001, 2002; Lasker et al. 1999, 2000). This observation is based on studies in monkeys and humans, which facilitated the development of a mathematical model of the VOR consisting of two parallel signal processing pathways: a velocity-sensitive pathway with tonic signal characteristics and an acceleration-sensitive pathway with phasic signal characteristics (Clendaniel et al. 2001, 2002; Lasker et al. 1999, 2000; Migliaccio et al. 2003, 2004, 2008; Minor et al. 1999b). The response dynamics of these tonic and phasic pathways resemble those of regularly and irregularly discharging vestibular primary afferents (Hullar and Minor 1999; Hullar et al. 2005) as well as type A and type B second-order vestibular neurons (2° VN) in the brain stem (Dickman and Angelaki 2004). The phasic pathway is highly modifiable and predominantly mediates VOR changes observed in response to spectacle-induced adaptation (Clendaniel et al. 2001, 2002), viewing of targets at close distances (a vergence-mediated gain change) (Migliaccio et al. 2004, 2008), and VOR compensation after vestibular canal plugging and UL (Lasker et al. 1999, 2000).

It is well established that information from contralesional primary vestibular afferents and 2° VN substantially contributes to the restoration of VOR responses following a unilateral vestibular lesion (e.g., UL) (Cartwright and Curthoys 1996; Curthoys 2000; Dieringer 1995; Graham and Dutia 2001; Smith and Curthoys 1988a, Smith and Curthoys 1988b). Notably, long-term changes in support of the findings by Minor and colleagues have been observed in primary vestibular afferents as well as 2° VN following UL (Beraneck et al. 2004; Cullen et al. 2009; Sadeghi et al. 2007; Straka et al. 2005). In particular, the proportion of contralesional primary vestibular afferents with phasic response dynamics (i.e., irregularly discharging afferents) was shown to increase following UL, whereas the number of afferents with tonic response dynamics (i.e., regularly discharging afferents) decreased proportionally (Sadeghi et al. 2007). A similar transition toward more phasic signal properties was also observed in contralesional 2° VN following UL in guinea pigs (i.e., sensitivity and proportion of phasic type B neurons increase following a vestibular lesion) (Beraneck et al. 2004). α9-nAChRs and their utilization by the EVS could play a critical role in facilitating this increase in contribution (sensitivity and proportion) of phasic peripheral signals. Specifically, an efferent-mediated tuning of dimorphic afferents and their respective inputs from type I and type II vestibular hair cells could be the mechanism responsible for the proportional change observed in primary vestibular afferents (Beraneck and Idoux 2012; Cullen et al. 2009; Sadeghi et al. 2007; for more explanation see Hübner et al. 2015).

The goal of the present study was to determine if VOR compensation after unilateral vestibular lesion relies on the availability of α9-nAChRs. We investigated the time course of vestibular compensation in α9 knockout mice and compared findings with results in cba129 (control) mice. The functional implications of a compromised EVS were evaluated at 4 time points over the course of the study: before UL surgery and 1 day (24 h), 5 days, and 28 days after UL surgery. At each point we tested VOR responses by using a wide range of vestibular stimuli, including horizontal sinusoidal rotations (0.1 to 10 Hz, with peak velocities of 20, 50, and 100°/s) and transient steps of acceleration (1,500, 3,000, and 6,000°/s², reaching constant velocities of 100, 150, and 300°/s). As shown for VOR adaptation, our findings demonstrate that α9-nAChRs also play an essential role in facilitating VOR compensation after a unilateral vestibular lesion.

METHODS

Animals and Surgical Preparation

A total of 20 α9 knockout mice and 16 controls (both sexes, ages 11–14 wk) were used in this study. The VOR response for baseline, day 1, day 5, and day 28 after UL was measured in 13, 11, 16, and 11 α9 knockout mice, respectively. Similarly, the VOR response for baseline, day 1, day 5, and day 28 after UL was measured in 11, 11, 11, and 12 cba129 mice, respectively. The baseline VOR response of cba129 and α9 knockout mice was described in detail by Hübner et al. (2015). The mouse strain carrying the α9-knockout mutation has been maintained on a CBA/CaJ × 129/SvEv background line by The Jackson Laboratory (Bar Harbor, ME; stock no. 005696). We set up an independent colony of hybrid CBA/CaJ (Jackson Laboratory; stock no. 000654) × 129/SvEv (Taconic Biosciences; model no. 129SVE) (from here on referred to as “cba129”) that we used as the controls. When the homozygous α9 knockout breeders became old, new breeders were selected from different heterozygous (cba129 × α9 knockout) breeding pairs. To ensure that the cba129 mice we used as controls throughout this study were valid, we compared their baseline and post-UL data with that from five homozygous wild-type littermates obtained from heterozygous breeding pairs. Genotyping was performed externally by Garvan Molecular Genetics (Sydney, Australia) from ear punches using the same protocols for mouse genotyping as Jackson Laboratory. The genotyping was performed using real-time polymerase chain reaction (PCR) in a 384-well plate format. The primer sequences and combinations used were provided by Jackson Laboratory and were as follows: α9 knockout, CAC GAG ACT AGT GAG ACG TG; cba129 forward, TCT GGT GCT GGG AAT CAA AT; and common, AGC CCC AGA ACC TCT GTT TT. Samples were processed via liquid handlers and analyzed on a LightCycler 480 with a Sito9-based Meltcurve analysis. Sample peaks were compared with those of a cba129 control. The initial controls were from our independent colony of cba129 mice but were then substituted by homozygous wild-type littermates obtained from heterozygous breeding pairs after PCR confirmed they had two chrna9 alleles. One complex PCR analysis was performed to differentiate between α9 knockout, heterozygous, and wild-type littermate control mice.

All mice were implanted with a custom-built head immobilization device consisting of a lightweight low-profile metal adapter plate permanently attached to the skull and a removable head pedestal attached to the adapter plate before each experimental session. The exact implantation technique has been outlined previously (Hübner et al. 2014). Following adapter plate implantation, mice were allowed to recover from the surgery for at least 3 days before the first experimental session (baseline VOR recording before UL). Unilateral labyrinthectomy was performed during a second surgery 1–4 days after the pre-lesion (baseline) VOR response was measured. Following UL, animals recovered in a normal visual environment (Shinder et al. 2005) under close monitoring.

All surgical and experimental procedures were approved by the Animal Care and Ethics Committee of the University of New South Wales and were in strict compliance with the Australian Code of Practice for the Care & Use of Animals for Scientific Purposes.

Unilateral Labyrinthectomy

Preparation.

Unilateral labyrinthectomy (UL) procedures were performed under full aseptic technique to avoid postoperative infections. Similar to the adapter plate implantation procedure for the head immobilization system, mice were anesthetized in an induction chamber using a mix of isoflurane (1.5–3%) and oxygen (3 l/min). Once anesthetized, mice were transferred to a nonsterile preparation area where anesthesia was maintained via nose cone. The eyes were treated with an ophthalmic ointment, and a generous area around the incision site (~5 mm around the auricle) was razor shaved and disinfected using a swab of betadine and 70% ethyl alcohol (see Fig. 1A). A subcutaneous (SC) injection of 0.03 ml of carprofen (5 mg/ml) was given as analgesic and antibiotic treatment, and 0.5 ml of sterile saline was injected intraperitoneally (IP) to prevent dehydration of the animals during surgery. After preparations were complete, mice were transferred to the prepared surgical field. At this point nonsterile examination gloves were removed and sterile surgical gloves were worn. Mice were draped using a transparent film dressing (Opsite Flexigrid 4630; Smith & Nephew USA, Andover, MA) with a small window cut out at the incision site.

Fig. 1.

Surgical approach: unilateral labyrinthectomy (UL; performed only on left ear). A: anterior auricular incision. B: the external auditory meatus was visualized and sectioned close to the tympanic membrane. C: fractioning and curettage of the posterior, superior, and inferior aspects of the bony tympanic annulus and removal of the malleus and incus uncovered the tympanic bulla of the middle ear. D: the tympanic bony annulus was further enlarged until the oval window and stapes footplate, the entire round window niche, and the stapedial branch of the internal carotid artery could be clearly visualized. E: the stapes was carefully lifted off the oval window in a gentle twisting motion to expose the vestibule. F: visualization of the vestibular end organs. G: the vestibule was packed with Gelfoam soaked in gentamicin. H: the tympanic cavity of the middle ear was further packed with dry Gelfoam and tissue fragments, and the skin was closed using interrupted sutures.

Surgical procedure.

UL surgery was performed only on the left ear. The procedure was conducted using a partial transcanal approach. Lidocaine and bupivacaine (≤0.02 ml, mixed 1:1) were injected as a local field block, and an anterior auricular incision was made (Fig. 1A). The external auditory meatus was visualized and sectioned close to the tympanic membrane (Fig. 1B). The peripheral end of the sectioned external auditory meatus was closed using a purse-string suture to avoid entry of particles and bacteria into the body. A retractor system was used to keep the incision site open to allow better visualization. The tympanic membrane, bony tympanic annulus, and horizontal section of the facial nerve were further exposed, and the tympanic membrane was removed. Fractioning and curettage of the posterior, superior, and inferior aspects of the bony tympanic annulus and removal of the malleus and incus uncovered the tympanic bulla of the middle ear (Fig. 1C). Spontaneous bleeding from the fractured, highly vesiculated bone edges was controlled using an application of adrenaline and Gelfoam. The tympanic bony annulus was further enlarged until the oval window and stapes footplate, the entire round window niche, and the stapedial branch of the internal carotid artery could be clearly visualized (Fig. 1D). To gain access to the inner ear and vestibular sensory organs, the stapedial artery had to be sacrificed. A fine monopolar electric cautery tool was used to cauterize the artery on both sides of the stapes footplate before it was sectioned and removed. The stapes was then carefully lifted off the oval window in a gentle twisting motion to expose the vestibule (Fig. 1E). Care was taken not to fracture the stapes in the process of removing it. Gentle suction was used to aspirate endolymph and perilymph fluid exiting the vestibule. Every effort was made to avoid accidental aspiration of the vestibular end organs at this time. These last steps ensured optimal visualization of the oval window. Once efflux of endolymph and perilymph from the vestibule ceased, the oval window was carefully out-fractioned. This provided access to the inner ear and improved visualization of the vestibular end organs (Fig. 1F). At this point the utricle, a large oval epithelial patch with whitish color, was clearly visible. With the use of fine biological forceps (Moria MC40 ultra fine forceps), the sensory epithelia of the utricle, as well as those of the lateral and anterior semicircular canals, were carefully excised (Fig. 1F). The saccule was destroyed mechanically by aspiration of the medial aspect of the vestibule. Because of the location of the posterior canal cupula, direct access and extraction using forceps was not possible. Postmortem dissection of the inner ear, however, confirmed that suction applied to the openings of lateral and anterior canals destroyed the posterior crista, as well. All manipulations inside the vestibule, especially those close to the medial aspect, were performed with great care to avoid fracture of the very thin internal wall between vestibule and brain cavity.

After complete destruction of the vestibular end organs was confirmed visually, the vestibule was packed with Gelfoam soaked in 1 µl of gentamicin-buffered solution (20 mg/ml; Troy Laboratories, Glendenning, Australia) (Fig. 1G). This guaranteed destruction of the residual neuroepithelium, especially in areas that were hard to access. The tympanic cavity of the middle ear was further packed with dry Gelfoam and tissue fragments, and the skin was closed using interrupted sutures (Fig. 1H). A liquid tissue adhesive was applied to the suture to prevent entry of bacteria. Before the animal was taken off the anesthetic gas and returned to its cage for recovery, it was given buprenorphine (0.02 ml SC; 32.4 μg/ml) as analgesic treatment and another dose of sterile saline (0.5 ml IP) to aid in hydration. Analgesic treatment using SC injection of buprenorphine (twice a day) was continued for up to 3 days postsurgery or until the animal recovered. Mice regained consciousness 10–15 min after inhalation anesthesia was discontinued. They immediately displayed a strong head tilt with the labyrinthectomized ear down, clearly deviated posture, and a strong tendency to body roll and circle toward the affected ear. In addition, mice displayed clearly visible spontaneous nystagmus with quick-phase eye movements “beating” away from the lesioned side. When picked up by the tail, these mice started to vigorously spin about the main axis of their body. The degree of postoperative vestibular disturbance was used as a measure of successful UL surgery. Body roll, circling, and spontaneous nystagmus usually ceased 1–2 days postsurgery. Head tilt, deviated posture, and spinning when picked up by the tail persisted and were still present several months after surgery.

Data Acquisition and Analysis

The method of recording three-dimensional binocular eye movements with the use of high-speed video-oculography and the technique used for offline analysis of VOR responses have been detailed in several previous studies (Hübner et al. 2013, 2014, 2015; Migliaccio et al. 2011). VOR responses were measured before vestibular organs were surgically lesioned using the UL approach (pre-lesion baseline recording) and following vestibular lesion at three time points, 1 day (24 h), 5 days, and 28 days postsurgery, to study the acute impaired, acute compensated, and chronic compensated VOR responses, respectively. At each individual time point during the recovery process, we measured VOR responses in darkness over the complete range of naturally occurring mouse head movement frequencies, velocities, and accelerations (Beraneck et al. 2008) using horizontal, whole body sinusoidal rotation at 0.2, 0.4, 0.5, 0.8, 1, 1.6, 2, 5, and 10 Hz with peak velocities of 20, 50, and 100°/s, and transient acceleration stimuli at 1,500, 3,000, and 6,000°/s² reaching a constant velocity plateau of 100, 150, and 300°/s, respectively (in the text, we refer to transient stimulus conditions as 1.5k100, 3k150, and 6k300). For a more detailed explanation of the rotational stimuli, see Hübner et al. (2015).

To analyze the three-dimensional VOR data, we converted eye movements acquired in eye coordinates into rotation vectors in head coordinates. Eye velocity traces with quick phases removed were inverted so that an ideal VOR would yield a gain (eye velocity/head velocity) of +1 and a phase of 0°. Positive phase lead denotes eye velocity leading head velocity. VOR responses to sinusoidal rotations and transient steps of acceleration were analyzed separately for rotations toward the operated ear (ipsilesional) and rotations toward the healthy ear (contralesional). To measure the slow-phase VOR response to sinusoidal rotations, least-square pure sine waves were fit to each individual cycle of the head velocity stimulus and eye velocity response. A least-square pure sine wave was fit to the eye velocity trace using the model shown in Eq. 1 to determine sinusoidal amplitude, phase, and constant offset.

$$M_{\text{Sine}}=a\cdot \sin \left(2\pi ft\right)+b\cdot \cos \left(2\pi ft\right)+c$$ (1)

where a = amplitude·cos(phase), b = amplitude·sin(phase) and c = offset.

The eye and head velocity trace amplitudes were used to calculate the sinusoidal VOR gain. Because VOR gain responses were expected to vary between ipsilesional and contralesional rotations, eye velocity traces were divided into a positive and negative half-wave, representing the VOR response to rightward and leftward head rotations. The traces were split at the point where the full-cycle sinusoidal head velocity fit intersected the zero-velocity axis, corresponding to a direction change in position. For half-wave sinusoidal analysis, Eq. 1 was modified such that the offset c was fixed to the value determined during the full-cycle fit. This was necessary because sinusoidal half-waves do not carry enough information to reliably estimate this parameter.

For transient steps of acceleration, we fit least-square linear regressions to the constant-acceleration and constant-velocity part of eye and head velocity traces. Using these fits, we calculated three parameters: acceleration gain (G_A), constant-velocity gain (G_V), and latency. G_A was calculated as the average ratio of eye-to-head acceleration (using the slopes of the constant-acceleration fit). G_V was calculated as the average ratio of eye-to-head velocity (using the point-by-point offset of the constant-velocity fit) during the 200- to 400-ms interval after stimulus onset.

During the initial 2–3 days after UL, animals exhibited a marked spontaneous nystagmus, which typically offset the sinusoidal slow-phase VOR response. We estimated the velocity offset c caused by spontaneous nystagmus by calculating the average slow-phase velocity over an interval of 10 to 50 ms before onset of the vestibular sinusoidal stimulus. This is a common approach to estimating the VOR response offset caused by spontaneous nystagmus (e.g., Beraneck et al. 2008; Fetter and Zee 1988; Sadeghi et al. 2006).

Statistical Analysis

Data were analyzed in R (R Core Team 2013), using a general linear mixed model with two-factor interaction (nlme package; Pinheiro et al. 2014). Post hoc pairwise comparisons of group means were performed using Bonferroni’s correction for two-sample t-statistics. All statistical tests were performed using a significance level of P = 0.05. Unless stated otherwise, all measurement results are means ± SD.

RESULTS

VOR Response Before UL

Figure 2A shows the baseline sinusoidal VOR gains from 0.2 to 10 Hz for cba129 control mice (dashed black lines) and wild-type littermates (dashed gray lines) obtained from heterozygous breeding pairs. There was no difference in baseline sinusoidal VOR between the cba129 control mice used throughout our analysis and wild-type littermates across frequencies (gain: F_1,377 = 0.64, P = 0.43; phase: F_1,376 = 2.97, P = 0.09) and peak velocities (gain: F_1,389 = 1.31, P = 0.26; phase: F_1,388 = 1.32, P = 0.25).

Fig. 2.

Vestibulo-ocular reflex (VOR) response for cba129 and wild-type littermate control mice. A: baseline sinusoidal VOR gains from 0.2 to 10 Hz for cba129 control mice (dashed black lines) and wild-type littermates (dashed gray lines) obtained from heterozygous breeding pairs. There was no difference in the baseline sinusoidal VOR gain between mouse types. The same pre-lesion gains are included in each panel. VOR gains measured on days 5 (top) and 28 (bottom) after UL (solid lines) show there was no difference in sinusoidal VOR gain recovery between mouse types. B: baseline transient VOR gain in response to the initial constant acceleration of transient step stimuli (G_A) and gain in response to the constant-velocity plateau of transient step stimuli (G_V) for cba129 control mice (black columns to left of vertical dashed line) and wild-type littermates (gray columns to left of vertical dashed lines). The data shown to the right of the vertical dashed lines represent the gains on days 1, 5, and 28 after UL. There was no difference in transient VOR gain recovery between mouse types.

The baseline VOR response of cba129 control and α9 knockout mice in this study are essentially the same as those described in Hübner et al. (2015). In brief, both mouse types showed a velocity-dependent gain increase (α9 knockout: F_2,281 = 70.2, P < 0.001; cba129: F_2,181 = 78.8, P < 0.001; difference between mice: F_1,1201 = 102.9, P < 0.001), whereas cba129 mice showed a frequency-dependent gain increase (peak velocity 20°/s: F_8,72 = 5.11, P < 0.001; peak velocity 50°/s: F_8,62 = 6.1, P < 0.001) that was absent in α9 knockout mice (20°/s: F_8,89 = 0.65, P = 0.73; 50°/s: F_8,97 = 1.42, P = 0.19). The VOR gain of α9 knockout mice was significantly lower than that of cba129 controls for frequencies >2 Hz, especially for peak velocities of 50 and 100°/s (F_1,87 = 12.2, P < 0.001 and F_1,83 = 23.5, P < 0.001, respectively). The overall VOR phase was similar for α9 knockout and cba129 mice (F_6,138 = 1.1, P = 0.36), averaging 5.07 ± 2.59° at 0.2 Hz and increasing to −6.22 ± 4.35° at 10 Hz when stimulus peak velocity was 100°/s (F_1,275 = 520.7, P < 0.001).

Figure 2B shows the baseline transient VOR G_A (gain in response to the initial constant acceleration of transient step stimuli) and G_V (gain in response to the constant velocity plateau of transient step stimuli) for cba129 control mice (black columns to left of vertical dashed line) and wild-type littermates (gray columns to left of vertical dashed lines) obtained from heterozygous breeding pairs. There was no difference in baseline transient VOR between cba129 controls and wild-type littermates for G_A (F_1,191 = 1.91, P = 0.18), G_V (F_1,190 = 0.93, P = 0.34), and latency (F_1,191 = 1.31, P = 0.26). In α9 knockout mice, G_A was significantly reduced at 1.02 ± 0.21 compared with cba129 control mice, which had an average G_A of 1.14 ± 0.19 (F_1,24 = 4.44, P < 0.05). In contrast, G_V was similar between the two mouse types (F_2,72 = 0.45, P = 0.64). Gains were symmetrical (similar for rotations to either side) for both the initial acceleration and constant velocity part of the VOR response under all tested stimulus conditions.

Sinusoidal VOR Response After UL

Figure 2A shows the sinusoidal VOR gains from 0.2 to 10 Hz for cba129 control mice (black lines) and wild-type littermates (gray lines) on days 5 (top row) and 28 (bottom row) after UL. There was no difference in sinusoidal VOR between cba129 controls and wild-type littermates across days after UL (gain: F_1,1653 = 0.62, P = 0.43; phase: F_1,1652 = 0.06, P = 0.80), across frequencies (gain: F_1,1641 = 0.17, P = 0.68; phase: F_1,1640 = 1.17, P = 0.28) and peak velocities (gain: F_1,1653 = 0.001, P = 0.99; phase: F_1,1652 = 0.21, P = 0.65).

Slow-phase eye velocity bias.

VOR gain and phase were measured separately for sinusoidal rotation half-cycles directed toward the ipsilesional and contralesional ear for stimulus frequencies ranging from 0.2 to 10 Hz with peak velocities of 20, 50, and 100°/s. Figure 3A shows the typical VOR response of a cba129 control mouse, evoked by sinusoidal rotations (1 Hz, with a peak velocity of 50°/s) on day 1 after UL. Figure 3B shows the head and eye velocity traces of the same mouse for >10 stimulus cycles superimposed with quick phases removed. Note the considerable slow-phase velocity bias toward the intact ear and the response asymmetry between ipsilesional and contralesional rotations. This nonzero bias in eye velocity was most pronounced on day 1 after UL and decreased substantially as VOR compensation progressed (F_1,31 = 20.8, P < 0.001). In controls, the average eye velocity bias during sinusoidal vestibular stimulation on day 1 [measured as the average slow-phase eye velocity offset as the stimulus changed direction (zero-cross of stimulus velocity trace)] was 7.87 ± 4.32°/s. Five days after UL, this bias mostly resolved with an average offset of 0.12 ± 0.29°/s. On day 28, the average offset was 0.07 ± 0.37°/s.

Fig. 3.

VOR response after UL. A: typical VOR response of a cba129 (control) mouse, evoked by sinusoidal rotations (1 Hz, with a peak velocity of 50°/s) on day 1 after UL. B: head and eye velocity traces of >10 stimulus cycles were superimposed and quick phases removed (inv, inverted eye velocity traces). Slow-phase eye movement responses were fit using individual least-squares sinusoidal half-cycles for ipsilesional (dashed filled squares) and contralesional rotations (dashed open squares), respectively. A significant asymmetry between ipsilesional vs. contralesional VOR gains was observed on day 1 following UL surgery. Note that ipsilesional VOR responses were virtually absent and that the slow-phase eye velocity traces had a nonzero bias due to spontaneous nystagmus. C: typical VOR response of an α9 knockout mouse, evoked by sinusoidal rotations (1 Hz, with a peak velocity of 50°/s) on day 1 after UL. D: head and eye velocity traces of >10 stimulus cycles were superimposed and quick phases removed. Compared with cba129 mice, α9 knockout mice demonstrated very low VOR gains and only minimal asymmetry between ipsilesional vs. contralesional rotations.

Figure 3, C and D, shows the typical VOR response of an α9 knockout mouse under the same test and analysis conditions as shown in Fig. 3, A and B. Compared with cba129 mice, α9 knockout mice developed a significantly lower slow-phase velocity bias after UL surgery (F_1,28 = 9.3, P < 0.01). The average bias in α9 knockout mice when measured on day 1 was only 2.12 ± 2.76°/s, ~70% smaller compared with that in controls (T₁₁₀₀ = 26, P < 0.001). Similarly to that in controls, this bias substantially decreased over subsequent days and was minimal when evaluated on day 5 (0.06 ± 1.01°/s) and day 28 (0.05 ± 0.27°/s). In both mouse types, the observed slow-phase velocity bias was consistent across stimulus velocities (F_1,123 = 0.0, P = 0.863) but increased with stimulus frequency (F_1,1294 = 10.0, P < 0.005).

Time course of VOR compensation.

Figure 4 shows VOR responses to sinusoidal rotations (0.2 to 10 Hz at 100°/s) measured on days 1, 5, and 28 following UL (solid lines). These VOR gains were compared with cba129 control (filled symbols) and α9 knockout mice (open symbols) pre-lesion responses (dashed lines). Figure 5 shows ipsilesional (black) and contralesional (gray) VOR gains for cba129 (solid lines) and α9 knockout mice (dashed lines) for peak velocities of 20, 50, and 100°/s (data were pooled for stimuli ≥1 Hz). On day 1 (24 ± 2 h) after UL, gains were significantly reduced in both mouse types and for all test stimulus frequencies and velocities. In cba129 mice, the average gain on day 1, pooled across frequencies and velocities, was 0.22 ± 0.13 for ipsilesional and 0.42 ± 0.22 for contralesional rotations. An even larger decrease in gain was observed in α9 knockout mice, with average gain of 0.10 ± 0.08 for ipsilesional and 0.13 ± 0.11 for contralesional rotations. Moreover, gain asymmetry between stimulus directions was less pronounced in α9 knockout compared with cba129 mice. Post-lesion VOR gains on day 1 did not change with stimulus peak velocity, as was the case before UL (F_1,42 = 2.748, P = 0.105).

Fig. 4.

VOR gain recovery across sinusoidal frequencies. VOR gains in response to sinusoidal rotations (0.2 to 10 Hz at 100°/s) were measured on days 1, 5 (acute compensated), and 28 (chronic compensated) after UL (solid lines). Dashed lines show the pre-lesion VOR response. After 28 days of recovery, VOR gains in cba129 mice recovered ≤80% of pre-lesion function, whereas VOR gains in α9 knockout mice remained significantly impaired at ≤30% of pre-lesion function.

Fig. 5.

VOR gain recovery across sinusoidal peak velocities. Comparison of ipsilesional (black) and contralesional (gray) VOR gains between cba129 (solid lines) and α9 knockout mice (thick dashed lines). Gains are shown for peak velocities 20, 50, and 100°/s (data were pooled for stimuli ≥1 Hz). VOR gains of cba129 mice recovered most for a stimulus peak velocity of 50°/s. α9 knockout mice showed only minimal VOR gain recovery compared with cba129 mice. VOR gain asymmetry in cba129 mice was maximal on day 1 but recovered successively until day 28. In contrast, α9 knockout mice showed no VOR gain asymmetry on day 1. VOR gain asymmetry on day 28 was similar between α9 knockout and cba129 mice.

For both mouse types, the VOR gain in both directions was significantly improved on day 5 compared with day 1 (F_1,1279 = 59, P < 0.001); however, it remained low compared with pre-lesion values. For cba129 mice, the average ipsilesional gains on day 5 pooled across stimulus peak velocities ranged from 0.12 ± 0.06 at 0.2 Hz to 0.49 ± 0.13 at 10 Hz. Similar recovery of gain was also observed for rotations toward the contralesional side, with gains ranging from 0.34 ± 0.22 at 0.2 Hz to 0.64 ± 0.18 at 10 Hz. For cba129 mice, there was a significant effect of stimulus frequency on both ipsilesional and contralesional VOR gains (F_1,47 = 8.30, P < 0.01). VOR gain recovery was minimal at peak velocity 20°/s and most pronounced at peak velocity 100°/s (F_1,47 = 51.97, P < 0.001; Fig. 5). The average gain increase (pooling ipsilesional and contralesional directions) between days 1 and 5 at 20°/s (for frequencies ≥2 Hz) was only 0.05 ± 0.03, whereas the average gain increase at 100°/s was 0.20 ± 0.03. For α9 knockout mice, the average ipsilesional VOR gain ranged from 0.07 ± 0.03 at 0.2 Hz to 0.33 ± 0.12 at 10 Hz, and the average contralesional gain ranged from 0.18 ± 0.10 at 0.2 Hz to 0.40 ± 0.13 at 10 Hz. This means that 5 days after UL, the VOR gain of α9 knockout mice remained on average 0.24 ± 0.01 lower than that of cba129 mice.

Cba129 mice continued to show significant recovery of VOR response gains between days 5 and 28 after UL (F_1,1390 = 173.6, P < 0.001). Both ipsilesional and contralesional gains recovered most for frequencies between 1 and 5 Hz. The average gain for ipsilesional rotations (pooled across 1 to 5 Hz) ranged from 0.54 ± 0.13 at peak velocity 20°/s to 0.69 ± 0.13 at peak velocity 100^o/s. VOR gains for contralesional rotations averaged 0.59 ± 0.13 and 0.80 ± 0.10 at peak velocities 20 and 100°/s, respectively. α9 knockout mice also had VOR recovery. Between days 5 and 28, ipsilesional gains improved to 0.14 ± 0.07 at 0.2 Hz and 0.36 ± 0.07 at 10 Hz, and contralesional gains improved to 0.27 ± 0.13 at 0.2 Hz and 0.37 ± 0.08 at 10 Hz. The average gain increase between days 5 and 28, pooled across all frequencies and peak velocities, was only 0.09 ± 0.01 in α9 knockout mice compared with 0.20 ± 0.01 in cba129 controls, indicating that the rate of recovery was significantly lower in α9 knockout mice (F_1,47 = 43.89, P < 0.001). On day 28, the difference in VOR gains between mouse types increased with frequency when the data at 10 Hz were excluded (F_8,247 = 11.6, P < 0.001). The apparent decrease in gain at 10 Hz between days 5 and 28 was due to additional animals being included at each time point. When only animals tested at both time points were included, the difference between days was no longer significant (cba129: F_1,51 = 0.001, P = 0.97; α9 knockout: F_1,51 = 0.57, P = 0.45).

Figure 6 shows the VOR phase on day 5 after UL (acute compensated), plotted across frequencies and days (pooling 0.2-, 1-, and 10-Hz phases) post-lesion in cba129 (solid lines) and α9 knockout mice (dashed lines). Phase leads decreased with increasing frequency. VOR phase in response to ipsilesional and contralesional rotations was significantly affected by UL (F_8,838 = 2.97, P < 0.001). On day 1 after UL, there was a marked increase in phase lead at all test frequencies and velocities. In cba129 mice, the average phase at 0.2 Hz was 51.75 ± 4.88° for ipsilesional and 15.95 ± 3.10° for contralesional rotations. This phase lead was even larger in α9 knockout mice, which had an average phase of 75.06 ± 25.13° and 49.23 ± 42.38° for ipsilesional and contralesional rotations, respectively. Smaller increases in phase were observed at 10 Hz. In cba129 mice, phase at 10 Hz was similar to pre-lesion values at −0.68 ± 8.58° for ipsilesional and −2.52 ± 6.02° for contralesional rotations (F_1,116 = 2.3, P = 0.10 and F_1,116 = 2.43: P = 0.14, respectively). In contrast, α9 knockout mice displayed significantly increased phase lag compared with pre-lesion values, with an average phase lag of 11.46 ± 19.13° and 14.86 ± 19.59° for ipsilesional (F_1,155 = 2.61, P = 0.11) and contralesional (F_1,155 = 35.5, P < 0.001) rotations, respectively.

Fig. 6.

VOR phase recovery across frequencies. A: VOR phase on day 5 after UL (acute compensated), plotted across frequencies. Changes in VOR phase following UL were similar in cba129 and α9 knockout mice. Only at 10 Hz did we observe a difference between the two mouse types. B: VOR phase recovery in cba129 (solid lines) and α9 knockout mice (dashed lines) at 3 frequencies. Phase leads decreased with increasing frequency. VOR phase was significantly more positive on day 1 for α9 knockout mice compared with controls. At 10 Hz, a larger phase lead for α9 knockout mice was still present 28 days after UL.

For cba129 mice, the VOR phase in response to ipsilesional rotations minimally normalized between days 1 and 5 after UL. On average, the phase lead decreased by 5.15 ± 2.73° for frequencies <1 Hz (T_65.9 = 1.89, P = 0.063) and remained unchanged for frequencies ≥1 Hz (T_99.8 = 0.53, P = 0.592). Similarly, VOR phase recovery in response to contralesional rotations was limited. For frequencies <1 Hz, contralesional phase lead decreased by 6.93 ± 1.33° between days 1 and 5 (T_69.9 = 5.19, P < 0.001). At frequencies ≥1 Hz, phase did not improve (T_101.3 = 1.24, P = 0.217). VOR phase showed continued recovery toward pre-lesion responses so that at 28 days after UL, phase was close to normal for frequencies ≥5 Hz (ipsilesional: T_136.94 = 2.55, P < 0.01; contralesional: T_145.4 = 1.01, P = 0.315). At frequencies <5 Hz, VOR phase also significantly improved, effectively halving the VOR phase lead compared with that on day 5; however, phase remained considerably increased compared with pre-lesion values. VOR phase leads at frequencies <1 Hz were similar between α9 knockout and cba129 mice. For ipsilesional rotations, only α9 knockout phase responses were slightly larger at 4.90 ± 2.38° on day 5 and 2.73 ± 1.42° on day 28, compared with controls (T_118.7 = 2.06, P < 0.05 and T_66.90 = 1.92, P = 0.059, respectively). However, phase in α9 knockout mice was significantly more positive than that in cba129 mice at frequencies >1 Hz for both ipsilesional and contralesional rotations (Fig. 6). The largest difference in phase between cba129 and α9 knockout mice was observed at 10 Hz (ipsilesional: 9.80° on day 5 and 11.66° on day 28; contralesional: 6.78° on day 5 and 10.20° on day 28).

In summary, VOR compensation in α9 knockout mice was severely impaired compared with that in cba129 controls. Figure 7 compares VOR gain recovery between cba129 (filled circles) and α9 knockout mice (open circles). Response gains were normalized to pre-lesion values for each vestibular stimulus condition to compute the percentage of recovery. For mid-range frequencies (between 2 and 5 Hz), the ipsilesional VOR response recovered to ~75% of its pre-lesion function in cba129 mice compared with ~30% recovery in α9 knockout mice. For cba129 mice, the VOR response to contralesional rotations recovered to ~90% of its pre-lesion function. The initially large asymmetry between ipsilesional and contralesional cba129 VOR responses continuously reduced from ~50% on day 1 after UL to ~20% on day 5 and <8% on day 28. However, VOR responses to both ipsilesional and contralesional rotations remained significantly impaired for low and high frequency extremes (<1 Hz and >5 Hz) and for the lowest peak velocity tested (20°/s). In contrast, the rate of recovery in α9 knockout mice was significantly lower than in controls, and VOR recovery was minimal for frequencies ≥5 Hz.

Fig. 7.

Percentage of function relative to pre-lesion. VOR gain recovery was compared between cba129 (filled triangles) and α9 knockout mice (open circles). Response gains were normalized to pre-lesion values for each vestibular stimulus condition to compute the percentage of recovery. Ipsilesional VOR responses on day 1 were similar between mouse types. However, α9 knockout mice showed only minimal functional recovery 28 days after UL, leaving the ipsilesional VOR significantly impaired. Contralesional VOR responses on day 1 were significantly lower in α9 knockout mice compared with controls. Although the time course of contralesional VOR recovery was similar between cba129 and α9 knockout mice, VOR responses after 28 days remained significantly lower in the latter.

Transient VOR Response After UL

Figure 2B shows the transient VOR G_A and G_V for cba129 control mice (black columns to right of vertical dashed line) and wild-type littermates (gray columns to right of vertical dashed lines) on days 1, 5, and 28 after UL. There was no difference in transient VOR between cba129 controls and wild-type littermates across days after UL (G_A: F_1,366 = 1.21, P = 0.27; G_V: F_1,377 = 0.001, P = 0.99; latency: F_1,374 = 0.01, P = 0.92).

Slow-phase eye velocity bias.

The post-lesion eye velocity bias due to spontaneous nystagmus was defined as the average slow-phase eye velocity 50–300 ms before transient stimulus onsets. A significant bias was only observed on day 1 after UL (F_1,226 = 50.71, P < 0.001), with average slow-phase velocities of 9.25 ± 7.15°/s for cba129 mice and 5.55 ± 7.73°/s for α9 knockout mice (F_1,22 = 4.06, P = 0.056). Five days after UL this bias completely resolved. Before the VOR gain and latency were calculated, the slow-phase bias for each eye velocity trace was subtracted so that VOR gain and latency described the dynamic VOR response to head rotation.

Time course of VOR compensation.

Figure 8 shows an example of the acute compensated VOR response after UL in a cba129 control mouse (top row). A large asymmetry between ipsilesional and contralesional VOR gain was observed during the initial acceleration part of the stimulus (G_A), but not during the subsequent constant-velocity part (G_V). The deacceleration after ipsilesional rotations also triggered a large VOR eye movement response. Measurements on day 1 (~24 h) after UL surgery confirmed severely impaired VOR responses for all mice tested. VOR gain reduction was most pronounced for rotations toward the lesioned ear. The average G_A in cba129 (control) mice was 0.25 ± 0.09 for ipsilesional rotations and 0.81 ± 0.36 for contralesional rotations. In comparison, α9 knockout mice had an average G_A of 0.24 ± 0.07 for ipsilesional rotations and 0.57 ± 0.26 for contralesional rotations. Ipsilesional G_A was similar for both mouse types (F_1,30 = 0.0, P = 0.528), whereas the contralesional VOR gain was significantly lower in α9 knockout mice (F_1,24 = 21, P < 0.001). This resulted in a significantly lower asymmetry between ipsilesional and contralesional rotations compared with cba129 mice. In cba129 mice, we observed a significant increase in G_A as contralesional stimulus acceleration increased (F_1,10 = 8.5, P < 0.02). At 6,000°/s², the highest acceleration tested, G_A was similar to the response before UL. A similar increase with contralesional acceleration, however, was absent in α9 knockout mice (F_1,13 = 2.6, P = 0.133). We did not observe an acceleration effect on ipsilesional VOR responses of either mouse type (F_1,31 = 0.0, P = 0.577).

Fig. 8.

Transient VOR response on day 5 after UL. Typical VOR response is shown for a cba129 mouse (top row) and α9 knockout mouse (bottom row) on day 5 after UL. A large asymmetry between ipsilesional and contralesional VOR gain was observed during the initial acceleration part of the stimulus (G_A), but not during the subsequent constant-velocity part (G_V).

Figure 9 shows the acceleration gain (G_A; top row) and constant-velocity gain (G_V; bottom row) measured in cba129 (black) and α9 knockout mice (gray) before and 1, 5, and 28 days after UL (stimulus: 3,000°/s² reaching 150°/s constant-velocity plateau). Transient VOR recovery showed a similar time course to that observed for sinusoidal stimuli. The contralesional acceleration gain G_A of cba129 mice decreased between days 1 and 5, particularly in response to 6,000°/s² stimuli (average G_A on day 5: 0.65 ± 0.16; F_1,10 = 4.7, P = 0.054). This was not the case for α9 knockout mice, which showed no change in G_A (F_1,20 = 0.3, P = 0.596). Following this initial decrease, the VOR response G_A in cba129 mice started to increase significantly (F_1,22 = 32, P < 0.001). By day 28, contralesional VOR responses were near normal with G_A measuring between 0.78 ± 0.22 at 1,500°/s² and 1.01 ± 0.18 at 6,000°/s (interaction with stimulus acceleration: F_1,22 = 10, P < 0.005). G_A in α9 knockout mice also started to increase (F_1,21 = 18.6, P < 0.001), but at a reduced rate. After 28 days of recovery, contralesional VOR gains in α9 knockout mice remained significantly impaired compared with values in cba129 mice, with an average G_A of 0.63 ± 0.16 (pooled across acceleration stimuli). Stimulus acceleration had no significant effect (F_1,29 = 3.2, P = 0.083).

Fig. 9.

VOR compensation during transient steps of acceleration. Acceleration gain (G_A; top row) and constant-velocity gain (G_V; bottom row) measured in cba129 (black) and α9 knockout mice (gray) before and after UL (stimulus: 3,000°/s² reaching 150°/s constant-velocity plateau). Transient VOR recovery showed a similar time course to that observed for sinusoidal stimuli. In contrast to sinusoidal rotations, responses to ipsilesional rotations remained significantly reduced relative to pre-lesion values even after 28 days of recovery.

The ipsilesional VOR response of cba129 mice showed significant recovery between days 1 and 28. On day 28, the ipsilesional VOR response recovered to 0.52 ± 0.15 (~50% of pre-lesion response gain) and almost double the response to the initial acceleration component of the transient step stimuli from day 1. The recovery of ipsilesional responses was less pronounced in α9 knockout mice. Changes between days 1 and 5 after UL were insignificant with an average G_A of 0.34 ± 0.05 on day 5 (F_1,23 = 2.7, P = 0.111). VOR responses continued to recover only slowly, resulting in a significantly lower G_A on day 28 (0.37 ± 0.24) compared with that in cba129 mice (F_1,20 = 6.5, P < 0.02). Figure 10 shows the relative difference of G_A between ipsilesional and contralesional transient steps of acceleration at 3,000°/s². Cba129 mice developed a VOR gain asymmetry of ~60% on day 1 after UL. In contrast, α9 knockout mice only showed an asymmetry of only ~20% on day 1. The asymmetry in cba129 mice significantly improved between days 1 and 28. However, even after 28 days, a considerable asymmetry between ipsilesional and contralesional rotations remained. VOR gain asymmetry of α9 knockout mice continued to increase following UL and was maximal after 28 days of recovery.

Fig. 10.

VOR gain asymmetry. Relative difference of acceleration gain (G_A) between ipsilesional and contralesional transient steps of acceleration at 3,000°/s² is shown. Cba129 mice developed an ~60% VOR gain asymmetry on day 1 after UL. In contrast, α9 knockout mice only had an ~20% asymmetry on day 1. The asymmetry in cba129 mice significantly improved between days 1 and 28. However, even after 28 days, a considerable asymmetry between ipsilesional and contralesional rotations remained. VOR gain asymmetry of α9 knockout mice continued to increase following UL and was maximal after 28 days of recovery.

In contrast to G_A, the velocity gain G_V was severely reduced for both ipsilesional and contralesional rotations. Although the reduction in G_V was less in response to contralesional compared with ipsilesional rotations (F_1,100 = 26, P < 0.001), the overall decrease in contralesional G_V was large compared with the decrease observed in contralesional G_A. For both directions, there was a marked difference in G_V between cba129 and α9 knockout mice (F_1,3 = 42, P < 0.001). For rotations toward the ipsilesional side, G_V was 0.130 ± 0.073 for cba129 mice and 0.06 ± 0.05 for α9 knockout mice. Similarly, for rotations toward the contralesional side, G_V was 0.30 ± 0.07 for cba129 mice and 0.05 ± 0.09 in α9 knockout mice. For α9 knockout mice, the VOR response during constant-velocity rotation (G_V) was completely absent for rotations in both directions, whereas in cba129 mice, G_V was reduced for both directions but was not completely abolished. G_V for both cba129 and α9 knockout mice recovered slowly and remained severely impaired throughout the 28-day recovery period (Fig. 9). In cba129 mice, G_V recovered to 0.45 ± 0.08 for ipsilesional rotations and 0.51 ± 0.10 for contralesional rotations by day 28 after UL. This recovery was less in α9 knockout mice, which only increased to 0.31 ± 0.08 and 0.34 ± 0.14 for ipsilesional and contralesional rotations, respectively. Note that for both mouse types, G_V was near symmetrical on day 28. The latency of VOR responses to transient steps of acceleration was not affected by UL (F_1,21 = 0.1, P = 0.792), and there was no difference between the two mouse types (F_1,65 = 0.0, P = 0.926).

DISCUSSION

In this study we compared the time course of vestibular compensation between cba129 (controls) and α9 knockout mice. The α9 knockout mouse has a compromised efferent vestibular system (EVS) due to deletion of most of the gene encoding α9 nicotinic acetylcholine receptors (α9-nAChRs) on efferent targets in the vestibular periphery. Our findings suggest that α9-nAChRs are an essential part of VOR compensation following UL. If utilization of α9-nAChRs by the EVS is impaired, as is the case for α9 knockout mice, then vestibular compensation is significantly reduced and less able to restore the VOR. Notably, a compromised EVS affects the recovery of both ipsilesional and contralesional VOR responses. Differences in the VOR response of α9 knockout mice were evident as early as ~24 h after UL. The most prominent difference was a substantially lower gain of the contralesional VOR response. Whereas cba129 mice retained ~50% of their initial function for rotations toward the contralesional side, α9 knockout mice only retained ~20%. Chronic compensated VOR gains (on day 28 after UL) in α9 knockout mice were on average ~48% lower for ipsilesional and ~45% lower for contralesional rotations compared with cba129 mice. Cba129 mice regained up to ~75% function (relative to pre-lesion values) for ipsilesional and up to ~90% of function for contralesional rotations (depending on the sinusoidal vestibular stimulus). In contrast, α9 knockout mice only regained ~30% and ~50% function, respectively, leaving the VOR severely impaired for rotations in both directions. Our findings are in contrast to those from a previous study by Eron et al. (2015) that examined the time course of VOR compensation in α9 knockout mice up to 9 days after UL. That study did not report significant differences in gain recovery after UL between α9 knockout mice and controls, but that could be due to the small number (n = 3) of α9 knockout mice they tested. The post-UL differences we observed between mouse types are unlikely due to significant loss of vision and/or generation of a retinal image slip signal to drive compensation in the α9 knockout, although there could be subtle losses. We did not measure optokinetic function in these mice; however, there are several lines of evidence suggesting that α9 knockout mice have adequate vision and slip signal for compensation. First, qualitatively, these mice react to light and visual movement in the same way as other mice we have tested. The fact that α9 knockout mice were able to perform a baseline visual discrimination task described by Terreros et al. (2015) just as well as wild-type mice also suggests they have normal vision. Visual problems could have been associated with the cba129 strain we employed during backcross breeding, but both wild-type littermates and cba129 controls had similar levels of robust compensation, suggesting adequate vision and slip signals. Second, we have previously shown that visual-vestibular mismatch VOR gain training results in VOR adaptation in α9 knockout mice, albeit ~70% less than in controls, suggesting they generate an image slip signal sufficiently large enough to drive adaptation (Hübner et al. 2015).

Vestibular Compensation via Central and Peripheral Mechanisms

Previous studies have focused efforts primarily on the identification and characterization of central vestibular plasticity mechanisms that are involved in the process of vestibular compensation after unilateral vestibular lesion (for a review see Beraneck and Idoux 2012; Curthoys and Halmagyi 1995; Straka et al. 2005). Identified processes include (among others) major synaptic reorganization within ipsi- and contralesional vestibular nuclei and changes in the intrinsic properties of 2° VN (Beraneck et al. 2004; Him and Dutia 2001; Straka et al. 2005; Vibert et al. 2000). Only hours after a UL has deprived ipsilesional 2° VN from sensory input, these neurons begin to spontaneously discharge, probably in an effort to restore a balanced state between vestibular nuclei on both sides and reduce static deficits that cause spontaneous nystagmus and vertigo (Ris et al. 1995, 1997; Vibert et al. 1999a). This spontaneous discharge appears to be independent of commissural innervation from the contralesional side (Anastasio 1992; Cartwright and Curthoys 1996; Fetter and Zee 1988). Interestingly, increases in spontaneous activity are predominantly seen in ipsilesional type B (phasic) neurons, whereas changes in type A (tonic) neurons only begin to appear 7–11 days after UL (Him and Dutia 2001). Furthermore, long-term changes of intrinsic signal properties of ipsi-and contralesional 2° VN result in an increased proportion of phasic (type B) neurons in contralesional vestibular nuclei that is paralleled by an increase of tonic (type A) neurons in ipsilesional vestibular nuclei (Beraneck et al. 2003, 2004; Him and Dutia 2001).

In contrast to the extensive body of work on central plasticity mechanisms, relatively few studies have investigated changes in peripheral vestibular signals following UL. However, emerging evidence suggests that vestibular compensation is a distributed process that requires not only changes in the various structures associated directly with vestibular function, including the aforementioned changes in the vestibular nuclei, but also changes involving the cerebellum (Beraneck et al. 2008; Dutia 2010; Faulstich et al. 2006; Goto et al. 1997) and changes at the level of the peripheral vestibular organs (Beraneck and Idoux 2012; Cullen et al. 2009; Lasker et al. 2000; Llinas and Walton 1979). Single-unit recordings of vestibular primary afferents in chronically compensated macaque monkeys (1–12 mo after UL) suggest that changes at the level of the vestibular periphery occur alongside central changes to facilitate VOR compensation (Sadeghi et al. 2007). After UL, the proportion of contralesional irregularly discharging afferents increased, whereas the proportion of regularly discharging afferents decreased. This resulted in a shift toward more phasic signal properties in the peripheral input to central 2° VN (Sadeghi et al. 2007). A potential mechanism responsible for these changes is EVS-mediated modulation of the response dynamics of regularly discharging afferents with dimorphic terminals. Dimorphic afferents receive inputs from type I and type II hair cells and make up the majority (~80%) of afferent inputs to brain stem vestibular nuclei. Note that the changes in primary vestibular afferents are quantitatively and qualitatively similar to changes in 2° VN (Beraneck et al. 2003, 2004; Beraneck and Idoux 2012).

Additional evidence suggesting changes in the proportion and sensitivity of vestibular primary afferents after UL has been produced by a series of studies investigating VOR signal processing along tonic (velocity sensitive) and phasic (acceleration sensitive) signal pathways (Lasker et al. 2000; Minor et al. 1999a). Morphologically these tonic and phasic pathways resemble the response dynamics (latency and sensitivity) of regularly and irregularly discharging vestibular primary afferents and 2° VNs, respectively (Hullar et al. 2005). The tonic pathway has low sensitivity and therefore unilaterally can encode a wide range of ipsilateral and contralateral head rotations before being driven into saturation. It is primarily active during stimuli with low-acceleration components (i.e., low-frequency, low-velocity sinusoids and the constant-velocity part of the transient step stimuli). The phasic pathway has high sensitivity and therefore unilaterally best encodes ipsilateral head rotations. The asymmetry of VOR responses following UL is thought to be caused by the phasic pathway because it predominantly encodes short-latency, high-acceleration head rotations toward the contralesional side but is easily driven into inhibitory cutoff for rotations toward the ipsilesional side. As a result, stimuli that preferentially activate the phasic pathway, for example, the rapid acceleration at the beginning of transient step stimuli, produce maximal asymmetry between contralesional and ipsilesional VOR response gains (G_A). In contrast, VOR responses that predominantly rely on the velocity-sensitive tonic pathway, for example, the constant-velocity response (G_V) of transient step stimuli, produce minimal asymmetry between ipsilesional and contralesional rotations (Lasker et al. 2000). The reduced post-lesion asymmetry we observed in α9 knockout compared with cba129 mice is consistent with this hypothesis. Furthermore, Lasker et al. (2000) concluded that VOR compensation depends on an increase in contribution of the contralesional highly modifiable phasic pathways, which is consistent with findings from single-unit recordings of vestibular afferents and 2° VN that showed increased populations of irregular afferents and type B 2° VN in contralesional vestibular nuclei (Beraneck et al. 2003, 2004; Sadeghi et al. 2007).

VOR Compensation in α9 Knockout Mice

It is important to note that before the vestibular lesion is surgically induced, the vestibular primary afferent signal is significantly different in α9 knockout mice compared with controls (more tonic with increased proportion and sensitivity of regularly discharging afferents; Han et al. 2007; see explanation in Hübner et al. 2015). This shift in afferent signaling was shown in a single-unit vestibular afferent study in the α9 knockout mouse (Han et al. 2007) and could be occurring because EVS activation, specifically the cholinergic component, is thought to have a dual effect. One effect of EVS activation is the inhibition of type II hair cells (i.e., strictly a reduction of resting discharge rate and attenuation of sensitivity/gain) via α9-nAChRs coupled to calcium-activated potassium (SK) channels (Holt et al. 2006; Poppi et al. 2014). The other effect is the excitation of afferents (Boyle and Highstein 1990; Goldberg and Fernández 1980), through nAChRs that contain α4-, α6-, and β2-subunits (Holt et al. 2015). In α9 knockout mice this inhibition/excitation dual effect would be partially compromised. Loss of α9-nAChRs would prevent EVS inhibition of type II hair cells, allowing normally suppressed type II hair cells to “contribute” to overall afferent activity (particularly dimorphs). Simultaneously, due to the presence of alternative types of nAChRs (i.e., α4-, α6-, and β2-subunits) on calyx-bearing afferents, the excitatory EVS effect would still be operating in α9 knockout mice. In short, the predicted overall effect of EVS activation on dimorphic afferents in α9 knockout mice is increased afferent discharge, but with an additional input from normally EVS-suppressed type II hair cells leading to an increase in afferent regularity and corresponding shift in afferent dynamic response. It is possible that α9 knockout mice also have other compensatory changes in their hair cell membrane properties, synapses, and vestibular neurons (for a review see Straka et al. 2005). Remarkably, only moderate changes can be observed in the baseline VOR response of α9 knockout mice before UL (Hübner et al. 2015). However, if the vestibular system is challenged by UL, the effects of a loss of α9-nAChRs become more apparent.

Only ~24 h after UL we observed clear differences between α9 knockout and control mice. Among them was a significantly lower asymmetry between ipsilesional and contralesional VOR response gains for all test stimuli. The VOR gain asymmetry after UL is thought to be caused by the phasic pathway because irregularly discharging afferents go into inhibitory cutoff more readily than regularly discharging afferents during contralateral rotations (Lasker et al. 2000). In α9 knockout mice, this phasic pathway is minimal (Han et al. 2007), which may explain why VOR response gains are more symmetrical. In addition, α9 knockout mice do not have a mechanism to control the proportion of the phasic pathway contribution, which might explain why contralesional VOR responses were significantly depressed in α9 knockout mice compared with controls. During the constant-velocity phase of transient step stimuli, a stimulus that predominantly activates the tonic pathways, we found absent VOR responses (G_V: 0.05 ± 0.09) in α9 knockout mice, but not in controls (G_V: 0.30 ± 0.07). On the basis of our observations in α9 knockout mice, we hypothesize that chronic compensation after UL relies on two changes in peripheral signal processing. First, the sensitivity of the phasic pathway is reduced, which could occur centrally (i.e., does not require changes in afferent sensitivity) so as to minimize inhibitory cutoff during ipsilesional head rotations and reduce asymmetry of the VOR response. Second, the proportional contribution of the phasic pathway is increased so as to aid central compensation mechanisms that rely on this highly modifiable pathway. If the sensitivity of the phasic pathway remains unchanged, one would predict larger asymmetry. If the proportion of the phasic pathway remains unchanged, one would predict reduced VOR gain compensation. The α9 knockout mouse appears to have control of the sensitivity (first change) but not the proportional contribution (second change) of the phasic pathway, resulting in reduced asymmetry but with smaller contralesional VOR gain and impaired long-term VOR compensation.

Could Other Central Mechanisms Be Affecting Compensation in α9 Knockout Mice?

Another possible mechanism that could account for reduced gains on the contralesional side is that ipsilesional commissures do not release contralesional 2° VN from inhibition. These commissural pathways generally originate from tonic (type A) neurons on the ipsilateral side but rely on contralateral phasic (type B) interneurons to modulate vestibular signals of the opposite ear (Camp et al. 2006; Malinvaud et al. 2010). If in α9 knockout mice these contralateral type B interneurons are affected by the loss of α9-nAChRs, then one might expect that contralesional 2° VN are not released from inhibition (disinhibited) when ipsilesional input is silenced, resulting in lower ipsilesional and contralesional VOR gain. This line of reasoning is supported by a recent study that examined vestibular function in three α9 knockout mice and reported differences in the generation of quick phases and the vestibular time constant compared with controls, which they attributed to possible changes in the commissural inhibitory system (Eron et al. 2015). However, in our previous study we found no difference in the amplitude, velocity, and number of quick phases per cycle between α9 knockout and control mice, whereas the quick-phase duration was ~10% faster in α9 knockout mice (Hübner et al. 2015).

Depressed VOR compensation is also observed in mice that lack cerebellar Purkinje cells (i.e., Lurcher mutant mice; Aleisa et al. 2007; Beraneck et al. 2008). Those “cerebellum-deficient” mice demonstrated normal recovery of static deficits and unaffected VOR compensation during the initial period of ~10 days after UL (acute compensation). However, unlike control mice, VOR gain recovery in cerebellum-deficient mice plateaued after this initial period, and mice were unable to regain functionally effective VOR responses >20 days after UL. This suggests that cerebellar pathways are not involved in the acute phase of compensation but are critical for long-term restoration of VOR responses. Notably, a majority of Purkinje cells make contact exclusively with phasic (type B) neurons within the vestibular nuclei (Shin et al. 2011). This could explain why in α9 knockout mice both VOR adaptation and VOR compensation, two forms of vestibular plasticity that rely on cerebellar pathways, are equally impaired.

In summary, our data suggest that the mammalian EVS plays an important role in VOR compensation, just as it does for VOR adaptation. VOR compensation was ~50% less than in controls, whereas VOR adaptation was ~70% less than in controls, suggesting that central mechanisms play a bigger role during VOR compensation compared with adaptation. Our findings also suggest that the converse of our findings could be true such that upon stimulation the EVS could potentially boost vestibular recovery after a peripheral vestibular lesion.

GRANTS

This work was supported by National Health and Medical Research Council of Australia (NHMRC) Biomedical Career Development Award CDA-568736 (to A. A. Migliaccio) and NHMRC Project Grant APP1010896 (to A. A. Migliaccio). P. P. Hübner was supported by a University of New South Wales International Research Scholarship and a Neuroscience Research Australia supplementary scholarship.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

P.P.H. and S.I.K. performed experiments; P.P.H., S.I.K., and A.A.M. analyzed data; P.P.H. and A.A.M. interpreted results of experiments; P.P.H., S.I.K., and A.A.M. prepared figures; P.P.H. and A.A.M. drafted manuscript; P.P.H., S.I.K., and A.A.M. approved final version of manuscript; A.A.M. conceived and designed research; A.A.M. edited and revised manuscript.