Vestibular Jn.01049.2015

J Neurophysiol 117: 1553–1568, 2017.
First published January 11, 2017; doi:10.1152/jn.01049.2015.
RESEARCH ARTICLE Sensory Processing
The mammalian efferent vestibular system plays a crucial role in

vestibulo-ocular reflex compensation after unilateral labyrinthectomy
Patrick P. Hübner,1,2 Serajul I. Khan,1,2 and Americo A. Migliaccio1,2,3

1
Balance and Vision Laboratory, Neuroscience Research Australia, Sydney, New South Wales, Australia; 2University of New
South Wales, Sydney, New South Wales, Australia; and 3Department of Otolaryngology-Head and Neck Surgery, Johns
Hopkins University, Baltimore, Maryland
Submitted 19 November 2015; accepted in final form 10 January 2017
Hübner PP, Khan SI, Migliaccio AA. The mammalian efferent mammalian efferent vestibular system; vestibular compensation; ves-
vestibular system plays a crucial role in vestibulo-ocular reflex compen- tibular plasticity; vestibulo-ocular reflex; ␣9 knockout mice
sation after unilateral labyrinthectomy. J Neurophysiol 117: 1553–1568,
2017. First published January 11, 2017; doi:10.1152/jn.01049.2015.—
The ␣9-nicotinic acetylcholine receptor (␣9-nAChR) subunit is ex- IN A RECENT STUDY, we investigated whether the ␣9-nicotinic
pressed in the vestibular and auditory periphery, and its loss of acetylcholine receptor (␣9-nAChR), important for the mam-
function could compromise peripheral input from the predominantly malian efferent vestibular system (EVS), plays a role in ves-
cholinergic efferent vestibular system (EVS). A recent study has tibular plasticity mechanisms that drive vestibulo-ocular reflex
shown that ␣9-nAChRs play an important role in short-term ves- (VOR) adaptation (Hübner et al. 2015). We compared the VOR
tibulo-ocular reflex (VOR) adaptation. We hypothesize that ␣9- response of ␣9 knockout mice, a mouse strain that lacks
nAChRs could also be important for other forms of vestibular plas- ␣9-nAChRs due to deletion of most of the gene (chrna9)
ticity, such as that needed for VOR recovery after vestibular organ encoding that receptor (Vetter et al. 1999), with data obtained
injury. We measured the efficacy of VOR compensation in ␣9 in cba129 (control) mice. In cba129 mice, ␣9-nAChRs interact
knockout mice. These mice have deletion of most of the gene (chrna9) with acetylcholine (ACh), the main neurotransmitter of the
encoding the nAChR and thereby lack ␣9-nAChRs. We measured the EVS, to alter the output of the vestibular peripheral organs
VOR gain (eye velocity/head velocity) in 20 ␣9 knockout mice and 16 (Gacek and Lyon 1974; Goldberg and Fernández 1980; High-
cba129 controls. We measured the sinusoidal (0.2–10 Hz, 20 –100°/s) stein 1991; Marco et al. 1993; Purcell and Perachio 1997). In
and transient (1,500 – 6,000°/s2) VOR in complete darkness before our previous study in ␣9 knockout mice, we demonstrated that
(baseline) unilateral labyrinthectomy (UL) and then 1, 5, and 28 days loss of the ␣9-nAChR subunit causes a significant reduction
after UL. On day 1 after UL, cba129 mice retained ~50% of their
(⬎70%) in the capacity for VOR adaptation compared with
initial function for contralesional rotations, whereas ␣9 knockout mice
controls. Thus the utilization of ␣9-nAChRs by the mammalian
only retained ~20%. After 28 days, ␣9 knockout mice had ~50%
lower gain for both ipsilesional and contralesional rotations compared
EVS appears to be essential for plasticity mechanisms involved
with cba129 mice. Cba129 mice regained ~75% of their baseline in VOR adaptation. However, it is unclear if VOR compensa-
function for ipsilesional and ~90% for contralesional rotations. In tion, another form of vestibular plasticity involved in the
contrast, ␣9 knockout mice only regained ~30% and ~50% function, (partial) restoration of static and dynamic deficits after vestib-
respectively, leaving the VOR severely impaired for rotations in both ular organ injury, is similarly dependent on the EVS (Curthoys
directions. Our results show that loss of ␣9-nAChRs severely affects 2000; Dieringer 1995, 2003; Vibert et al. 1997).
VOR compensation, suggesting that complimentary central and pe- Historically, the process of vestibular compensation was
ripheral EVS-mediated adaptive mechanisms might be affected by thought to be independent from plasticity mechanisms in-
this loss. volved in the adaptive modification of the VOR response (e.g.,
during visual-vestibular mismatch adaptation and vergence-
NEW & NOTEWORTHY Loss of the ␣9-nicotinic acetylcholine
receptor (␣9-nAChR) subunit utilized by the efferent vestibular sys-
mediated gain changes). Increasing evidence, however, sug-
tem (EVS) has been shown to significantly affect vestibulo-ocular gests that vestibular compensation is a distributed process
reflex (VOR) adaptation. In our present study we have shown that loss involving numerous parallel events at various sites in the brain
of ␣9-nAChRs also affects VOR compensation, suggesting that the and vestibular periphery. Some of these processes are closely
mammalian EVS plays an important role in vestibular plasticity, in related to mechanisms that are active during VOR adaptation
general, and that VOR compensation is a more distributed process (Beraneck et al. 2004; Dieringer 1995, 2003; Dutia 2010;
than previously thought, relying on both central and peripheral Straka et al. 2005; Vibert et al. 1999a, Vibert et al. 1999b). For
changes. example, in mice with cerebellar dysfunction (Lurcher mu-
tants) it was shown that long-term cerebellum-dependent motor
learning is just as important for VOR compensation after
Address for reprint requests and other correspondence: A. A. Migliaccio,
Balance and Vision Laboratory, Neuroscience Research Australia, Cnr Barker
unilateral labyrinthectomy (UL) (Aleisa et al. 2007; Beraneck
Street and Easy Street, Randwick 2031, NSW, Australia (e-mail: a.migliaccio et al. 2008; Faulstich et al. 2006) as it is for VOR adaptation
@neura.edu.au). (Koekkoek et al. 1997; Van Alphen et al. 2002). The restora-
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1554 THE EVS ROLE IN VOR COMPENSATION
tion of dynamic deficits (e.g., reduced VOR response gains; findings with results in cba129 (control) mice. The functional
gain ⫽ eye/head velocity) seems to be especially dependent on implications of a compromised EVS were evaluated at 4 time
intact cerebellar innervation of brain stem vestibular nuclei points over the course of the study: before UL surgery and 1
(Beraneck et al. 2004). Furthermore, both forms of vestibular day (24 h), 5 days, and 28 days after UL surgery. At each point
plasticity (VOR adaptation and compensation) were found to we tested VOR responses by using a wide range of vestibular
rely on an increase in gain of phasic peripheral signals (i.e., stimuli, including horizontal sinusoidal rotations (0.1 to 10 Hz,
irregularly discharging primary vestibular afferents) as well as with peak velocities of 20, 50, and 100°/s) and transient steps
an increase in the proportion and sensitivity of phasic central of acceleration (1,500, 3,000, and 6,000°/s2, reaching constant
neurons in the brain stem vestibular nuclei (i.e., type-B neu- velocities of 100, 150, and 300°/s). As shown for VOR adap-
rons) (Clendaniel et al. 2001, 2002; Lasker et al. 1999, 2000). tation, our findings demonstrate that ␣9-nAChRs also play an
This observation is based on studies in monkeys and humans, essential role in facilitating VOR compensation after a unilat-
which facilitated the development of a mathematical model of eral vestibular lesion.
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 char- METHODS
acteristics (Clendaniel et al. 2001, 2002; Lasker et al. 1999,
2000; Migliaccio et al. 2003, 2004, 2008; Minor et al. 1999b). Animals and Surgical Preparation
The response dynamics of these tonic and phasic pathways A total of 20 ␣9 knockout mice and 16 controls (both sexes, ages
resemble those of regularly and irregularly discharging vestib- 11–14 wk) were used in this study. The VOR response for baseline,
ular primary afferents (Hullar and Minor 1999; Hullar et al. day 1, day 5, and day 28 after UL was measured in 13, 11, 16, and 11
2005) as well as type A and type B second-order vestibular ␣9 knockout mice, respectively. Similarly, the VOR response for
neurons (2° VN) in the brain stem (Dickman and Angelaki baseline, day 1, day 5, and day 28 after UL was measured in 11, 11,
2004). The phasic pathway is highly modifiable and predom- 11, and 12 cba129 mice, respectively. The baseline VOR response of
inantly mediates VOR changes observed in response to spec- cba129 and ␣9 knockout mice was described in detail by Hübner et al.
tacle-induced adaptation (Clendaniel et al. 2001, 2002), view- (2015). The mouse strain carrying the ␣9-knockout mutation has been
maintained on a CBA/CaJ ⫻ 129/SvEv background line by The
ing of targets at close distances (a vergence-mediated gain Jackson Laboratory (Bar Harbor, ME; stock no. 005696). We set up
change) (Migliaccio et al. 2004, 2008), and VOR compensa- an independent colony of hybrid CBA/CaJ (Jackson Laboratory; stock
tion after vestibular canal plugging and UL (Lasker et al. 1999, no. 000654) ⫻ 129/SvEv (Taconic Biosciences; model no. 129SVE)
2000). (from here on referred to as “cba129”) that we used as the controls.
It is well established that information from contralesional When the homozygous ␣9 knockout breeders became old, new breed-
primary vestibular afferents and 2° VN substantially contrib- ers were selected from different heterozygous (cba129 ⫻ ␣9 knock-
utes to the restoration of VOR responses following a unilateral out) breeding pairs. To ensure that the cba129 mice we used as
vestibular lesion (e.g., UL) (Cartwright and Curthoys 1996; controls throughout this study were valid, we compared their baseline
Curthoys 2000; Dieringer 1995; Graham and Dutia 2001; and post-UL data with that from five homozygous wild-type litter-
Smith and Curthoys 1988a, Smith and Curthoys 1988b). No- mates obtained from heterozygous breeding pairs. Genotyping was
performed externally by Garvan Molecular Genetics (Sydney, Aus-
tably, long-term changes in support of the findings by Minor tralia) from ear punches using the same protocols for mouse geno-
and colleagues have been observed in primary vestibular af- typing as Jackson Laboratory. The genotyping was performed using
ferents as well as 2° VN following UL (Beraneck et al. 2004; real-time polymerase chain reaction (PCR) in a 384-well plate format.
Cullen et al. 2009; Sadeghi et al. 2007; Straka et al. 2005). In The primer sequences and combinations used were provided by
particular, the proportion of contralesional primary vestibular Jackson Laboratory and were as follows: ␣9 knockout, CAC GAG
afferents with phasic response dynamics (i.e., irregularly dis- ACT AGT GAG ACG TG; cba129 forward, TCT GGT GCT GGG
charging afferents) was shown to increase following UL, AAT CAA AT; and common, AGC CCC AGA ACC TCT GTT TT.
whereas the number of afferents with tonic response dynamics Samples were processed via liquid handlers and analyzed on a
(i.e., regularly discharging afferents) decreased proportionally LightCycler 480 with a Sito9-based Meltcurve analysis. Sample peaks
(Sadeghi et al. 2007). A similar transition toward more phasic were compared with those of a cba129 control. The initial controls
were from our independent colony of cba129 mice but were then
signal properties was also observed in contralesional 2° VN substituted by homozygous wild-type littermates obtained from
following UL in guinea pigs (i.e., sensitivity and proportion of heterozygous breeding pairs after PCR confirmed they had two
phasic type B neurons increase following a vestibular lesion) chrna9 alleles. One complex PCR analysis was performed to differ-
(Beraneck et al. 2004). ␣9-nAChRs and their utilization by the entiate between ␣9 knockout, heterozygous, and wild-type littermate
EVS could play a critical role in facilitating this increase in control mice.
contribution (sensitivity and proportion) of phasic peripheral All mice were implanted with a custom-built head immobilization
signals. Specifically, an efferent-mediated tuning of dimorphic device consisting of a lightweight low-profile metal adapter plate
afferents and their respective inputs from type I and type II permanently attached to the skull and a removable head pedestal
vestibular hair cells could be the mechanism responsible for the attached to the adapter plate before each experimental session. The
proportional change observed in primary vestibular afferents exact implantation technique has been outlined previously (Hübner et
al. 2014). Following adapter plate implantation, mice were allowed to
(Beraneck and Idoux 2012; Cullen et al. 2009; Sadeghi et al. recover from the surgery for at least 3 days before the first experi-
2007; for more explanation see Hübner et al. 2015). mental session (baseline VOR recording before UL). Unilateral lab-
The goal of the present study was to determine if VOR yrinthectomy was performed during a second surgery 1– 4 days after
compensation after unilateral vestibular lesion relies on the the pre-lesion (baseline) VOR response was measured. Following UL,
availability of ␣9-nAChRs. We investigated the time course of animals recovered in a normal visual environment (Shinder et al.
vestibular compensation in ␣9 knockout mice and compared 2005) under close monitoring.
J Neurophysiol • doi:10.1152/jn.01049.2015 • www.jn.org

THE EVS ROLE IN VOR COMPENSATION 1555
All surgical and experimental procedures were approved by the transferred to the prepared surgical field. At this point nonsterile
Animal Care and Ethics Committee of the University of New South examination gloves were removed and sterile surgical gloves were
Wales and were in strict compliance with the Australian Code of worn. Mice were draped using a transparent film dressing (Opsite
Practice for the Care & Use of Animals for Scientific Purposes. Flexigrid 4630; Smith & Nephew USA, Andover, MA) with a small
window cut out at the incision site.
Unilateral Labyrinthectomy Surgical procedure. UL surgery was performed only on the left ear.
The procedure was conducted using a partial transcanal approach.
Preparation. Unilateral labyrinthectomy (UL) procedures were
Lidocaine and bupivacaine (ⱕ0.02 ml, mixed 1:1) were injected as a
performed under full aseptic technique to avoid postoperative infec-
local field block, and an anterior auricular incision was made (Fig.
tions. Similar to the adapter plate implantation procedure for the head
immobilization system, mice were anesthetized in an induction cham- 1A). The external auditory meatus was visualized and sectioned close
ber using a mix of isoflurane (1.5–3%) and oxygen (3 l/min). Once to the tympanic membrane (Fig. 1B). The peripheral end of the
anesthetized, mice were transferred to a nonsterile preparation area sectioned external auditory meatus was closed using a purse-string
where anesthesia was maintained via nose cone. The eyes were treated suture to avoid entry of particles and bacteria into the body. A
with an ophthalmic ointment, and a generous area around the incision retractor system was used to keep the incision site open to allow better
site (~5 mm around the auricle) was razor shaved and disinfected visualization. The tympanic membrane, bony tympanic annulus, and
using a swab of betadine and 70% ethyl alcohol (see Fig. 1A). A horizontal section of the facial nerve were further exposed, and the
subcutaneous (SC) injection of 0.03 ml of carprofen (5 mg/ml) was tympanic membrane was removed. Fractioning and curettage of the
given as analgesic and antibiotic treatment, and 0.5 ml of sterile saline posterior, superior, and inferior aspects of the bony tympanic annulus
was injected intraperitoneally (IP) to prevent dehydration of the and removal of the malleus and incus uncovered the tympanic bulla of
animals during surgery. After preparations were complete, mice were the middle ear (Fig. 1C). Spontaneous bleeding from the fractured,
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 vestib-
ular 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.

highly vesiculated bone edges was controlled using an application of baseline recording) and following vestibular lesion at three time
adrenaline and Gelfoam. The tympanic bony annulus was further points, 1 day (24 h), 5 days, and 28 days postsurgery, to study the
enlarged until the oval window and stapes footplate, the entire round acute impaired, acute compensated, and chronic compensated VOR
window niche, and the stapedial branch of the internal carotid artery responses, respectively. At each individual time point during the
could be clearly visualized (Fig. 1D). To gain access to the inner ear recovery process, we measured VOR responses in darkness over the
and vestibular sensory organs, the stapedial artery had to be sacrificed. complete range of naturally occurring mouse head movement frequen-
A fine monopolar electric cautery tool was used to cauterize the artery cies, velocities, and accelerations (Beraneck et al. 2008) using hori-
on both sides of the stapes footplate before it was sectioned and zontal, whole body sinusoidal rotation at 0.2, 0.4, 0.5, 0.8, 1, 1.6, 2,
removed. The stapes was then carefully lifted off the oval window in 5, and 10 Hz with peak velocities of 20, 50, and 100°/s, and transient
a gentle twisting motion to expose the vestibule (Fig. 1E). Care was acceleration stimuli at 1,500, 3,000, and 6,000°/s2 reaching a constant
taken not to fracture the stapes in the process of removing it. Gentle velocity plateau of 100, 150, and 300°/s, respectively (in the text, we
suction was used to aspirate endolymph and perilymph fluid exiting refer to transient stimulus conditions as 1.5k100, 3k150, and 6k300).
the vestibule. Every effort was made to avoid accidental aspiration of For a more detailed explanation of the rotational stimuli, see Hübner
the vestibular end organs at this time. These last steps ensured optimal et al. (2015).
visualization of the oval window. Once efflux of endolymph and To analyze the three-dimensional VOR data, we converted eye
perilymph from the vestibule ceased, the oval window was carefully movements acquired in eye coordinates into rotation vectors in head
out-fractioned. This provided access to the inner ear and improved coordinates. Eye velocity traces with quick phases removed were
visualization of the vestibular end organs (Fig. 1F). At this point the inverted so that an ideal VOR would yield a gain (eye velocity/head
utricle, a large oval epithelial patch with whitish color, was clearly velocity) of ⫹1 and a phase of 0°. Positive phase lead denotes eye
visible. With the use of fine biological forceps (Moria MC40 ultra fine velocity leading head velocity. VOR responses to sinusoidal rotations
forceps), the sensory epithelia of the utricle, as well as those of the and transient steps of acceleration were analyzed separately for
lateral and anterior semicircular canals, were carefully excised (Fig. rotations toward the operated ear (ipsilesional) and rotations toward
1F). The saccule was destroyed mechanically by aspiration of the the healthy ear (contralesional). To measure the slow-phase VOR
medial aspect of the vestibule. Because of the location of the posterior response to sinusoidal rotations, least-square pure sine waves were fit
canal cupula, direct access and extraction using forceps was not to each individual cycle of the head velocity stimulus and eye velocity
possible. Postmortem dissection of the inner ear, however, confirmed response. A least-square pure sine wave was fit to the eye velocity
that suction applied to the openings of lateral and anterior canals trace using the model shown in Eq. 1 to determine sinusoidal ampli-
destroyed the posterior crista, as well. All manipulations inside the tude, phase, and constant offset.
vestibule, especially those close to the medial aspect, were performed
M Sine ⫽ a · sin共2␲ ft兲 ⫹ b · cos共2␲ ft兲 ⫹ c (1)
with great care to avoid fracture of the very thin internal wall between
vestibule and brain cavity. where a ⫽ amplitude·cos(phase), b ⫽ amplitude·sin(phase) and c ⫽
After complete destruction of the vestibular end organs was con- offset.
firmed visually, the vestibule was packed with Gelfoam soaked in 1 ␮l The eye and head velocity trace amplitudes were used to
of gentamicin-buffered solution (20 mg/ml; Troy Laboratories, Glen- calculate the sinusoidal VOR gain. Because VOR gain responses
denning, Australia) (Fig. 1G). This guaranteed destruction of the were expected to vary between ipsilesional and contralesional
residual neuroepithelium, especially in areas that were hard to access. rotations, eye velocity traces were divided into a positive and
The tympanic cavity of the middle ear was further packed with dry negative half-wave, representing the VOR response to rightward and
Gelfoam and tissue fragments, and the skin was closed using inter- leftward head rotations. The traces were split at the point where the
rupted sutures (Fig. 1H). A liquid tissue adhesive was applied to the full-cycle sinusoidal head velocity fit intersected the zero-velocity
suture to prevent entry of bacteria. Before the animal was taken off the axis, corresponding to a direction change in position. For half-wave
anesthetic gas and returned to its cage for recovery, it was given sinusoidal analysis, Eq. 1 was modified such that the offset c was fixed
buprenorphine (0.02 ml SC; 32.4 ␮g/ml) as analgesic treatment and to the value determined during the full-cycle fit. This was necessary
another dose of sterile saline (0.5 ml IP) to aid in hydration. Analgesic because sinusoidal half-waves do not carry enough information to
treatment using SC injection of buprenorphine (twice a day) was reliably estimate this parameter.
continued for up to 3 days postsurgery or until the animal recovered. For transient steps of acceleration, we fit least-square linear regres-
Mice regained consciousness 10 –15 min after inhalation anesthesia sions to the constant-acceleration and constant-velocity part of eye
was discontinued. They immediately displayed a strong head tilt with and head velocity traces. Using these fits, we calculated three param-
the labyrinthectomized ear down, clearly deviated posture, and a eters: acceleration gain (GA), constant-velocity gain (GV), and latency.
strong tendency to body roll and circle toward the affected ear. In GA was calculated as the average ratio of eye-to-head acceleration
addition, mice displayed clearly visible spontaneous nystagmus with (using the slopes of the constant-acceleration fit). GV was calculated
quick-phase eye movements “beating” away from the lesioned side. as the average ratio of eye-to-head velocity (using the point-by-point
When picked up by the tail, these mice started to vigorously spin offset of the constant-velocity fit) during the 200- to 400-ms interval
about the main axis of their body. The degree of postoperative after stimulus onset.
vestibular disturbance was used as a measure of successful UL During the initial 2–3 days after UL, animals exhibited a marked
surgery. Body roll, circling, and spontaneous nystagmus usually spontaneous nystagmus, which typically offset the sinusoidal slow-
ceased 1–2 days postsurgery. Head tilt, deviated posture, and spinning phase VOR response. We estimated the velocity offset c caused by
when picked up by the tail persisted and were still present several spontaneous nystagmus by calculating the average slow-phase veloc-
months after surgery. ity over an interval of 10 to 50 ms before onset of the vestibular
sinusoidal stimulus. This is a common approach to estimating the
Data Acquisition and Analysis VOR response offset caused by spontaneous nystagmus (e.g., Bera-
neck et al. 2008; Fetter and Zee 1988; Sadeghi et al. 2006).
The method of recording three-dimensional binocular eye move-
ments with the use of high-speed video-oculography and the tech- Statistical Analysis
nique used for offline analysis of VOR responses have been detailed
in several previous studies (Hübner et al. 2013, 2014, 2015; Migliac- Data were analyzed in R (R Core Team 2013), using a general linear
cio et al. 2011). VOR responses were measured before vestibular mixed model with two-factor interaction (nlme package; Pinheiro et al.
organs were surgically lesioned using the UL approach (pre-lesion 2014). Post hoc pairwise comparisons of group means were performed

using Bonferroni’s correction for two-sample t-statistics. All statistical wild-type littermates (dashed gray lines) obtained from
tests were performed using a significance level of P ⫽ 0.05. Unless stated heterozygous breeding pairs. There was no difference in base-
otherwise, all measurement results are means ⫾ SD. line sinusoidal VOR between the cba129 control mice used
throughout our analysis and wild-type littermates across fre-
RESULTS quencies (gain: F1,377 ⫽ 0.64, P ⫽ 0.43; phase: F1,376 ⫽ 2.97,
P ⫽ 0.09) and peak velocities (gain: F1,389 ⫽ 1.31, P ⫽ 0.26;
VOR Response Before UL phase: F1,388 ⫽ 1.32, P ⫽ 0.25).
Figure 2A shows the baseline sinusoidal VOR gains from 0.2 The baseline VOR response of cba129 control and ␣9
to 10 Hz for cba129 control mice (dashed black lines) and knockout mice in this study are essentially the same as those
Fig. 2. Vestibulo-ocular reflex (VOR) response for

cba129 and wild-type littermate control mice. A: base-
line sinusoidal VOR gains from 0.2 to 10 Hz for cba129
control mice (dashed black lines) and wild-type litter-
mates (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 (GA) and gain in
response to the constant-velocity plateau of transient
step stimuli (GV) for cba129 control mice (black col-
umns 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.

described in Hübner et al. (2015). In brief, both mouse types Sinusoidal VOR Response After UL
showed a velocity-dependent gain increase (␣9 knockout:
F2,281 ⫽ 70.2, P ⬍ 0.001; cba129: F2,181 ⫽ 78.8, P ⬍ 0.001; Figure 2A shows the sinusoidal VOR gains from 0.2 to 10
difference between mice: F1,1201 ⫽ 102.9, P ⬍ 0.001), whereas Hz for cba129 control mice (black lines) and wild-type litter-
cba129 mice showed a frequency-dependent gain increase mates (gray lines) on days 5 (top row) and 28 (bottom row)
(peak velocity 20°/s: F8,72 ⫽ 5.11, P ⬍ 0.001; peak velocity after UL. There was no difference in sinusoidal VOR between
cba129 controls and wild-type littermates across days after UL
50°/s: F8,62 ⫽ 6.1, P ⬍ 0.001) that was absent in ␣9 knockout
(gain: F1,1653 ⫽ 0.62, P ⫽ 0.43; phase: F1,1652 ⫽ 0.06, P ⫽
mice (20°/s: F8,89 ⫽ 0.65, P ⫽ 0.73; 50°/s: F8,97 ⫽ 1.42, P ⫽
0.80), across frequencies (gain: F1,1641 ⫽ 0.17, P ⫽ 0.68;
0.19). The VOR gain of ␣9 knockout mice was significantly phase: F1,1640 ⫽ 1.17, P ⫽ 0.28) and peak velocities (gain:
lower than that of cba129 controls for frequencies ⬎2 Hz, F1,1653 ⫽ 0.001, P ⫽ 0.99; phase: F1,1652 ⫽ 0.21, P ⫽ 0.65).
especially for peak velocities of 50 and 100°/s (F1,87 ⫽ 12.2, Slow-phase eye velocity bias. VOR gain and phase were
P ⬍ 0.001 and F1,83 ⫽ 23.5, P ⬍ 0.001, respectively). The measured separately for sinusoidal rotation half-cycles directed
overall VOR phase was similar for ␣9 knockout and cba129 toward the ipsilesional and contralesional ear for stimulus
mice (F6,138 ⫽ 1.1, P ⫽ 0.36), averaging 5.07 ⫾ 2.59° at 0.2 frequencies ranging from 0.2 to 10 Hz with peak velocities of
Hz and increasing to ⫺6.22 ⫾ 4.35° at 10 Hz when stimulus 20, 50, and 100°/s. Figure 3A shows the typical VOR response
peak velocity was 100°/s (F1,275 ⫽ 520.7, P ⬍ 0.001). of a cba129 control mouse, evoked by sinusoidal rotations (1
Figure 2B shows the baseline transient VOR GA (gain in Hz, with a peak velocity of 50°/s) on day 1 after UL. Figure 3B
response to the initial constant acceleration of transient step shows the head and eye velocity traces of the same mouse for
stimuli) and GV (gain in response to the constant velocity ⬎10 stimulus cycles superimposed with quick phases re-
plateau of transient step stimuli) for cba129 control mice moved. Note the considerable slow-phase velocity bias toward
(black columns to left of vertical dashed line) and wild-type the intact ear and the response asymmetry between ipsilesional
littermates (gray columns to left of vertical dashed lines) and contralesional rotations. This nonzero bias in eye velocity
obtained from heterozygous breeding pairs. There was no was most pronounced on day 1 after UL and decreased sub-
difference in baseline transient VOR between cba129 con- stantially as VOR compensation progressed (F1,31 ⫽ 20.8, P ⬍
trols and wild-type littermates for GA (F1,191 ⫽ 1.91, P ⫽ 0.001). In controls, the average eye velocity bias during sinu-
0.18), GV (F1,190 ⫽ 0.93, P ⫽ 0.34), and latency (F1,191 ⫽ soidal vestibular stimulation on day 1 [measured as the average
1.31, P ⫽ 0.26). In ␣9 knockout mice, GA was significantly slow-phase eye velocity offset as the stimulus changed direc-
reduced at 1.02 ⫾ 0.21 compared with cba129 control mice, tion (zero-cross of stimulus velocity trace)] was 7.87 ⫾
which had an average GA of 1.14 ⫾ 0.19 (F1,24 ⫽ 4.44, P ⬍ 4.32°/s. Five days after UL, this bias mostly resolved with an
0.05). In contrast, GV was similar between the two mouse average offset of 0.12 ⫾ 0.29°/s. On day 28, the average offset
types (F2,72 ⫽ 0.45, P ⫽ 0.64). Gains were symmetrical was 0.07 ⫾ 0.37°/s.
(similar for rotations to either side) for both the initial Figure 3, C and D, shows the typical VOR response of an ␣9
acceleration and constant velocity part of the VOR response knockout mouse under the same test and analysis conditions as
under all tested stimulus conditions. shown in Fig. 3, A and B. Compared with cba129 mice, ␣9
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. contral-
esional 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 non-
zero bias due to spontaneous nystagmus. C:
typical VOR response of an ␣9 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, ␣9 knockout mice demonstrated very
low VOR gains and only minimal asymmetry
between ipsilesional vs. contralesional rota-
tions.

knockout mice developed a significantly lower slow-phase 50, and 100°/s (data were pooled for stimuli ⱖ1 Hz). On day
velocity bias after UL surgery (F1,28 ⫽ 9.3, P ⬍ 0.01). The 1 (24 ⫾ 2 h) after UL, gains were significantly reduced in both
average bias in ␣9 knockout mice when measured on day 1 was mouse types and for all test stimulus frequencies and veloci-
only 2.12 ⫾ 2.76°/s, ~70% smaller compared with that in ties. In cba129 mice, the average gain on day 1, pooled across
controls (T1100 ⫽ 26, P ⬍ 0.001). Similarly to that in controls, frequencies and velocities, was 0.22 ⫾ 0.13 for ipsilesional
this bias substantially decreased over subsequent days and was and 0.42 ⫾ 0.22 for contralesional rotations. An even larger
minimal when evaluated on day 5 (0.06 ⫾ 1.01°/s) and day 28 decrease in gain was observed in ␣9 knockout mice, with
(0.05 ⫾ 0.27°/s). In both mouse types, the observed slow- average gain of 0.10 ⫾ 0.08 for ipsilesional and 0.13 ⫾ 0.11
phase velocity bias was consistent across stimulus velocities for contralesional rotations. Moreover, gain asymmetry be-
(F1,123 ⫽ 0.0, P ⫽ 0.863) but increased with stimulus fre- tween stimulus directions was less pronounced in ␣9 knockout
quency (F1,1294 ⫽ 10.0, P ⬍ 0.005). compared with cba129 mice. Post-lesion VOR gains on day 1
Time course of VOR compensation. Figure 4 shows VOR did not change with stimulus peak velocity, as was the case
responses to sinusoidal rotations (0.2 to 10 Hz at 100°/s) before UL (F1,42 ⫽ 2.748, P ⫽ 0.105).
measured on days 1, 5, and 28 following UL (solid lines). For both mouse types, the VOR gain in both directions was
These VOR gains were compared with cba129 control (filled significantly improved on day 5 compared with day 1
symbols) and ␣9 knockout mice (open symbols) pre-lesion (F1,1279 ⫽ 59, P ⬍ 0.001); however, it remained low compared
responses (dashed lines). Figure 5 shows ipsilesional (black) with pre-lesion values. For cba129 mice, the average ipsile-
and contralesional (gray) VOR gains for cba129 (solid lines) sional gains on day 5 pooled across stimulus peak velocities
and ␣9 knockout mice (dashed lines) for peak velocities of 20, ranged from 0.12 ⫾ 0.06 at 0.2 Hz to 0.49 ⫾ 0.13 at 10 Hz.
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 ⱕ80% of pre-lesion
function, whereas VOR gains in ␣9 knockout mice remained
significantly impaired at ⱕ30% 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 ␣9 knock-
out mice (thick dashed lines). Gains are
shown for peak velocities 20, 50, and 100°/s
(data were pooled for stimuli ⱖ1 Hz). VOR
gains of cba129 mice recovered most for a
stimulus peak velocity of 50°/s. ␣9 knockout
mice showed only minimal VOR gain recov-
ery compared with cba129 mice. VOR gain
asymmetry in cba129 mice was maximal on
day 1 but recovered successively until day
28. In contrast, ␣9 knockout mice showed no
VOR gain asymmetry on day 1. VOR gain
asymmetry on day 28 was similar between
␣9 knockout and cba129 mice.
Similar recovery of gain was also observed for rotations toward Figure 6 shows the VOR phase on day 5 after UL (acute
the contralesional side, with gains ranging from 0.34 ⫾ 0.22 at compensated), plotted across frequencies and days (pooling
0.2 Hz to 0.64 ⫾ 0.18 at 10 Hz. For cba129 mice, there was a 0.2-, 1-, and 10-Hz phases) post-lesion in cba129 (solid lines)
significant effect of stimulus frequency on both ipsilesional and and ␣9 knockout mice (dashed lines). Phase leads decreased
contralesional VOR gains (F1,47 ⫽ 8.30, P ⬍ 0.01). VOR gain with increasing frequency. VOR phase in response to ipsile-
recovery was minimal at peak velocity 20°/s and most pro- sional and contralesional rotations was significantly affected by
nounced at peak velocity 100°/s (F1,47 ⫽ 51.97, P ⬍ 0.001; UL (F8,838 ⫽ 2.97, P ⬍ 0.001). On day 1 after UL, there was
Fig. 5). The average gain increase (pooling ipsilesional and a marked increase in phase lead at all test frequencies and
contralesional directions) between days 1 and 5 at 20°/s (for velocities. In cba129 mice, the average phase at 0.2 Hz was
frequencies ⱖ2 Hz) was only 0.05 ⫾ 0.03, whereas the aver- 51.75 ⫾ 4.88° for ipsilesional and 15.95 ⫾ 3.10° for contral-
age gain increase at 100°/s was 0.20 ⫾ 0.03. For ␣9 knockout esional rotations. This phase lead was even larger in ␣9
mice, the average ipsilesional VOR gain ranged from knockout mice, which had an average phase of 75.06 ⫾ 25.13°
0.07 ⫾ 0.03 at 0.2 Hz to 0.33 ⫾ 0.12 at 10 Hz, and the average and 49.23 ⫾ 42.38° for ipsilesional and contralesional rota-
contralesional gain ranged from 0.18 ⫾ 0.10 at 0.2 Hz to tions, respectively. Smaller increases in phase were observed at
0.40 ⫾ 0.13 at 10 Hz. This means that 5 days after UL, the 10 Hz. In cba129 mice, phase at 10 Hz was similar to pre-
VOR gain of ␣9 knockout mice remained on average lesion values at ⫺0.68 ⫾ 8.58° for ipsilesional and ⫺2.52 ⫾
0.24 ⫾ 0.01 lower than that of cba129 mice. 6.02° for contralesional rotations (F1,116 ⫽ 2.3, P ⫽ 0.10 and
Cba129 mice continued to show significant recovery of VOR F1,116 ⫽ 2.43: P ⫽ 0.14, respectively). In contrast, ␣9 knock-
response gains between days 5 and 28 after UL (F1,1390 ⫽ 173.6, out mice displayed significantly increased phase lag compared
P ⬍ 0.001). Both ipsilesional and contralesional gains recov- with pre-lesion values, with an average phase lag of
ered most for frequencies between 1 and 5 Hz. The average 11.46 ⫾ 19.13° and 14.86 ⫾ 19.59° for ipsilesional (F1,155 ⫽
gain for ipsilesional rotations (pooled across 1 to 5 Hz) ranged 2.61, P ⫽ 0.11) and contralesional (F1,155 ⫽ 35.5, P ⬍ 0.001)
from 0.54 ⫾ 0.13 at peak velocity 20°/s to 0.69 ⫾ 0.13 at peak rotations, respectively.
velocity 100o/s. VOR gains for contralesional rotations aver- For cba129 mice, the VOR phase in response to ipsilesional
aged 0.59 ⫾ 0.13 and 0.80 ⫾ 0.10 at peak velocities 20 and rotations minimally normalized between days 1 and 5 after UL.
100°/s, respectively. ␣9 knockout mice also had VOR recov- On average, the phase lead decreased by 5.15 ⫾ 2.73° for
ery. Between days 5 and 28, ipsilesional gains improved to frequencies ⬍1 Hz (T65.9 ⫽ 1.89, P ⫽ 0.063) and remained
0.14 ⫾ 0.07 at 0.2 Hz and 0.36 ⫾ 0.07 at 10 Hz, and contral- unchanged for frequencies ⱖ1 Hz (T99.8 ⫽ 0.53, P ⫽ 0.592).
esional gains improved to 0.27 ⫾ 0.13 at 0.2 Hz and Similarly, VOR phase recovery in response to contralesional
0.37 ⫾ 0.08 at 10 Hz. The average gain increase between days rotations was limited. For frequencies ⬍1 Hz, contralesional
5 and 28, pooled across all frequencies and peak velocities, was phase lead decreased by 6.93 ⫾ 1.33° between days 1 and 5
only 0.09 ⫾ 0.01 in ␣9 knockout mice compared with (T69.9 ⫽ 5.19, P ⬍ 0.001). At frequencies ⱖ1 Hz, phase did not
0.20 ⫾ 0.01 in cba129 controls, indicating that the rate of improve (T101.3 ⫽ 1.24, P ⫽ 0.217). VOR phase showed
recovery was significantly lower in ␣9 knockout mice (F1,47 ⫽ continued recovery toward pre-lesion responses so that at 28
43.89, P ⬍ 0.001). On day 28, the difference in VOR gains days after UL, phase was close to normal for frequencies ⱖ5
between mouse types increased with frequency when the data Hz (ipsilesional: T136.94 ⫽ 2.55, P ⬍ 0.01; contralesional:
at 10 Hz were excluded (F8,247 ⫽ 11.6, P ⬍ 0.001). The T145.4 ⫽ 1.01, P ⫽ 0.315). At frequencies ⬍5 Hz, VOR phase
apparent decrease in gain at 10 Hz between days 5 and 28 was also significantly improved, effectively halving the VOR phase
due to additional animals being included at each time point. lead compared with that on day 5; however, phase remained
When only animals tested at both time points were included, considerably increased compared with pre-lesion values. VOR
the difference between days was no longer significant (cba129: phase leads at frequencies ⬍1 Hz were similar between ␣9
F1,51 ⫽ 0.001, P ⫽ 0.97; ␣9 knockout: F1,51 ⫽ 0.57, P ⫽ knockout and cba129 mice. For ipsilesional rotations, only ␣9
0.45). knockout phase responses were slightly larger at 4.90 ⫾ 2.38°
Fig. 6. VOR phase recovery across frequen-

cies. A: VOR phase on day 5 after UL (acute
compensated), plotted across frequencies.
Changes in VOR phase following UL were
similar in cba129 and ␣9 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 ␣9
knockout mice (dashed lines) at 3 frequen-
cies. Phase leads decreased with increasing
frequency. VOR phase was significantly
more positive on day 1 for ␣9 knockout mice
compared with controls. At 10 Hz, a larger
phase lead for ␣9 knockout mice was still
present 28 days after UL.
on day 5 and 2.73 ⫾ 1.42° on day 28, compared with controls rotations recovered to ~90% of its pre-lesion function. The
(T118.7 ⫽ 2.06, P ⬍ 0.05 and T66.90 ⫽ 1.92, P ⫽ 0.059, initially large asymmetry between ipsilesional and contral-
respectively). However, phase in ␣9 knockout mice was sig- esional cba129 VOR responses continuously reduced from
nificantly more positive than that in cba129 mice at frequencies ~50% on day 1 after UL to ~20% on day 5 and ⬍8% on day
⬎1 Hz for both ipsilesional and contralesional rotations (Fig. 28. However, VOR responses to both ipsilesional and contral-
6). The largest difference in phase between cba129 and ␣9 esional rotations remained significantly impaired for low and
knockout mice was observed at 10 Hz (ipsilesional: 9.80° on high frequency extremes (⬍1 Hz and ⬎5 Hz) and for the
day 5 and 11.66° on day 28; contralesional: 6.78° on day 5 and lowest peak velocity tested (20°/s). In contrast, the rate of
10.20° on day 28). recovery in ␣9 knockout mice was significantly lower than in
In summary, VOR compensation in ␣9 knockout mice was controls, and VOR recovery was minimal for frequencies
severely impaired compared with that in cba129 controls. ⱖ5 Hz.
Figure 7 compares VOR gain recovery between cba129 (filled
circles) and ␣9 knockout mice (open circles). Response gains Transient VOR Response After UL
were normalized to pre-lesion values for each vestibular stim-
ulus condition to compute the percentage of recovery. For Figure 2B shows the transient VOR GA and GV for cba129
mid-range frequencies (between 2 and 5 Hz), the ipsilesional control mice (black columns to right of vertical dashed line)
VOR response recovered to ~75% of its pre-lesion function in and wild-type littermates (gray columns to right of vertical
cba129 mice compared with ~30% recovery in ␣9 knockout dashed lines) on days 1, 5, and 28 after UL. There was no
mice. For cba129 mice, the VOR response to contralesional difference in transient VOR between cba129 controls and
Fig. 7. Percentage of function relative to pre-lesion. VOR gain

recovery was compared between cba129 (filled triangles) and
␣9 knockout mice (open circles). Response gains were normal-
ized to pre-lesion values for each vestibular stimulus condition
to compute the percentage of recovery. Ipsilesional VOR re-
sponses on day 1 were similar between mouse types. However,
␣9 knockout mice showed only minimal functional recovery 28
days after UL, leaving the ipsilesional VOR significantly im-
paired. Contralesional VOR responses on day 1 were signifi-
cantly lower in ␣9 knockout mice compared with controls.
Although the time course of contralesional VOR recovery was
similar between cba129 and ␣9 knockout mice, VOR responses
after 28 days remained significantly lower in the latter.

wild-type littermates across days after UL (GA: F1,366 ⫽ 1.21, P ⬍ 0.02). At 6,000°/s2, the highest acceleration tested, GA was
P ⫽ 0.27; GV: F1,377 ⫽ 0.001, P ⫽ 0.99; latency: F1,374 ⫽ 0.01, similar to the response before UL. A similar increase with
P ⫽ 0.92). contralesional acceleration, however, was absent in ␣9 knock-
Slow-phase eye velocity bias. The post-lesion eye velocity out mice (F1,13 ⫽ 2.6, P ⫽ 0.133). We did not observe an
bias due to spontaneous nystagmus was defined as the average acceleration effect on ipsilesional VOR responses of either
slow-phase eye velocity 50 –300 ms before transient stimulus mouse type (F1,31 ⫽ 0.0, P ⫽ 0.577).
onsets. A significant bias was only observed on day 1 after UL Figure 9 shows the acceleration gain (GA; top row) and
(F1,226 ⫽ 50.71, P ⬍ 0.001), with average slow-phase veloc- constant-velocity gain (GV; bottom row) measured in cba129
ities of 9.25 ⫾ 7.15°/s for cba129 mice and 5.55 ⫾ 7.73°/s for (black) and ␣9 knockout mice (gray) before and 1, 5, and 28
␣9 knockout mice (F1,22 ⫽ 4.06, P ⫽ 0.056). Five days after days after UL (stimulus: 3,000°/s2 reaching 150°/s constant-
UL this bias completely resolved. Before the VOR gain and velocity plateau). Transient VOR recovery showed a similar
latency were calculated, the slow-phase bias for each eye time course to that observed for sinusoidal stimuli. The con-
velocity trace was subtracted so that VOR gain and latency tralesional acceleration gain GA of cba129 mice decreased
described the dynamic VOR response to head rotation. between days 1 and 5, particularly in response to 6,000°/s2
Time course of VOR compensation. Figure 8 shows an stimuli (average GA on day 5: 0.65 ⫾ 0.16; F1,10 ⫽ 4.7, P ⫽
example of the acute compensated VOR response after UL in 0.054). This was not the case for ␣9 knockout mice, which
a cba129 control mouse (top row). A large asymmetry between showed no change in GA (F1,20 ⫽ 0.3, P ⫽ 0.596). Following
ipsilesional and contralesional VOR gain was observed during this initial decrease, the VOR response GA in cba129 mice
the initial acceleration part of the stimulus (GA), but not during started to increase significantly (F1,22 ⫽ 32, P ⬍ 0.001). By
the subsequent constant-velocity part (GV). The deacceleration day 28, contralesional VOR responses were near normal with
after ipsilesional rotations also triggered a large VOR eye GA measuring between 0.78 ⫾ 0.22 at 1,500°/s2 and 1.01 ⫾
movement response. Measurements on day 1 (~24 h) after UL 0.18 at 6,000°/s (interaction with stimulus acceleration: F1,22 ⫽
surgery confirmed severely impaired VOR responses for all 10, P ⬍ 0.005). GA in ␣9 knockout mice also started to
mice tested. VOR gain reduction was most pronounced for increase (F1,21 ⫽ 18.6, P ⬍ 0.001), but at a reduced rate. After
rotations toward the lesioned ear. The average GA in cba129 28 days of recovery, contralesional VOR gains in ␣9 knockout
(control) mice was 0.25 ⫾ 0.09 for ipsilesional rotations and mice remained significantly impaired compared with values in
0.81 ⫾ 0.36 for contralesional rotations. In comparison, ␣9 cba129 mice, with an average GA of 0.63 ⫾ 0.16 (pooled
knockout mice had an average GA of 0.24 ⫾ 0.07 for ipsile- across acceleration stimuli). Stimulus acceleration had no sig-
sional rotations and 0.57 ⫾ 0.26 for contralesional rotations. nificant effect (F1,29 ⫽ 3.2, P ⫽ 0.083).
Ipsilesional GA was similar for both mouse types (F1,30 ⫽ 0.0, The ipsilesional VOR response of cba129 mice showed
P ⫽ 0.528), whereas the contralesional VOR gain was signif- significant recovery between days 1 and 28. On day 28, the
icantly lower in ␣9 knockout mice (F1,24 ⫽ 21, P ⬍ 0.001). ipsilesional VOR response recovered to 0.52 ⫾ 0.15 (~50% of
This resulted in a significantly lower asymmetry between pre-lesion response gain) and almost double the response to the
ipsilesional and contralesional rotations compared with cba129 initial acceleration component of the transient step stimuli from
mice. In cba129 mice, we observed a significant increase in GA day 1. The recovery of ipsilesional responses was less pro-
as contralesional stimulus acceleration increased (F1,10 ⫽ 8.5, nounced in ␣9 knockout mice. Changes between days 1 and 5
Fig. 8. Transient VOR response on day 5 after UL.

Typical VOR response is shown for a cba129 mouse
(top row) and ␣9 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 (GA), but not during the
subsequent constant-velocity part (GV).

Fig. 9. VOR compensation during transient

steps of acceleration. Acceleration gain (GA;
top row) and constant-velocity gain (GV;
bottom row) measured in cba129 (black) and
␣9 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 con-
trast to sinusoidal rotations, responses to
ipsilesional rotations remained significantly
reduced relative to pre-lesion values even
after 28 days of recovery.
after UL were insignificant with an average GA of 0.34 ⫾ 0.05 toward the contralesional side, GV was 0.30 ⫾ 0.07 for cba129
on day 5 (F1,23 ⫽ 2.7, P ⫽ 0.111). VOR responses continued mice and 0.05 ⫾ 0.09 in ␣9 knockout mice. For ␣9 knockout
to recover only slowly, resulting in a significantly lower GA on mice, the VOR response during constant-velocity rotation (GV)
day 28 (0.37 ⫾ 0.24) compared with that in cba129 mice was completely absent for rotations in both directions, whereas
(F1,20 ⫽ 6.5, P ⬍ 0.02). Figure 10 shows the relative difference
of GA 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, ␣9
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 contral-
esional rotations remained. VOR gain asymmetry of ␣9 knock-
out mice continued to increase following UL and was maximal
after 28 days of recovery.
In contrast to GA, the velocity gain GV was severely reduced
for both ipsilesional and contralesional rotations. Although the
reduction in GV was less in response to contralesional com- Fig. 10. VOR gain asymmetry. Relative difference of acceleration gain (GA)
pared with ipsilesional rotations (F1,100 ⫽ 26, P ⬍ 0.001), the between ipsilesional and contralesional transient steps of acceleration at
overall decrease in contralesional GV was large compared with 3,000°/s2 is shown. Cba129 mice developed an ~60% VOR gain asymmetry on
the decrease observed in contralesional GA. For both direc- day 1 after UL. In contrast, ␣9 knockout mice only had an ~20% asymmetry
tions, there was a marked difference in GV between cba129 and on day 1. The asymmetry in cba129 mice significantly improved between days
1 and 28. However, even after 28 days, a considerable asymmetry between
␣9 knockout mice (F1,3 ⫽ 42, P ⬍ 0.001). For rotations toward ipsilesional and contralesional rotations remained. VOR gain asymmetry of ␣9
the ipsilesional side, GV was 0.130 ⫾ 0.073 for cba129 mice knockout mice continued to increase following UL and was maximal after 28
and 0.06 ⫾ 0.05 for ␣9 knockout mice. Similarly, for rotations days of recovery.

in cba129 mice, GV was reduced for both directions but was not suggests they have normal vision. Visual problems could have
completely abolished. GV for both cba129 and ␣9 knockout been associated with the cba129 strain we employed during
mice recovered slowly and remained severely impaired backcross breeding, but both wild-type littermates and cba129
throughout the 28-day recovery period (Fig. 9). In cba129 controls had similar levels of robust compensation, suggesting
mice, GV recovered to 0.45 ⫾ 0.08 for ipsilesional rotations adequate vision and slip signals. Second, we have previously
and 0.51 ⫾ 0.10 for contralesional rotations by day 28 after shown that visual-vestibular mismatch VOR gain training
UL. This recovery was less in ␣9 knockout mice, which only results in VOR adaptation in ␣9 knockout mice, albeit ~70%
increased to 0.31 ⫾ 0.08 and 0.34 ⫾ 0.14 for ipsilesional and less than in controls, suggesting they generate an image slip
contralesional rotations, respectively. Note that for both mouse signal sufficiently large enough to drive adaptation (Hübner et
types, GV was near symmetrical on day 28. The latency of al. 2015).
VOR responses to transient steps of acceleration was not
affected by UL (F1,21 ⫽ 0.1, P ⫽ 0.792), and there was no Vestibular Compensation via Central and Peripheral
difference between the two mouse types (F1,65 ⫽ 0.0, P ⫽ Mechanisms
0.926).
Previous studies have focused efforts primarily on the iden-
tification and characterization of central vestibular plasticity
DISCUSSION mechanisms that are involved in the process of vestibular
In this study we compared the time course of vestibular compensation after unilateral vestibular lesion (for a review see
compensation between cba129 (controls) and ␣9 knockout Beraneck and Idoux 2012; Curthoys and Halmagyi 1995;
mice. The ␣9 knockout mouse has a compromised efferent Straka et al. 2005). Identified processes include (among others)
vestibular system (EVS) due to deletion of most of the gene major synaptic reorganization within ipsi- and contralesional
encoding ␣9 nicotinic acetylcholine receptors (␣9-nAChRs) on vestibular nuclei and changes in the intrinsic properties of 2°
efferent targets in the vestibular periphery. Our findings sug- VN (Beraneck et al. 2004; Him and Dutia 2001; Straka et al.
gest that ␣9-nAChRs are an essential part of VOR compensa- 2005; Vibert et al. 2000). Only hours after a UL has deprived
tion following UL. If utilization of ␣9-nAChRs by the EVS is ipsilesional 2° VN from sensory input, these neurons begin to
impaired, as is the case for ␣9 knockout mice, then vestibular spontaneously discharge, probably in an effort to restore a
compensation is significantly reduced and less able to restore balanced state between vestibular nuclei on both sides and
the VOR. Notably, a compromised EVS affects the recovery of reduce static deficits that cause spontaneous nystagmus and
both ipsilesional and contralesional VOR responses. Differ- vertigo (Ris et al. 1995, 1997; Vibert et al. 1999a). This
ences in the VOR response of ␣9 knockout mice were evident spontaneous discharge appears to be independent of commis-
as early as ~24 h after UL. The most prominent difference was sural innervation from the contralesional side (Anastasio 1992;
a substantially lower gain of the contralesional VOR response. Cartwright and Curthoys 1996; Fetter and Zee 1988). Interest-
Whereas cba129 mice retained ~50% of their initial function ingly, increases in spontaneous activity are predominantly seen
for rotations toward the contralesional side, ␣9 knockout mice in ipsilesional type B (phasic) neurons, whereas changes in
only retained ~20%. Chronic compensated VOR gains (on day type A (tonic) neurons only begin to appear 7–11 days after UL
28 after UL) in ␣9 knockout mice were on average ~48% lower (Him and Dutia 2001). Furthermore, long-term changes of
for ipsilesional and ~45% lower for contralesional rotations intrinsic signal properties of ipsi-and contralesional 2° VN
compared with cba129 mice. Cba129 mice regained up to result in an increased proportion of phasic (type B) neurons in
~75% function (relative to pre-lesion values) for ipsilesional contralesional vestibular nuclei that is paralleled by an increase
and up to ~90% of function for contralesional rotations (de- of tonic (type A) neurons in ipsilesional vestibular nuclei
pending on the sinusoidal vestibular stimulus). In contrast, ␣9 (Beraneck et al. 2003, 2004; Him and Dutia 2001).
knockout mice only regained ~30% and ~50% function, re- In contrast to the extensive body of work on central plastic-
spectively, leaving the VOR severely impaired for rotations in ity mechanisms, relatively few studies have investigated
both directions. Our findings are in contrast to those from a changes in peripheral vestibular signals following UL. How-
previous study by Eron et al. (2015) that examined the time ever, emerging evidence suggests that vestibular compensation
course of VOR compensation in ␣9 knockout mice up to 9 days is a distributed process that requires not only changes in the
after UL. That study did not report significant differences in various structures associated directly with vestibular function,
gain recovery after UL between ␣9 knockout mice and con- including the aforementioned changes in the vestibular nuclei,
trols, but that could be due to the small number (n ⫽ 3) of ␣9 but also changes involving the cerebellum (Beraneck et al.
knockout mice they tested. The post-UL differences we ob- 2008; Dutia 2010; Faulstich et al. 2006; Goto et al. 1997) and
served between mouse types are unlikely due to significant loss changes at the level of the peripheral vestibular organs (Bera-
of vision and/or generation of a retinal image slip signal to neck and Idoux 2012; Cullen et al. 2009; Lasker et al. 2000;
drive compensation in the ␣9 knockout, although there could Llinas and Walton 1979). Single-unit recordings of vestibular
be subtle losses. We did not measure optokinetic function in primary afferents in chronically compensated macaque mon-
these mice; however, there are several lines of evidence sug- keys (1–12 mo after UL) suggest that changes at the level of
gesting that ␣9 knockout mice have adequate vision and slip the vestibular periphery occur alongside central changes to
signal for compensation. First, qualitatively, these mice react to facilitate VOR compensation (Sadeghi et al. 2007). After UL,
light and visual movement in the same way as other mice we the proportion of contralesional irregularly discharging affer-
have tested. The fact that ␣9 knockout mice were able to ents increased, whereas the proportion of regularly discharging
perform a baseline visual discrimination task described by afferents decreased. This resulted in a shift toward more phasic
Terreros et al. (2015) just as well as wild-type mice also signal properties in the peripheral input to central 2° VN
(Sadeghi et al. 2007). A potential mechanism responsible for reduction of resting discharge rate and attenuation of sensitiv-
these changes is EVS-mediated modulation of the response ity/gain) via ␣9-nAChRs coupled to calcium-activated potas-
dynamics of regularly discharging afferents with dimorphic sium (SK) channels (Holt et al. 2006; Poppi et al. 2014). The
terminals. Dimorphic afferents receive inputs from type I and other effect is the excitation of afferents (Boyle and Highstein
type II hair cells and make up the majority (~80%) of afferent 1990; Goldberg and Fernández 1980), through nAChRs that
inputs to brain stem vestibular nuclei. Note that the changes in contain ␣4-, ␣6-, and ␤2-subunits (Holt et al. 2015). In ␣9
primary vestibular afferents are quantitatively and qualitatively knockout mice this inhibition/excitation dual effect would be
similar to changes in 2° VN (Beraneck et al. 2003, 2004; partially compromised. Loss of ␣9-nAChRs would prevent
Beraneck and Idoux 2012). EVS inhibition of type II hair cells, allowing normally sup-
Additional evidence suggesting changes in the proportion pressed type II hair cells to “contribute” to overall afferent
and sensitivity of vestibular primary afferents after UL has activity (particularly dimorphs). Simultaneously, due to the
been produced by a series of studies investigating VOR signal presence of alternative types of nAChRs (i.e., ␣4-, ␣6-, and
processing along tonic (velocity sensitive) and phasic (accel- ␤2-subunits) on calyx-bearing afferents, the excitatory EVS
eration sensitive) signal pathways (Lasker et al. 2000; Minor et effect would still be operating in ␣9 knockout mice. In short,
al. 1999a). Morphologically these tonic and phasic pathways the predicted overall effect of EVS activation on dimorphic
resemble the response dynamics (latency and sensitivity) of afferents in ␣9 knockout mice is increased afferent discharge,
regularly and irregularly discharging vestibular primary affer- but with an additional input from normally EVS-suppressed
ents and 2° VNs, respectively (Hullar et al. 2005). The tonic type II hair cells leading to an increase in afferent regularity
pathway has low sensitivity and therefore unilaterally can and corresponding shift in afferent dynamic response. It is
encode a wide range of ipsilateral and contralateral head possible that ␣9 knockout mice also have other compensatory
rotations before being driven into saturation. It is primarily changes in their hair cell membrane properties, synapses, and
active during stimuli with low-acceleration components (i.e., vestibular neurons (for a review see Straka et al. 2005).
low-frequency, low-velocity sinusoids and the constant-veloc- Remarkably, only moderate changes can be observed in the
ity part of the transient step stimuli). The phasic pathway has baseline VOR response of ␣9 knockout mice before UL (Hüb-
high sensitivity and therefore unilaterally best encodes ipsilat- ner et al. 2015). However, if the vestibular system is chal-
eral head rotations. The asymmetry of VOR responses follow- lenged by UL, the effects of a loss of ␣9-nAChRs become
ing UL is thought to be caused by the phasic pathway because more apparent.
it predominantly encodes short-latency, high-acceleration head Only ~24 h after UL we observed clear differences between
rotations toward the contralesional side but is easily driven into ␣9 knockout and control mice. Among them was a signifi-
inhibitory cutoff for rotations toward the ipsilesional side. As a cantly lower asymmetry between ipsilesional and contral-
result, stimuli that preferentially activate the phasic pathway, esional VOR response gains for all test stimuli. The VOR gain
for example, the rapid acceleration at the beginning of transient asymmetry after UL is thought to be caused by the phasic
step stimuli, produce maximal asymmetry between contral- pathway because irregularly discharging afferents go into in-
esional and ipsilesional VOR response gains (GA). In contrast, hibitory cutoff more readily than regularly discharging affer-
VOR responses that predominantly rely on the velocity-sensi- ents during contralateral rotations (Lasker et al. 2000). In ␣9
tive tonic pathway, for example, the constant-velocity response knockout mice, this phasic pathway is minimal (Han et al.
(GV) of transient step stimuli, produce minimal asymmetry 2007), which may explain why VOR response gains are more
between ipsilesional and contralesional rotations (Lasker et al. symmetrical. In addition, ␣9 knockout mice do not have a
2000). The reduced post-lesion asymmetry we observed in ␣9 mechanism to control the proportion of the phasic pathway
knockout compared with cba129 mice is consistent with this contribution, which might explain why contralesional VOR
hypothesis. Furthermore, Lasker et al. (2000) concluded that responses were significantly depressed in ␣9 knockout mice
VOR compensation depends on an increase in contribution of compared with controls. During the constant-velocity phase of
the contralesional highly modifiable phasic pathways, which is transient step stimuli, a stimulus that predominantly activates
consistent with findings from single-unit recordings of vestib- the tonic pathways, we found absent VOR responses (GV:
ular afferents and 2° VN that showed increased populations of 0.05 ⫾ 0.09) in ␣9 knockout mice, but not in controls (GV:
irregular afferents and type B 2° VN in contralesional vestib- 0.30 ⫾ 0.07). On the basis of our observations in ␣9 knockout
ular nuclei (Beraneck et al. 2003, 2004; Sadeghi et al. 2007). mice, we hypothesize that chronic compensation after UL
relies on two changes in peripheral signal processing. First, the
VOR Compensation in ␣9 Knockout Mice sensitivity of the phasic pathway is reduced, which could occur
centrally (i.e., does not require changes in afferent sensitivity)
It is important to note that before the vestibular lesion is so as to minimize inhibitory cutoff during ipsilesional head
surgically induced, the vestibular primary afferent signal is rotations and reduce asymmetry of the VOR response. Second,
significantly different in ␣9 knockout mice compared with the proportional contribution of the phasic pathway is in-
controls (more tonic with increased proportion and sensitivity creased so as to aid central compensation mechanisms that rely
of regularly discharging afferents; Han et al. 2007; see expla- on this highly modifiable pathway. If the sensitivity of the
nation in Hübner et al. 2015). This shift in afferent signaling phasic pathway remains unchanged, one would predict larger
was shown in a single-unit vestibular afferent study in the ␣9 asymmetry. If the proportion of the phasic pathway remains
knockout mouse (Han et al. 2007) and could be occurring unchanged, one would predict reduced VOR gain compensa-
because EVS activation, specifically the cholinergic compo- tion. The ␣9 knockout mouse appears to have control of the
nent, is thought to have a dual effect. One effect of EVS sensitivity (first change) but not the proportional contribution
activation is the inhibition of type II hair cells (i.e., strictly a (second change) of the phasic pathway, resulting in reduced
asymmetry but with smaller contralesional VOR gain and DISCLOSURES

impaired long-term VOR compensation. No conflicts of interest, financial or otherwise, are declared by the authors.
Could Other Central Mechanisms Be Affecting

Compensation in ␣9 Knockout Mice? AUTHOR CONTRIBUTIONS
P.P.H. and S.I.K. performed experiments; P.P.H., S.I.K., and A.A.M.
Another possible mechanism that could account for reduced analyzed data; P.P.H. and A.A.M. interpreted results of experiments; P.P.H.,
gains on the contralesional side is that ipsilesional commis- S.I.K., and A.A.M. prepared figures; P.P.H. and A.A.M. drafted manuscript;
sures do not release contralesional 2° VN from inhibition. P.P.H., S.I.K., and A.A.M. approved final version of manuscript; A.A.M.
These commissural pathways generally originate from tonic conceived and designed research; A.A.M. edited and revised manuscript.
(type A) neurons on the ipsilateral side but rely on contralateral
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First published January 11, 2017; doi:10.1152/jn.01049.2015.

RESEARCH ARTICLE Sensory Processing

The mammalian efferent vestibular system plays a crucial role in

Patrick P. Hübner,1,2 Serajul I. Khan,1,2 and Americo A. Migliaccio1,2,3

J Neurophysiol • doi:10.1152/jn.01049.2015 • www.jn.org

Fig. 1. Surgical approach: unilateral labyrinthectomy (UL;

J Neurophysiol • doi:10.1152/jn.01049.2015 • www.jn.org

J Neurophysiol • doi:10.1152/jn.01049.2015 • www.jn.org

Fig. 2. Vestibulo-ocular reflex (VOR) response for

J Neurophysiol • doi:10.1152/jn.01049.2015 • www.jn.org

Fig. 3. VOR response after UL. A: typical

J Neurophysiol • doi:10.1152/jn.01049.2015 • www.jn.org

Fig. 4. VOR gain recovery across sinusoidal frequencies. VOR

J Neurophysiol • doi:10.1152/jn.01049.2015 • www.jn.org

Fig. 5. VOR gain recovery across sinusoidal

Fig. 6. VOR phase recovery across frequen-

Fig. 7. Percentage of function relative to pre-lesion. VOR gain

J Neurophysiol • doi:10.1152/jn.01049.2015 • www.jn.org

Fig. 8. Transient VOR response on day 5 after UL.

J Neurophysiol • doi:10.1152/jn.01049.2015 • www.jn.org

Fig. 9. VOR compensation during transient

J Neurophysiol • doi:10.1152/jn.01049.2015 • www.jn.org

asymmetry but with smaller contralesional VOR gain and DISCLOSURES

Could Other Central Mechanisms Be Affecting

J Neurophysiol • doi:10.1152/jn.01049.2015 • www.jn.org

J Neurophysiol • doi:10.1152/jn.01049.2015 • www.jn.org

J Neurophysiol • doi:10.1152/jn.01049.2015 • www.jn.org

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