Understanding the Pathophysiology of Congenital Vestibular Disorders: Current Challenges and Future Directions.

In congenital vestibular disorders (CVDs), children develop an abnormal inner ear before birth and face postnatal challenges to maintain posture, balance, walking, eye-hand coordination, eye tracking, or reading. Only limited information on inner ear pathology is acquired from clinical imaging of the temporal bone or studying histological slides of the temporal bone. A more comprehensive and precise assessment and determination of the underlying mechanisms necessitate analyses of the disorders at the cellular level, which can be achieved using animal models. Two main criteria for a suitable animal model are first, a pathology that mirrors the human disorder, and second, a reproducible experimental outcome leading to statistical power. With over 40 genes that affect inner ear development, the phenotypic abnormalities resulting from congenital vestibular disorders (CVDs) are highly variable. Nonetheless, there is a large subset of CVDs that form a common phenotype of a sac-like inner ear with the semicircular canals missing or dysplastic, and discrete abnormalities in the vestibular sensory organs. We have focused the review on this subset, but to advance research on CVDs we have added other CVDs not forming a sac-like inner ear. We have included examples of animal models used to study these CVDs. Presently, little is known about the central pathology resulting from CVDs at the cellular level in the central vestibular neural network, except for preliminary studies on a chick model that show significant loss of second-order, vestibular reflex projection neurons.

Implementing the chick embryo model to study vestibular developmental disorders.

Children with congenital vestibular disorders show delayed motor development and challenges in maintaining posture and balance. Computed tomography images reveal that these children have abnormal inner ears in the form of a sac, with the semicircular canals missing or truncated. Little is known about how this inner ear abnormality affects central vestibular development. At present, mice with the chromodomain helicase DNA-binding protein 7 mutation are the most common model for studying congenital vestibular disorders, despite forming multiple diverse inner ear phenotypes and inducing abnormal cerebellar and visual system development. To identify the effects of a sac-like inner ear on central vestibular development, we have designed and implemented a new model, the anterior-posterior axis rotated otocyst (ARO) chick, which forms a sac-like inner ear in 85% of cases. The ARO chick is produced by anterior-posterior rotation of the otocyst at embryonic day 2. Here, we describe for the first time the 15% of ARO chicks that form three small semicircular canals and rename the ARO chicks forming sacs (ARO/s chicks). The basic features of the vestibular sensory organs in ARO/s chicks are similar to those found in patients' sacs, and ARO/s hatchlings experience balance and walking problems like patients. Thus, ARO/s chicks have a reproducible inner ear phenotype without abnormalities in vestibular-related structures, making the model a relatively simple one to evaluate the relationship between the sac-like inner ear pathology and formation of the central vestibular neural circuitry. Here, we describe unpublished details on the surgical approaches to produce ARO chicks, including pitfalls and difficulties to avoid.NEW & NOTEWORTHY This paper describes simple techniques for chick otocyst rotation resulting in a sac-like inner ear (85%), the common phenotype in congenital vestibular disorders. We now describe anterior-posterior axis rotated otocyst chicks, which form three small canals (15%), and rename chicks forming a sac (ARO/s chicks). Basic protocols and potential complications of otocyst rotation are described. With the use of ARO/s chicks, it will be possible to determine how the vestibular neural circuit is modified by sac-like inner ear formation.

A New Model for Congenital Vestibular Disorders.

Many developmental disorders of the inner ear are manifested clinically as delayed motor development and challenges in maintaining posture and balance, indicating involvement of central vestibular circuits. How the vestibular circuitry is rewired in pediatric cases is poorly understood due to lack of a suitable animal model. Based on this, our lab designed and validated a chick embryo model to study vestibular development in congenital vestibular disorders. The developing inner ear or "otocyst" on the right side of 2-day-old chick embryos (E2) was surgically rotated 180° in the anterior-posterior axis, forming the "anterior-posterior axis rotated otocyst chick" or ARO chick. The ARO chick has a reproducible pathology of a sac with truncated or missing semicircular canals. A sac is the most common inner ear defect found in children with congenital vestibular disorders. In E13 ARO chicks, the sac contained all three cristae and maculae utriculi and sacculi, but the superior crista and macula utriculi were shortened in anterior-posterior extent. Also, the number of principal cells of the tangential vestibular nucleus, a major avian vestibular nucleus, was decreased 66 % on the rotated side. After hatching, no difference was detected between ARO and normal chicks in their righting reflex times. However, unlike normal chicks, ARO hatchlings had a constant, right head tilt, and after performing the righting reflex, ARO chicks stumbled and walked with a widened base. Identifying the structure and function of abnormally developed brain regions in ARO chicks may assist in improving treatments for patients with congenital vestibular disorder.

Basic Concepts in Understanding Recovery of Function in Vestibular Reflex Networks during Vestibular Compensation.

Unilateral peripheral vestibular lesions produce a syndrome of oculomotor and postural deficits with the symptoms at rest, the static symptoms, partially or completely normalizing shortly after the lesion due to a process known as vestibular compensation. The symptoms are thought to result from changes in the activity of vestibular sensorimotor reflexes. Since the vestibular nuclei must be intact for recovery to occur, many investigations have focused on studying these neurons after lesions. At present, the neuronal plasticity underlying early recovery from the static symptoms is not fully understood. Here we propose that knowledge of the reflex identity and input-output connections of the recorded neurons is essential to link the responses to animal behavior. We further propose that the cellular mechanisms underlying vestibular compensation can be sorted out by characterizing the synaptic responses and time course for change in morphologically defined subsets of vestibular reflex projection neurons. Accordingly, this review focuses on the perspective gained by performing electrophysiological and immunolabeling studies on a specific subset of morphologically defined, glutamatergic vestibular reflex projection neurons, the principal cells of the chick tangential nucleus. Reference is made to pertinent findings from other studies on vestibular nuclei neurons, but no comprehensive review of the literature is intended since broad reviews already exist. From recording excitatory and inhibitory spontaneous synaptic activity in principal cells, we find that the rebalancing of excitatory synaptic drive bilaterally is essential for vestibular compensation to proceed. This work is important for it defines for the first time the excitatory and inhibitory nature of the changing synaptic inputs and the time course for changes in a morphologically defined subset of vestibular reflex projection neurons during early stages of vestibular compensation.

Plasticity of spontaneous excitatory and inhibitory synaptic activity in morphologically defined vestibular nuclei neurons during early vestibular compensation.

After unilateral peripheral vestibular lesions, the brain plasticity underlying early recovery from the static symptoms is not fully understood. Principal cells of the chick tangential nucleus offer a subset of morphologically defined vestibular nuclei neurons to study functional changes after vestibular lesions. Chickens show posture and balance deficits immediately after unilateral vestibular ganglionectomy (UVG), but by 3 days most subjects begin to recover, although some remain uncompensated. With the use of whole cell voltage-clamp, spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) and miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) were recorded from principal cells in brain slices 1 and 3 days after UVG. One day after UVG, sEPSC frequency increased on the lesion side and remained elevated at 3 days in uncompensated chickens only. Also by 3 days, sIPSC frequency increased on the lesion side in all operated chickens due to major increases in GABAergic events. Significant change also occurred in decay time of the events. To determine whether fluctuations in frequency and kinetics influenced overall excitatory or inhibitory synaptic drive, synaptic charge transfer was calculated. Principal cells showed significant increase in excitatory synaptic charge transfer only on the lesion side of uncompensated chickens. Thus compensation continues when synaptic charge transfer is in balance bilaterally. Furthermore, excessive excitatory drive in principal cells on the lesion side may prevent vestibular compensation. Altogether, this work is important for it defines the time course and excitatory and inhibitory nature of changing spontaneous synaptic inputs to a morphologically defined subset of vestibular nuclei neurons during critical early stages of recovery after UVG.

Expression of glutamate receptors and potassium channels in vestibular nuclei neurons during development of signal processing.

The principal cells of the chick tangential vestibular nucleus offer a simple neuron model to study signal processing in second-order, vestibular reflex projection neurons. The principal cells represent a relatively uniform population of vestibular nuclei neurons which receive a major input from the primary vestibular fibers and send axons to targets mainly involved in the vestibuloocular reflexes. Here, studies performed on ion channels involved in the emergence and establishment of signal processing in this morphologically-identified subset of vestibular nuclei neurons are reviewed, including the AMPA glutamate receptor subunits GluR1, GluR2, GluR3, and GluR4 and the potassium channel subunits Kv1.1 and Kv1.2.

Identification of vestibuloocular projection neurons in the developing chicken medial vestibular nucleus.

Biocytin was injected into the oculomotor, trochlear, or abducens nucleus on one side using isolated chicken brainstem preparations or brain slices to identify the medial vestibular nucleus (MVN) neurons projecting to these targets. Oculomotor nucleus injections produced retrogradely labeled neurons in the contralateral ventrolateral MVN (MVN(VL)), with few labeled neurons in the ipsilateral MVN(VL) and rarely in the dorsomedial MVN on either side. Labeled MVN(VL) neurons were identified as stellate (95%) and elongate (5%) cells. Trochlear nucleus injections produced a similar pattern of MVN neuron labeling. Abducens nucleus injections resulted in retrogradely labeled stellate (87%) and elongate (13%) neurons in the MVN(VL), which had smaller cell bodies than those projecting to the oculomotor nucleus. Anteroposteriorly, labeled MVN(VL) neurons were coextensive with the tangential nucleus, with neurons projecting to the oculomotor nucleus distributed lateral to and intermixed with the more medially situated neurons projecting to the abducens nucleus. The fundamental pattern of vestibuloocular projecting neurons was similar at both embryonic ages studied, E16 and E13. In contrast to the case in mammals, where most vestibuloocular projection neurons reside within the MVN, most retrogradely labeled neurons in these chicken preparations were found within the ventrolateral vestibular, descending vestibular, and tangential nuclei. The morphological identification and mapping of vestibuloocular projection neurons in the chicken MVN described here represents the first step in a systematic evaluation of the relationship between avian vestibuloocular neuron structure and function.

Presynaptic and postsynaptic ion channel expression in vestibular nuclei neurons after unilateral vestibular deafferentation.

Vestibular compensation refers to the recovery of function occurring after unilateral vestibular deafferentation, but some patients remain uncompensated. Similarly, more than half of the operated chickens compensate three days after unilateral vestibular ganglionectomy (UVG), but the rest remain uncompensated. This review focuses on the studies performed on the principal cells of the chick tangential nucleus after UVG. The tangential nucleus is a major avian vestibular nucleus whose principal cells are all second-order, vestibular reflex projection neurons participating in the vestibuloocular and vestibulocollic reflexes controlling posture, balance, and eye movements. Using whole-cell patch-clamp approach in brain slice preparations, spontaneous spike firing, ionic conductances, and spontaneous excitatory postsynaptic currents (sEPSCs) are recorded in principal cells from controls and operated chickens three days after UVG. In compensated chickens, the proportion of spontaneous spike firing principal cells and their spike discharge rate are symmetric on the lesion and intact sides, with the rates increased over controls. However, in the uncompensated chickens, the spike discharge rate increases on the lesion side, but not on the intact side, where only silent principal cells are recorded. In all the experimental groups, including controls, silent principal cells are distinguished from spontaneous spiking cells by smaller persistent sodium conductances and higher activation thresholds for the fast sodium channel. In addition, silent principal cells on the intact side of uncompensated chickens have larger dendrotoxin-sensitive potassium conductances, with a higher ratio of immunolabeling for surface/cytoplasmic expression of a dendrotoxin-sensitive, potassium channel subunit, Kv1.1. Finally, in compensated chickens, sEPSC frequency is symmetric bilaterally, but in uncompensated chickens sEPSC frequency increased only on the lesion side, where the expression of Kv1.2 decreased in synaptotagmin-labeled terminal profiles on the principal cell bodies. Altogether, the specific sodium and potassium channels important for the development of spike firing pattern and/or presynaptic glutamate release on vestibular reflex projection neurons may be critically involved in changing postsynaptic neuron excitability after vestibular deafferentation.

Otolith fibers and terminals in chick vestibular nuclei.

The distribution of gravity-sensing, otolith afferent fibers and terminals was studied in the vestibular nuclei of 4-5-day hatchling chicks by using single and double labeling of fibers and terminals with biocytin conjugated to Alexa Fluor and confocal imaging. The vestibular nuclei are represented in a series of five transverse sections of the brainstem immunolabeled with MAP2. Saccular fibers entered the medulla posterior to and at the level of the posterior tangential vestibular nucleus and coursed through ventral parts, producing ascending and descending branches. Small saccular terminals contacted a few dendrites in the tangential nucleus. In contrast, small saccular terminals contacted many dendrites and a few neuron cell bodies in the ventrolateral vestibular nucleus, vestibulocerebellar nucleus, and descending vestibular nuclei. Utricular fibers coursed through ventral parts of the central tangential nucleus before bifurcating into ascending and descending branches. In the tangential nucleus, utricular fibers formed a few large axosomatic terminals (spoon terminals) and a few small terminals on dendrites. In addition, small utricular terminals contacted numerous dendrites and a few neuron cell bodies in the ventrolateral, vestibulocerebellar, and descending vestibular nuclei. Thus, there was negligible overlap in the distribution of the otolith nerves, although each otolith afferent shared common regions with the canal afferents, previously shown, suggesting that some second-order vestibular neurons process convergent inputs from otolith and canal afferents. Taken together with previous results, the present findings identify discrete regions of the chick vestibular nuclei where second-order vestibular neurons likely process directly convergent otolith and canal inputs.

Emergence of action potential generation and synaptic transmission in vestibular nucleus neurons.

Principal cells of the chick tangential nucleus are vestibular nucleus neurons in the hindbrain. Although detailed information is available on the morphogenesis of principal cells and synaptogenesis of primary vestibular fibers, this is the first study of their early functional development, when vestibular terminals emerge at embryonic days 10 and 13 (E10 and E13). At E10, 60% of principal cells generated spikes on depolarization, whereas 50% exhibited excitatory postsynaptic currents (EPSCs) on vestibular-nerve stimulation. The frequency was 0.2 Hz for glutamatergic spontaneous EPSCs (sEPSCs) at -60 mV, and 0.6 Hz for spontaneous inhibitory postsynaptic current (sIPSC) at +10 mV and completely GABAergic. All of these synaptic events were TTX-insensitive, miniature events. At E13, 50% of principal cells generated spikes on depolarization and 82% exhibited EPSCs on vestibular-nerve stimulation. The frequency was 0.7 Hz for sEPSCs at -60 mV, and 0.8 Hz for sIPSCs at +10 mV. Most principal cells had sIPSCs composed of both GABAergic (75%) and glycinergic (25%) events, but a few cells had only GABAergic sIPSCs. TTX decreased the frequency of EPSCs by 12%, and the IPSCs by 17%. In summary, at E10, some principal cells generated immature spikes on depolarization and EPSCs on vestibular-nerve stimulation. At E10, GABAergic events predominated, AMPA events had low frequencies, and glycinergic activity was absent. By E13, glycinergic events first appeared. This data were compared systematically to that obtained from the late-term embryo and hatchling to reveal the long-term sequence of changes in synaptic events and excitability and offer a broader understanding of how the vestibular system is assembled during development.

Vestibular ganglionectomy and otolith nerve identification in the hatchling chicken.

Unilateral peripheral vestibular lesions are characterized by rapid recovery from the static symptoms, called vestibular compensation, a process likely involving brain plasticity. The hatchling chick offers a promising model for studies of this process. Ganglionectomy is performed, since it provides a reproducible lesion. Here, we describe a surgical approach for vestibular ganglionectomy and the identification of the otolith nerves, using drawings and digital images of the surgical field to assist in visualizing and accessing this small, complex, and highly vascular region of the inner ear. A retroauricular approach was used in 4-8-day-old hatchling chicks. Broad access and easy identification of the otolith nerves were achieved by cauterizing the caudal auricular artery and vein in the exoccipital bone and excising the surrounding exoccipital and squamosal bones. The vestibular ganglion was accessed by removing the bony medial wall of the vestibule. Dura mater covering the ganglion was opened, the primary vestibular fibers were cut at the lateral brain surface, and the anterior and posterior parts of the vestibular ganglion were extirpated. At 24 h after surgery, the survival rate was 87% and complete ganglionectomy was achieved in 85% of operated animals.

AMPA receptor subunit expression in chick vestibular nucleus neurons.

The principal cells of the chick tangential nucleus are vestibular nucleus neurons whose responses on vestibular nerve stimulation are abolished by glutamate receptor antagonists. Using confocal microscopy, we quantified immunolabeling for AMPA receptor subunits GluR1, GluR2, GluR2/3, and GluR4 in principal cells that were identified by the neuronal marker, microtubule-associated protein 2 (MAP2). This work was focused primarily on 9 days after hatching (H9) when the principal cells have acquired some important mature electrophysiologic properties. At H9, the principal cell bodies stained strongly with GluR2/3 and GluR4, whereas GluR1 and GluR2 produced weak signals. Moreover, GluR2/3 and GluR4 receptor subunit clusters in principal cell bodies and dendrites were localized at sites contacted by biocytin-labeled vestibular nerve terminals and synaptotagmin-labeled terminals. Developmental expression of AMPA receptor immunolabeling was studied in the principal cell bodies at embryonic day 16 (E16) and hatching (H1). At E16, labeling for GluR4 was already strong, and continued to increase at H1 and H9. In contrast, GluR2/3 labeling was weak at E16, but increased significantly at H1, and more so by H9. GluR1 and GluR2 were present at low levels at E16 and H1. From E16 to H9, overall AMPA receptor subunit expression increased steadily, with H9 showing the strongest labeling. Ultrastructural observations at E16 and H3 confirmed the presence of immunogold labeling for AMPA receptor subunits at the vestibular nerve and non-vestibular nerve synapses on the principal cell bodies. In summary, these results indicate that GluR3 and GluR4 are the major AMPA receptor subunits involved in excitatory synaptic transmission in principal cells during the perinatal period.

Spontaneous synaptic activity is primarily GABAergic in vestibular nucleus neurons of the chick embryo.

The principal cells of the chick tangential nucleus are vestibular nucleus neurons participating in the vestibular reflexes. In 16-day embryos, the application of glutamate receptor antagonists abolished the postsynaptic responses generated on vestibular-nerve stimulation, but spontaneous synaptic activity was largely unaffected. Here, spontaneous synaptic activity was characterized in principal cells from brain slices at E16 using whole cell voltage-clamp recordings. With KCl electrodes, the frequency of spontaneous inward currents was 3.1 Hz at -60 mV, and the reversal potential was +4 mV. Cs-gluconate pipette solution allowed the discrimination of glycine/GABA(A) versus glutamate receptor-mediated events according to their different reversal potentials. The ratio for spontaneous excitatory to inhibitory events was about 1:4. Seventy-four percent of the outward events were GABA(A), whereas 26% were glycine receptor-mediated events. Both pre- and postsynaptic GABA(B) receptor effects were shown, with presynaptic GABA(B) receptors inhibiting 40% of spontaneous excitatory postsynaptic currents (sEPSCs) and 53% of spontaneous inhibitory postsynaptic currents (sIPSCs). With TTX, the frequency decreased approximately 50% for EPSCs and 23% for IPSCs. These data indicate that the spontaneous synaptic activity recorded in the principal cells at E16 is primarily inhibitory, action potential-independent, and based on the activation of GABA(A) receptors that can be modulated by presynaptic GABA(B) receptors.

Developmental change in expression and subcellular localization of two shaker-related potassium channel proteins (Kv1.1 and Kv1.2) in the chick tangential vestibular nucleus.

The chick tangential nucleus is a major avian vestibular nucleus whose principal cells participate in two vestibular reflexes. Intracellular recordings have shown that the principal cells acquire their mature firing pattern gradually during development. At embryonic day 16 (E16), most principal cells fire a single spike, whereas shortly after hatching (H) the vast majority fire repetitively on depolarization. The transition in firing pattern was likely due in part to a downregulation of a low-threshold, sustained, dendrotoxin-sensitive (DTX) potassium current, I(DS). Since the DTX-sensitive potassium channel subunits Kv1.1 and Kv1.2 generate sustained currents, in the present study we applied fluorescence immunocytochemistry and confocal microscopy to characterize their developmental expression at E16, H1, and H9. At E16, both Kv1.1 and Kv1.2 staining were confined to the principal cell bodies. Immunolabeling decreased significantly for both proteins at H1, and more so by H9. Double-labeling with a monoclonal antibody against microtubule-associated protein 2 (MAP2) in hatchlings showed that some Kv1.1 remained as clusters within the cell body, at the base of the dendrites, and in the axon initial segment. In hatchlings, Kv1.2 staining decreased in the cell bodies and simultaneously appeared in the neuropil, colocalized with biocytin-labeled primary vestibular fibers and vestibular "spoon" terminals. Also, double-labeling with synaptotagmin showed that Kv1.2 colocalized with many nonvestibular terminals surrounding the principal cell bodies. These results identified developmental decreases in the staining of these two potassium channel protein subunits and changes in their subcellular localization corresponding to the downregulation of I(DS) defined electrophysiologically around hatching. Accordingly, both of these protein subunits could be involved in regulating excitability of the principal cells.

Differential expression of connexin 43 in the chick tangential vestibular nucleus.

The chick tangential nucleus is a major vestibular nucleus whose principal cells receive convergent inputs from primary vestibular and nonvestibular fibers and participate in the vestibular reflexes. During development, the principal cells gradually acquire the mature firing pattern in part by losing a specific potassium current around hatching (H). Here we focus on characterizing the expression of connexin 43 (Cx43), a gap junction protein found mainly between astrocytes in the mature brain. The astrocytic syncytium plays an important role in maintaining extracellular potassium ion balance in the brain. Accordingly, it is important to characterize the potential of this syncytium to communicate during the critical developmental age of hatching. Using fluorescence immunocytochemistry, we investigated whether Cx43 staining was concentrated in specific cellular compartments at H1 by applying well-known markers for astrocytes (glial fibrillary acidic protein; GFAP), oligodendrocytes (antimyelin), neurons (microtubule-associated protein 2), and synaptic terminals (synaptotagmin). GFAP-positive astrocytes and GFAP-negative nonneuronal cells around the principal cell bodies were labeled with Cx43, suggesting that Cx43 was expressed exclusively by nonneuronal cells near the neuronal elements. Next, the developmental pattern of expression of Cx43 was studied at embryonic day 16 (E16), H1, and H9. At E16, Cx43 was present weakly as random small clusters in the tangential nucleus, whereas, at H1, overall staining became localized, with increases in size, brightness, and number of immunostained clusters. Finally, at H9, Cx43 staining decreased, but cluster size and location remained unchanged. These results suggest that Cx43 is developmentally regulated with a peak at birth and is associated primarily with astrocytes and nonneuronal cells near the principal cell bodies.

Vestibular compensation after ganglionectomy: ultrastructural study of the tangential vestibular nucleus and behavioral study of the hatchling chick.

The tangential nucleus is a major part of the avian vestibular nuclear complex, and its principal cells are structurally distinctive neurons participating in the vestibuloocular and vestibulocollic reflexes. After unilateral peripheral vestibular lesion, a behavioral recovery of function defined as vestibular compensation is observed. Because sprouting and hypertrophy of synapses have been reported in other regions of immature animals after central nervous system injury, we investigated whether this also occurs in the vestibular nuclei during compensation. To test this hypothesis, unilateral vestibular ganglionectomy was performed on 4-6-day-old hatchlings and vestibular function was tested during the next 2 months. Degeneration and evidence for regeneration of synapses were studied in the tangential nucleus at 1, 3, 7, and 56 days after surgery. Spoon endings, large vestibular terminals on the principal somata, degenerated 1-3 days after surgery. However, the small synaptic terminals showed no significant change in the percentage or number covering the soma or in mean terminal lengths in the deafferented or contralateral tangential nucleus. Furthermore, there was no evidence of neuron death in the tangential nucleus. Vestibular compensation occurred in three stages: 0-3 days, when vestibular synapses degenerated and severe behavioral deficits were seen; 4-9 days, when primary vestibular fibers degenerated centrally and marked improvement in both the static and the dynamic symptoms were observed; and 10-56 days, when changes in neuronal morphology were not detected but the dynamic symptoms gradually improved. Accordingly, after unilateral vestibular ganglionectomy, vestibular compensation proceeded without ultrastructural evidence of sprouting or hypertrophy of axosomatic synapses in the hatchling tangential nucleus. This rapid behavioral recovery of function distinguishes the vestibular system from other sensory systems, which, in general, exhibit much less robust recovery after injury to their peripheral receptors.