Genetically eliminating Purkinje neuron GABAergic neurotransmission increases their response gain to vestibular motion.

Purkinje neurons in the caudal cerebellar vermis combine semicircular canal and otolith signals to segregate linear and gravitational acceleration, evidence for how the cerebellum creates internal models of body motion. However, it is not known which cerebellar circuit connections are necessary to perform this computation. We first showed that this computation is evolutionarily conserved and represented across multiple lobules of the rodent vermis. Then we tested whether Purkinje neuron GABAergic output is required for accurately differentiating linear and gravitational movements through a conditional genetic silencing approach. By using extracellular recordings from lobules VI through X in awake mice, we show that silencing Purkinje neuron output significantly alters their baseline simple spike variability. Moreover, the cerebellum of genetically manipulated mice continues to distinguish linear from gravitational acceleration, suggesting that the underlying computations remain intact. However, response gain is significantly increased in the mutant mice over littermate controls. Altogether, these data argue that Purkinje neuron feedback regulates gain control within the cerebellar circuit.

Zika virus can directly infect and damage the auditory and vestibular components of the embryonic chicken inner ear.

BACKGROUND: Sensorineural hearing loss is an understudied consequence of congenital Zika syndrome, and balance disorders are essentially unreported to date. Also lacking is information about the susceptibility and the pathogenesis of the developing inner ear following Zika virus (ZIKV) exposure. To address this, ZIKV was delivered directly into the otic cup/otocyst of chicken embryos and infection of inner ear tissues was evaluated using immunohistochemistry. RESULTS: After injections on embryonic days 2 to 5, ZIKV infection was observed in 90% of the samples harvested 2 to 8 days later; however, the degree of infection was highly variable across individuals. ZIKV was detected in all regions of the inner ear, associated ganglia, and in the surrounding periotic mesenchyme. Detection of virus peaked earlier in the ganglion and vestibular compartments, and later in the cochlea. ZIKV infection increased cell death robustly in the auditory ganglion, and modestly in the auditory sensory organ. Macrophage accumulation was found to overlap with dense viral infection in some tissues. Additionally, dysmorphogenesis of the semicircular canals and ganglion was observed for a subset of injection conditions. CONCLUSIONS: This article presents evidence of direct ZIKV infection of developing inner ear epithelium and shows previously unknown inner ear dysmorphogenesis phenotypes.

Distinct functions for netrin 1 in chicken and murine semicircular canal morphogenesis.

The vestibular system of the inner ear detects head position using three orthogonally oriented semicircular canals; even slight changes in their shape and orientation can cause debilitating behavioral defects. During development, the canals are sculpted from pouches that protrude from the otic vesicle, the embryonic anlage of the inner ear. In the center of each pouch, a fusion plate forms where cells lose their epithelial morphology and the basement membrane breaks down. Cells in the fusing epithelia intercalate and are removed, creating a canal. In mice, fusion depends on the secreted protein netrin 1 (Ntn1), which is necessary for basement membrane breakdown, although the underlying molecular mechanism is unknown. Using gain-of-function approaches, we found that overexpression of Ntn1 in the chick otic vesicle prevented canal fusion by inhibiting apoptosis. In contrast, ectopic expression of the same chicken Ntn1 in the mouse otic vesicle, where apoptosis is less prominent, resulted in canal truncation. These findings highlight the importance of apoptosis for tissue morphogenesis and suggest that Ntn1 may play divergent cellular roles despite its conserved expression during canal morphogenesis in chicken and mouse.

BMP regulates regional gene expression in the dorsal otocyst through canonical and non-canonical intracellular pathways.

The inner ear consists of two otocyst-derived, structurally and functionally distinct components: the dorsal vestibular and ventral auditory compartments. BMP signaling is required to form the vestibular compartment, but how it complements other required signaling molecules and acts intracellularly is unknown. Using spatially and temporally controlled delivery of signaling pathway regulators to developing chick otocysts, we show that BMP signaling regulates the expression of Dlx5 and Hmx3, both of which encode transcription factors essential for vestibular formation. However, although BMP regulates Dlx5 through the canonical SMAD pathway, surprisingly, it regulates Hmx3 through a non-canonical pathway involving both an increase in cAMP-dependent protein kinase A activity and the GLI3R to GLI3A ratio. Thus, both canonical and non-canonical BMP signaling establish the precise spatiotemporal expression of Dlx5 and Hmx3 during dorsal vestibular development. The identification of the non-canonical pathway suggests an intersection point between BMP and SHH signaling, which is required for ventral auditory development.

Ildr1b is essential for semicircular canal development, migration of the posterior lateral line primordium and hearing ability in zebrafish: implications for a role in the recessive hearing impairment DFNB42.

Immunoglobulin-like domain containing receptor 1 (ILDR1) is a poorly characterized gene that was first identified in lymphoma cells. Recently, ILDR1 has been found to be responsible for autosomal recessive hearing impairment DFNB42. Patients with ILDR1 mutations cause bilateral non-progressive moderate-to-profound sensorineural hearing impairment. However, the etiology and mechanism of ILDR1-related hearing loss remains to be elucidated. In order to uncover the pathology of DFNB42 deafness, we used the morpholino injection technique to establish an ildr1b-morphant zebrafish model. Ildr1b-morphant zebrafish displayed defective hearing and imbalanced swimming, and developmental delays were seen in the semicircular canals of the inner ear. The gene expression profile and real-time PCR revealed down-regulation of atp1b2b (encoding Na(+)/K(+) transporting, beta 2b polypeptide) in ildr1b-morphant zebrafish. We found that injection of atp1b2b mRNA into ildr1b-knockdown zebrafish could rescue the phenotype of developmental delay of the semicircular canals. Moreover, ildr1b-morphant zebrafish had reduced numbers of lateral line neuromasts due to the disruption of lateral line primordium migration. In situ hybridization showed the involvement of attenuated FGF signaling and the chemokine receptor 4b (cxcr4b) and chemokine receptor 7b (cxcr7b) in posterior lateral line primordium of ildr1b-morphant zebrafish. We concluded that Ildr1b is crucial for the development of the inner ear and the lateral line system. This study provides the first evidence for the mechanism of Ildr1b on hearing in vivo and sheds light on the pathology of DFNB42.

Expression of zebrafish anterior gradient 2 in the semicircular canals and supporting cells of otic vesicle sensory patches is regulated by Sox10.

AGR2 is a member of the protein disulfide isomerase (PDI) family, which is implicated in cancer cell growth and metastasis, asthma, and inflammatory bowel disease. Despite the contributions of this protein to several biological processes, the regulatory mechanisms controlling expression of the AGR2 gene in different organs remain unclear. Zebrafish anterior gradient 2 (agr2) is expressed in several organs, including the otic vesicles that contain mucus-secreting cells. To elucidate the regulatory mechanisms controlling agr2 expression in otic vesicles, we generated a Tg(-6.0 k agr2:EGFP) transgenic fish line that expressed EGFP in a pattern recapitulating that of agr2. Double immunofluorescence studies were used to demonstrate that Agr2 and GFP colocalize in the semicircular canals and supporting cells of all sensory patches in the otic vesicles of Tg(-6.0 k agr2:EGFP) embryos. Transient/stable transgenic analyses coupled with 5'-end deletion revealed that a 100 bp sequence within the -2.6 to -2.5 kbp region upstream of agr2 directs EGFP expression specifically in the otic vesicles. Two HMG-binding motifs were detected in this region. Mutation of these motifs prevented EGFP expression. Furthermore, EGFP expression in the otic vesicles was prevented by knockdown of the sox10 gene. This corresponded with decreased agr2 expression in the otic vesicles of sox10 morphants during different developmental stages. Electrophoretic mobility shift assays were used to show that Sox10 binds to HMG-binding motifs located within the -2.6 to -2.5 kbp region upstream of agr2. These results demonstrate that agr2 expression in the otic vesicles of zebrafish embryos is regulated by Sox10.

The cytokine macrophage migration inhibitory factor (MIF) acts as a neurotrophin in the developing inner ear of the zebrafish, Danio rerio.

Macrophage migration inhibitory factor (MIF) plays versatile roles in the immune system. MIF is also widely expressed during embryonic development, particularly in the nervous system, although its roles in neural development are only beginning to be understood. Evidence from frogs, mice and zebrafish suggests that MIF has a major role as a neurotrophin in the early development of sensory systems, including the auditory system. Here we show that the zebrafish mif pathway is required for both sensory hair cell (HC) and sensory neuronal cell survival in the ear, for HC differentiation, semicircular canal formation, statoacoustic ganglion (SAG) development, and lateral line HC differentiation. This is consistent with our findings that MIF is expressed in the developing mammalian and avian auditory systems and promotes mouse and chick SAG neurite outgrowth and neuronal survival, demonstrating key instructional roles for MIF in vertebrate otic development.

Expression and localization of ryanodine receptors in the frog semicircular canal.

Several experiments suggest an important role for store-released Ca²âº in hair cell organs: drugs targeting IP3 and ryanodine (RyRs) receptors affect release from hair cells, and stores are thought to be involved in vesicle recycling at ribbon synapses. In this work we investigated the semicircular canal distribution of RyRs by immunofluorescence, using slice preparations of the sensory epithelium (to distinguish cell types) and flat mounts of the simpler nonsensory regions. RyRs were present in hair cells, mostly in supranuclear spots, but not in supporting cells; as regards nonsensory regions, they were also localized in dark cells and cells from the ductus. No labeling was found in nerve terminals, although nerve branches could be observed in proximity to hair cell RyR spots. The differential expression of RyR isoforms was studied by RT-PCR and immunoblotting, showing the presence of RyRα in both ampulla and canal arm and RyRß in the ampulla only.

The mammalian efferent vestibular system utilizes cholinergic mechanisms to excite primary vestibular afferents.

Electrical stimulation of the mammalian efferent vestibular system (EVS) predominantly excites primary vestibular afferents along two distinct time scales. Although roles for acetylcholine (ACh) have been demonstrated in other vertebrates, synaptic mechanisms underlying mammalian EVS actions are not well-characterized. To determine if activation of ACh receptors account for efferent-mediated afferent excitation in mammals, we recorded afferent activity from the superior vestibular nerve of anesthetized C57BL/6 mice while stimulating EVS neurons in the brainstem, before and after administration of cholinergic antagonists. Using a normalized coefficient of variation (CV*), we broadly classified vestibular afferents as regularly- (CV* < 0.1) or irregularly-discharging (CV* > 0.1) and characterized their responses to midline or ipsilateral EVS stimulation. Afferent responses to efferent stimulation were predominantly excitatory, grew in amplitude with increasing CV*, and consisted of fast and slow components that could be identified by differences in rise time and post-stimulus duration. Both efferent-mediated excitatory components were larger in irregular afferents with ipsilateral EVS stimulation. Our pharmacological data show, for the first time in mammals, that muscarinic AChR antagonists block efferent-mediated slow excitation whereas the nicotinic AChR antagonist DHßE selectively blocks efferent-mediated fast excitation, while leaving the efferent-mediated slow component intact. These data confirm that mammalian EVS actions are predominantly cholinergic.

Transcriptomic characterization of dying hair cells in the avian cochlea.

Sensory hair cells are prone to apoptosis caused by various drugs including aminoglycoside antibiotics. In mammals, this vulnerability results in permanent hearing loss because lost hair cells are not regenerated. Conversely, hair cells regenerate in birds, making the avian inner ear an exquisite model for studying ototoxicity and regeneration. Here, we use single-cell RNA sequencing and trajectory analysis on control and dying hair cells after aminoglycoside treatment. Interestingly, the two major subtypes of avian cochlear hair cells, tall and short hair cells, respond differently. Dying short hair cells show a noticeable transient upregulation of many more genes than tall hair cells. The most prominent gene group identified is associated with potassium ion conductances, suggesting distinct physiological differences. Moreover, the dynamic characterization of >15,000 genes expressed in tall and short avian hair cells during their apoptotic demise comprises a resource for further investigations toward mammalian hair cell protection and hair cell regeneration.

Development of K(+) and Na(+) conductances in rodent postnatal semicircular canal type I hair cells.

The rodent vestibular system is immature at birth. During the first postnatal week, vestibular type I and type II hair cells start to acquire their characteristic morphology and afferent innervation. We have studied postnatal changes in the membrane properties of type I hair cells acutely isolated from the semicircular canals (SCC) of gerbils and rats using whole cell patch clamp and report for the first time developmental changes in ionic conductances in these cells. At postnatal day (P) 5 immature hair cells expressed a delayed rectifier K(+) conductance (G(DR)) which activated at potentials above approximately -50 mV in both species. Hair cells also expressed a transient Na(+) conductance (G(Na)) with a mean half-inactivation of approximately -90 mV. At P6 in rat and P7 in gerbil, a low-voltage activated K(+) conductance (G(K,L)) was first observed and conferred a low-input resistance, typical of adult type I hair cells, on SCC type I hair cells. G(K,L) expression in hair cells increased markedly during the second postnatal week and was present in all rat type I hair cells by P14. In gerbil hair cells, G(K,L) appeared later and was present in all type I hair cells by P19. During the third postnatal week, G(Na) expression declined and was absent by the fourth postnatal week in rat and the sixth postnatal week in gerbils. Understanding the ionic changes associated with hair cell maturation could help elucidate development and regeneration mechanisms in the inner ear.

Dopaminergic modulation of afferent synaptic transmission in the semicircular canals of frogs.

Using multiunit recording of action potentials from the whole nerve with the aid of external perfusion, we investigated the effects of dopamine (DOP) agonists that are involved in modulatory actions on synaptic transmission in the isolated labyrinth preparations of frogs. The external application of DOP (0.1-1 mM), the D(1) agonist chloro-APB hydrobromide (CAPB, 50-100 microM) and the D2 agonist quinerolane (QUI, 50-100 microM) induced a dose-dependent and reversible decline in the resting discharge frequency. In this concentration range, the potency of applied CAPB considerably exceeded that of QUI. AMPA, NMDA and ACPD responses were inhibited by the D1 and D2 agonists, implicating both subtypes of DOP receptors in the modulation of both ionotropic and metabotropic glutamate receptors. The inhibitory action of the DOP agonists on L-glutamate responses persisted in a high Mg2+ solution in conditions of selective activation of the postsynaptic membrane. The results obtained suggest that DOP may interact with both D1 and D2 receptor subtypes, most likely located postsynaptically on the afferent nerve fibers. This dopaminergic control mechanism may result in the reduction of the activated firing rate, thus preventing over-excitation and excitotoxic injury of the afferent dendrites after the external application of L-glutamate and excessive receptor stimulation.

Spontaneous mutations of the Zpld1 gene in mice cause semicircular canal dysfunction but do not impair gravity receptor or hearing functions.

The cupula is a gelatinous membrane overlying the crista ampullaris of the semicircular canal, important for sensing rotation of the head and critical for normal balance. Recently the zona pellucida like domain containing 1 protein (ZPLD1, also known as cupulin) was identified in the cupula of fish. Here, we describe two new spontaneous mutations in the mouse Zpld1 gene, which were discovered by the circling behavior of mutant mice, an indicator of balance dysfunction. The Zpld1 mutant mice exhibited normal hearing function as assessed by auditory brainstem response (ABR) measurements, and their otolithic organs appeared normal. In the inner ear, Zpld1 mRNA expression was detected only in the hair cells and supporting cells of the crista ampullaris. Normal vestibular sensory evoked potential (VsEP) responses and abnormal vestibulo-ocular reflex (VOR) responses demonstrated that the vestibular dysfunction of the Zpld1 mutant mice is caused by loss of sensory input for rotary head movements (detected by cristae ampullaris) and not by loss of input for linear head translations (detected by maculae of the utricle and saccule). Taken together, these results are consistent with ZPLD1 being an important functional component of the cupula, but not tectorial or otoconial membranes.

Cog4 is required for protrusion and extension of the epithelium in the developing semicircular canals.

The semicircular canals in the inner ear sense angular acceleration. In zebrafish, the semicircular canals develop from epithelial projections that grow toward each other and fuse to form pillars. The growth of the epithelial projections is driven by the production and secretion of extracellular matrix components by the epithelium. The conserved oligomeric Golgi 4 protein, Cog4, functions in retrograde vesicle transport within the Golgi and mutations can lead to sensory neural hearing loss. In zebrafish cog4 mutants, the inner ear is smaller and the number of hair cells is reduced. Here, we show that formation of the pillars is delayed and that secretion of extracellular matrix components (ECM) is impaired in cog4-/- mutants. These results show that Cog4 is required for secretion of ECM molecules essential to drive the growth of the epithelial projections and thus regulates morphogenesis of the semicircular canals.

Expression of marker genes during otolith development in medaka.

Little is known about the genes and processes involved in the development of otoliths. In this study, we isolated the biomineralization-related genes otolin and chondromodulin-1 (chm1) from medaka, and examined their spatiotemporal expression pattern as well as that of two other genes also related to biomineralization, i.e., sparc/osteonectin and type II collagen (col2a), during otic development in medaka. Our results demonstrated that all the tested genes were expressed in the otic vesicle, and that chm1 was exclusively expressed in the semicircular canal of the otic vesicle.

Ultrastructural co-localization of cochlin and type II collagen in the rat semicircular canal.

Cochlin and type II collagen are major constituents of the inner ear extracellular matrix. To investigate the morphological relation of cochlin and type II collagen in the rat semicircular canal, immuno-electronmicroscopic analysis was performed using the post-embedding immunogold method. Immunolabeling for cochlin was detected in the fibrillar substance underlying the supporting epithelium of the sensory cells and beneath the epithelial cells facing the endolymph in the semicircular canals. Immunolabeling for type II collagen was observed in the same fibrillar substance in the subepithelial area. The co-localization of cochlin and type II collagen in the fibrillar substance in the subepithelial area indicate that cochlin may play a role in the structural homeostasis of the vestibule acting in concert with the fibrillar type II collagen bundles.

Bone Metabolic Markers in the Clinical Assessment of Patients with Superior Semicircular Canal Dehiscence.

BACKGROUND: Superior semicircular canal dehiscence (SSCD) is a bony defect in the osseous shell of the petrous temporal bone. The pathophysiological association between osteoporosis and SSCD remains poorly understood. We investigated the relationship between bone metabolic markers and symptoms in patients with SSCD. METHODS: We collected patient demographics and clinical parameters for adult patients diagnosed with SSCD on high-resolution computed tomography scans. We used point-biserial correlation analysis to investigate the relationship between bone metabolic markers and symptoms in patients with SSCD. We compared clinical symptoms before and after surgical repair of SSCD through a middle fossa craniotomy using McNemar's test for paired comparisons of binary measures. RESULTS: We included a total of 99 patients (64 females and 35 males; average age 52 years; 118 surgeries). The level of serum calcium correlated with the need for a second surgery (rpb = -0.35, P = 0.001). Postoperative calcium supplementation negatively correlated with improvement in dizziness (rpb = -0.36, P = 0.01). The level of 25-hydroxyvitamin D correlated with preoperative hyperacusis (rpb = -0.98, P = 0.02) and postoperative autophony (rpb = 0.96, P = 0.04). Postoperative vitamin D supplementation positively correlated with hearing decline (rpb = 0.04, P = 0.04) The level of thyroid stimulating hormone correlated with preoperative autophony, amplification, and tinnitus (rpb = -0.71, rpb = -0.75, rpb = -0.70, all P < 0.001). CONCLUSIONS: Bone metabolic markers could be important in the clinical assessment of SSCD patients and could be potential targets for symptom management.

The role of defensins in the excitability of the peripheral vestibular system in the frog: evidence for the presence of communication between the immune and nervous systems.

Defensins are one of the major groups of endogenous peptides that are considered to be important antibiotic-like effectors of host innate and adaptive antimicrobial immunity. The current study investigated the electrophysiological effects of externally applied human and rabbit defensins (HNP-1 and RNP-1, correspondingly) on afferent neurotransmission in the frog semicircular canals (SCC). Application of HNP-1 and RNP-1 induces a concentration-dependent decrease in resting activity. Threshold concentrations for both substances were of the order of 0.0001 nM. The firing evoked by L-glutamate (L-Glu) and its agonists alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) and (1S, 3R)-1-aminocyclopentane-trans-1,3-dicarboxilic acid (ACPD) could be inhibited by HNP-1, suggesting that defensins exert inhibitory control over both ionotropic and metabotropic glutamate receptors. HNP-1 considerably inhibited the L-glutamate/high Mg2+ -induced increase in frequency, thus, demonstrating its postsynaptic site of action. Acetylcholine (ACh) responses under HNP-1 did not differ from the frequency increase induced by ACh alone, and the ACh antagonist atropine left the response to HNP-1 intact. The specific opioid receptor antagonist naloxone (Nal) antagonized the inhibitory response evoked by HNP-1. The results obtained support the evidence for the recruitment of defensins in communication between the immune and nervous systems, and on the potential of sensory receptors to participate in the inflammatory response.

[Comparative analysis of the effect of endogenous antibiotic defensin NP-1 and aminoglycoside antibiotic gentamicin on synaptic transmission in receptors of the frog vestibular apparatus].

The effect of human and rabbit neutrophilic defensins NP-1 and amonoglycoside antibiotic gentamicin on the synaptic transmission in the afferent synapse of isolated vestibular apparatus of the frog has been comparatively studied. Both defensins proved active in the concentration range of 0.0001 to 1 nM and efficiently decreased the impulse frequency in the afferent nerve fibers in a concentration-dependent manner. No significant differences in the efficiency of rabbit and human defensin NP-1 have been revealed in these experiments. Gentamicin also had an inhibitory effect on the afferent discharge in the concentration range of 10-500 microM (0.5-25 mg/kg). The inhibitory effect of gentamicin on the impulse activity of the vestibular nerve was observed at therapeutic doses. The excitatory effect of the putative neurotransmitter L-glutamate was considerably inhibited by defensin NP-1. These findings suggest that the mechanism of defensin action involves a modification of the synaptic transmission the hair receptor and is mediated by L-glutamate.

Numerical modeling of the cupular displacement and motion of otoconia particles in a semicircular canal.

Balance is achieved and maintained by a balance system called a labyrinth that is composed of three semicircular canals and the otolith organs that sense linear gravity and acceleration. Within each semicircular canal, there is a gelatinous structure called the cupula, which is deformed under the influence of the surrounding endolymph. One of the balance disorders is benign paroxysmal positional vertigo, and one of the pathological conditions that have been identified as possible causes of this syndrome is canalithiasis-disturbance of the endolymph flow and cupular displacement caused by the free-moving otoconia particles within the lumen of the canal. Analysis of phenomena occurring within the semicircular canal can help to explain some balance-related disorders and the response of the vestibular system to external perturbations under various pathological conditions. Numerical simulations allow a study of the influence of a wide range of factors, without the need to perform experiments and clinical examinations. In case of canalithiasis, an accurate explanation and tracking of the motion of otoconia particles in vivo is obviously nearly impossible. In this study, a numerical model was developed to predict the motion of otoconia particles within the semicircular canal and the effect of the endolymph flow and particles on the deformation of the cupula.