Expression of α4 and β2 nicotinic acetylcholine receptor subunit mRNA and localization of α-bungarotoxin binding proteins in the rat vestibular periphery

In situ hybridization histochemistry was used to map the distribution of α2, α3, α4, and β2 nAChR subunit mRNAs throughout the peripheral vestibular system of the rat. The α4 and β2 nAChR subunit genes were co-expressed by populations of primary afferent neurons within Scarpa's ganglion, while there was no expression of the α2, α3, α4, or β2 nAChR subunit genes by type I or type II vestibular hair cells. α-bungarotoxin binding to nAChRs in the vestibular end-organs was primarily limited to the afferent chalices surrounding type I hair cells and the basal aspect of type II hair cells. These data suggest that nAChRs composed of α4 and β2 subunits are localized on afferent chalices innervating the type I vestibular hair cells and that the direct cholinergic efferent innervation of the type II vestibular hair cells utilizes nAChR composed of other subunits.     

Choline acetyltransferase immunoreactivity in the human vestibular end-organs

Acetylcholine (ACh) is believed to play a major role in the efferent vestibular system in several animal models, however no information regarding the role of ACh in the human efferent vestibular system has been published. Post-embedding immunohistochemistry in a hydrophilic resin was used to investigate the choline acetyltransferase immunoreactivity (ChATi) and acetylcholinesterase (ACHE) histochemistry in human vestibular end-organs. ChATi and AChE activity was found in numerous bouton-type terminals at the basal area of the vestibular hair cells. These terminals were found to contact type II vestibular hair cells and the afferent chalices surrounding type I hair cells. This study provides the first evidence that the human efferent vestibular axons and terminals are cholinergic.     

Demonstration and localization of synapsin I in vestibular receptors in the cat: immunocytochemical study

The presence and localization of synapsin I, a neuron-specific phosphoprotein, was investigated in the cat vestibular epithelium, using a rabbit antisynapsin I anti-serum. The staining was performed by immunofluorescence or by a peroxidase-antiperoxidase (PAP) technique. A strong immunoreactivity was observed with both methods. This immunoreactivity appeared as spherical patches distributed in the lower part of the epithelium. This distribution pattern is very similar to that of the efferent synaptic endings which form axodendritic synapses with the afferent nerve chalice of type I hair cells, or axosomatic synapses with type II hair cells. Some of the nerve chalices were also labelled; in this case, the immunoreactivity was more evident with PAP staining. These results thus suggest the presence of large amounts of synapsin I in the vestibular efferent nerve endings. These endings are known to be filled with numerous synaptic vesicles. This localization of synapsin I is well correlated with previous work that report a close association between synapsin I and small synaptic vesicles. The presence of synapsin I in sensory endings such as the afferent nerve chalices was unexpected and is under investigation.     

Postnatal Expression of an Apamin-Sensitive K(Ca) Current in Vestibular Calyx Terminals

Afferent innervation patterns in the vestibular periphery are complex, and vestibular afferents show a large variation in their regularity of firing. Calyx fibers terminate on type I vestibular hair cells and have firing characteristics distinct from the bouton fibers that innervate type II hair cells. Whole-cell patch clamp was used to investigate ionic currents that could influence firing patterns in calyx terminals. Underlying K(Ca) conductances have been described in vestibular ganglion cells, but their presence in afferent terminals has not been investigated previously. Apamin, a selective blocker of SK-type calcium-activated K(+) channels, was tested on calyx afferent terminals isolated from gerbil semicircular canals during postnatal days 1-50. Lowering extracellular calcium or application of apamin (20-500?nM) reduced slowly activating outward currents in voltage clamp. Apamin also reduced the action potential afterhyperpolarization (AHP) in whole-cell current clamp, but only after the first two postnatal weeks. K(+) channel expression increased during the first postnatal month, and SK channels were found to contribute to the AHP, which may in turn influence discharge regularity in calyx vestibular afferents.     

Purinergic signaling in cochleovestibular hair cells and afferent neurons

Purinergic signaling in the mammalian cochleovestibular hair cells and afferent neurons is reviewed. The scope includes P2 and P1 receptors in the inner hair cells (IHCs) of the cochlea, the type I spiral ganglion neurons (SGNs) that convey auditory signals from IHCs, the vestibular hair cells (VHCs) in the vestibular end organs (macula in the otolith organs and crista in the semicircular canals), and the vestibular ganglion neurons (VGNs) that transmit postural and rotatory information from VHCs. Various subtypes of P2X ionotropic receptors are expressed in IHCs as well as P2Y metabotropic receptors that mobilize intracellular calcium. Their functional roles still remain speculative, but adenosine 5′-triphosphate (ATP) could regulate the spontaneous activity of the hair cells during development and the receptor potentials of mature hair cells during sound stimulation. In SGNs, P2Y metabotropic receptors activate a nonspecific cation conductance that is permeable to large cations as NMDG⁺ and TEA⁺. Remarkably, this depolarizing nonspecific conductance in SGNs can also be activated by other metabotropic processes evoked by acetylcholine and tachykinin. The molecular nature and the role of this depolarizing channel are unknown, but its electrophysiological properties suggest that it could lie within the transient receptor potential channel family and could regulate the firing properties of the afferent neurons. Studies on the vestibular partition (VHC and VGN) are sparse but have also shown the expression of P2X and P2Y receptors. There is still little evidence of functional P1 (adenosine) receptors in the afferent system of the inner ear.     

Striola magica. A functional explanation of otolith geometry

Otolith end organs of vertebrates sense linear accelerations of the head and gravitation. The hair cells on their epithelia are responsible for transduction. In mammals, the striola, parallel to the line where hair cells reverse their polarization, is a narrow region centered on a curve with curvature and torsion. It has been shown that the striolar region is functionally different from the rest, being involved in a phasic vestibular pathway. We propose a mathematical and computational model that explains the necessity of this amazing geometry for the striola to be able to carry out its function. Our hypothesis, related to the biophysics of the hair cells and to the physiology of their afferent neurons, is that striolar afferents collect information from several type I hair cells to detect the jerk in a large domain of acceleration directions. This predicts a mean number of two calyces for afferent neurons, as measured in rodents. The domain of acceleration directions sensed by our striolar model is compatible with the experimental results obtained on monkeys considering all afferents. Therefore, the main result of our study is that phasic and tonic vestibular afferents cover the same geometrical fields, but at different dynamical and frequency domains.     

Cellular distribution of myosin-V in the guinea pig cochlea

The importance of unconventional myosins to hearing has recently been revealed by the identification of myosins-VI and -VII as the defective genes in mouse mutations and in a human syndrome which lead to profound hearing loss. Another class of novel myosins (V) has been implicated in the trafficking of intracellular vesicles in neurons and other secretory cells. We used affinity-purified antibodies to determine the localization of myosin-V in the guinea pig inner ear. In the sensory epithelium of the cochlea, myosin-V epitopes were recognized in neuronal and supporting cells. Neuronal labelling was most intense in the afferent innervation of inner and outer hair cells. Supporting cells labelled were cells of Hensen and Deiters, and inner border, inner phalangeal, inner sulcus and interdental cells. In the vascular tissue of the cochlea, we observed staining of intermediate cells of the stria vascularis and of border cells between the stria and the spiral prominence. Staining of afferent chalice nerve endings was observed on type I vestibular hair cells. The results suggest that, like myosins VI and VII, myosin-V is localized in positions that may be critical to auditory function.     

Spatiotemporal definition of neurite outgrowth, refinement and retraction in the developing mouse cochlea

The adult mammalian cochlea receives dual afferent innervation: the inner sensory hair cells are innervated exclusively by type I spiral ganglion neurons (SGN), whereas the sensory outer hair cells are innervated by type II SGN. We have characterized the spatiotemporal reorganization of the dual afferent innervation pattern as it is established in the developing mouse cochlea. This reorganization occurs during the first postnatal week just before the onset of hearing. Our data reveal three distinct phases in the development of the afferent innervation of the organ of Corti: (1) neurite growth and extension of both classes of afferents to all hair cells (E18-P0); (2) neurite refinement, with formation of the outer spiral bundles innervating outer hair cells (P0-P3); (3) neurite retraction and synaptic pruning to eliminate type I SGN innervation of outer hair cells, while retaining their innervation of inner hair cells (P3-P6). The characterization of this developmental innervation pattern was made possible by the finding that tetramethylrhodamine-conjugated dextran (TMRD) specifically labeled type I SGN. Peripherin and choline-acetyltransferase immunofluorescence confirmed the type II and efferent innervation patterns, respectively, and verified the specificity of the type I SGN neurites labeled by TMRD. These findings define the precise spatiotemporal neurite reorganization of the two afferent nerve fiber populations in the cochlea, which is crucial for auditory neurotransmission. This reorganization also establishes the cochlea as a model system for studying CNS synapse development, plasticity and elimination.     

Hair cells in the inner ear of the pirouette and shaker 2 mutant mice

The shaker 2 (sh2) and pirouette (pi) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions “cytocauds.” The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.     

Glutamate Transporters EAAT4 and EAAT5 Are Expressed in Vestibular Hair Cells and Calyx Endings

Glutamate is the neurotransmitter released from hair cells. Its clearance from the synaptic cleft can shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear and in other sensory organs where there is a continually high rate of neurotransmitter release. In the case of most cochlear and type II vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glutamate transporter. A similar mechanism cannot work in vestibular type I hair cells as the presence of calyx endings separates supporting cells from hair-cell synapses. Because of this arrangement, it has been conjectured that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrate that a glutamate-activated anion current, attributable to a high-affinity glutamate transporter and blocked by DL-TBOA, is expressed in type I, but not in type II hair cells. Molecular investigations reveal that EAAT4 and EAAT5, two glutamate transporters that could underlie the anion current, are expressed in both type I and type II hair cells and in calyx endings. EAAT4 has been thought to be expressed almost exclusively in the cerebellum and EAAT5 in the retina. Our results show that these two transporters have a wider distribution in mice. This is the first demonstration of the presence of transporters in hair cells and provides one of the few examples of EAATs in presynaptic elements.     

GABA-Mediated Synaptic Interaction Between the Visual and Vestibular Pathways of Hermissenda

Abstract: The synaptic convergence of the eyes and the vestibular hair cells in the nudibranch mollusc Hermissenda has been shown previously to mediate the learning of simple visual-vestibular associations. The neurotransmitter mediating this interaction between the visual and vestibular organs was characterized. HPLC chromatography, confirmed by mass spectroscopic analysis, demonstrated endogenous GABA in the statocysts, in a concentration approximately 150 times greater than in the whole CMS. Additional confirmation was provided by immunocytochemical localization of GABA in hair cell axons and branches that converge with photoreceptor terminal branches. Depolarization of the hair cells in the caudal region of the statocyst in response to positive current injection or vibratory stimulation caused a hyperpolarization and a cessation of the type B photoreceptor impulse activity. The inhibition of the B cell was unaffected by addition to the artificial sea water bath of the adrenergic antagonist yohimbine (250 μM), the cholinergic antagonist atropine (250 μM), and the serotonergic antagonist imipramine (50 μM). In contrast, the GABA_A antagonist bicuculline (250 μM) significantly reduced the inhibitory interaction. Moreover, the GABA reuptake inhibitor guvisine (250 μM)M) increased the hyperpolarization. Pressure microapplication of GABA (12.5 or 25 μM) onto the terminal branches of the B cell resulted in a concentration-dependent hyperpolarization and cessation of spikes in the B cell. Depolarization of the caudal hair cell, or direct GABA application, decreased input resistance across the B cell soma membrane. Moreover, removal of chloride from the extracellular solution reduced inhibition of the B cell induced by GABA application or hair cell stimulation. Furthermore, application of the GABA_B agonist baclofen hyperpolarized the type B cell and reduced or eliminated spontaneous impulse activity at the resting membrane potential. The reversal potentials for inhibition induced in all three procedures ranged from ?70 to ?80 mV and were consistent with mixed Cl^- and K⁺ conductances. These results implicate GABA as the endogenous neurotransmitter mediating visual-vestibular interactions in this animal, and suggest a possible role of GABA in visual-vestibular associative learning.     

Glutamate excitatory effects on ampullar receptors of the frog

Summary The action of glutamate on frog ampullar receptors was investigated to assess the potential role of this excitatory amino acid as an afferent transmitter in the hair cell system. Intracellular recordings from single afferent units in the isolated labyrinth revealed that glutamate and the glutamate receptor agonists, N-methyl-D-aspartic acid, quisqualic acid and kainic acid increase dose-dependently the frequency of the resting afferent discharge of EPSPs and spikes and produce long lasting depolarizations. After blocking synaptic transmission by using 5 mM Co²⁺, the same compounds elicited only depolarizations of amplitude comparable to those observed in normal saline. Quisqualic acid and kainic acid were much more potent than N-methyl-D-aspartic acid in increasing the frequency of afferent discharge and in causing axonal depolarizations. The depolarization caused by glutamate was reduced dose-dependently by the competitive non-NMDA receptor antagonist 6-cyano-7-nitroquinaxoline-2,3 dione and disappeared almost completely in Na⁺-free Ringer solution. These results are consistent with the hypothesis that glutamate is the afferent transmitter in vestibular organs and indicate that receptors mainly of the non-NMDA type are present not only at postsynaptic level but also in hair cells. Presynaptic glutamate receptors may function as autoreceptors controlling by a positive feed-back mechanism the release of the afferent transmitter.     

Modalities of GABA and Glutamate Neurotransmission in the Vertebrate Inner Ear Vestibule

GABA and glutamate have been postulated as afferent neurotransmitters at the sensory periphery inner ear vestibule in vertebrates. GABA has fulfilled the main criteria to act as afferent neurotransmitter but may also be a putative efferent neurotransmitter, mainly due to cellular localization of its synthesizing enzyme glutamate decarboxylase derived from biochemical, immunocytochemical, in situ hybridization and molecular biological techniques, whereas glutamate afferent neurotransmission role is supported mainly by pharmacological evidences. GABA and Glu could also act as afferent co-neurotransmitters based upon immunocytochemical techniques. This multiplicity was not considered earlier and postulates a peripheral modulation of afferent information being sent to higher vestibular centers. In order to make a definitive cellular assignation to these putative neurotransmitters it is necessary to have evidences derived from immunocytochemical and pharmacological experiments in which both substances are tested simultaneously. Special issue article in honor of Dr. Ricardo Tapia.     

Structure and Function of the Hair Cell Ribbon Synapse

Faithful information transfer at the hair cell afferent synapse requires synaptic transmission to be both reliable and temporally precise. The release of neurotransmitter must exhibit both rapid on and off kinetics to accurately follow acoustic stimuli with a periodicity of 1 ms or less. To ensure such remarkable temporal fidelity, the cochlear hair cell afferent synapse undoubtedly relies on unique cellular and molecular specializations. While the electron microscopy hallmark of the hair cell afferent synapse — the electron-dense synaptic ribbon or synaptic body — has been recognized for decades, dissection of the synapse’s molecular make-up has only just begun. Recent cell physiology studies have added important insights into the synaptic mechanisms underlying fidelity and reliability of sound coding. The presence of the synaptic ribbon links afferent synapses of cochlear and vestibular hair cells to photoreceptors and bipolar neurons of the retina. This review focuses on major advances in understanding the hair cell afferent synapse molecular anatomy and function that have been achieved during the past years.     

Mice with altered KCNQ4 K+ channels implicate sensory outer hair cells in human progressive deafness

KCNQ4 is an M-type K+ channel expressed in sensory hair cells of the inner ear and in the central auditory pathway. KCNQ4 mutations underlie human DFNA2 dominant progressive hearing loss. We now generated mice in which the KCNQ4 gene was disrupted or carried a dominant negative DFNA2 mutation. Although KCNQ4 is strongly expressed in vestibular hair cells, vestibular function appeared normal. Auditory function was only slightly impaired initially. It then declined over several weeks in Kcnq4-/- mice and over several months in mice carrying the dominant negative allele. This progressive hearing loss was paralleled by a selective degeneration of outer hair cells (OHCs). KCNQ4 disruption abolished the I(K,n) current of OHCs. The ensuing depolarization of OHCs impaired sound amplification. Inner hair cells and their afferent synapses remained mostly intact. These cells were only slightly depolarized and showed near-normal presynaptic function. We conclude that the hearing loss in DFNA2 is predominantly caused by a slow degeneration of OHCs resulting from chronic depolarization.     

Cellular distribution of parvalbumin immunoreactivity in the peripheral vestibular system of three rodents

The cellular distribution of parvalbumin immunoreactivity in the vestibular peripheral system of mouse, rat, and guinea pig was investigated by light and electron microscopy. Parvalbumin was found in all neurons of the vestibular ganglia of these species but in the sensory epithelia immunoreactivity was restricted to type I hair cells localized exclusively in the central areas. The very intense staining pattern was similar in the cristae ampullares and utricles of all three species but a faint immunoreaction was also detectable in sensory cells of peripheral areas of rat cristae. The parvalbumin-immunoreactive type I sensory cells are connected by nerve fibres of the calyx unit type which are known selectively to contain calretinin.     

Responses of afferent and efferent neurons to auditory inputs in the vestibular nerve of the frog

Summary In the frog, the spontaneous discharges of afferent fibres from the horizontal semicircular canal (HC) and of efferent vestibular units were recorded by means of glass micropipettes filled with 2 mol/l NaCl as well as during acoustic stimulation; pure tones 300–2,000 Hz and clicks 150/s, 80–100 dB re 10^–5 N/m² were used. The activity of 56% of the efferent fibres recorded was increased by such stimulations while the discharge of the others was not modified. In intact preparations the activity of 34.4% of the afferent fibres recorded was either increased or decreased by sound stimulation depending on the unit; the discharge of the others (65.6%) was not modified (Fig. 3). Section of both saccular nerves did not change the percentage of the units modulated by sound showing that the saccules have probably no effect on this modulation (Fig. 4). In preparations where the contralateral auditory papillae were eliminated, 21.1% of the afferent units were facilitated and no unit was inhibited (Fig. 5), while in preparations where the ipsilateral auditory organs were eliminated 21.1% of the afferent units were inhibited and no unit was facilitated (Fig. 6). Therefore, in intact preparations one can assume that decrease and increase of the HC afferent fibre discharges were due to stimulation of the contralateral and the ipsilateral auditory organs, respectively. Such a modulation of canal afferent discharges being mediated by efferent vestibular fibres, it can be postulated that the efferent vestibular system has a double influence upon the hair cells of the vestibular epithelium: one inhibitory and the other facilitatory. Such a double effect is discussed.Abbreviations EVS efferent vestibular system - HC horizontal semicircular canal     

Expression of myosin VIIA in the developing chick inner ear neurons

The auditory-vestibular ganglion (AVG) is formed by the division of otic placode-derived neuroblasts, which then differentiate into auditory and vestibular afferent neurons. The developmental mechanisms that regulate neuronal cell fate determination, axonal pathfinding and innervation of otic neurons are poorly understood. The present study characterized the expression of myosin VIIA, along with the neuronal markers, Islet1, NeuroD1 and TuJ1, in the developing avian ear, during Hamburger–Hamilton (HH) stages 16–40. At early stages, when neuroblasts are delaminating from the otic epithelium, myosin VIIA expression was not observed. Myosin VIIA was initially detected in a subset of neurons during the early phase of neuronal differentiation (HH stage 20). As the AVG segregates into the auditory and vestibular portions, myosin VIIA was restricted to a subset of vestibular neurons, but was not present in auditory neurons. Myosin VIIA expression in the vestibular ganglion was maintained through HH stage 33 and was downregulated by stage 36. Myosin VIIA was also observed in the migrating processes of vestibular afferents as they begin to innervate the otic epithelium HH stage 22/23. Notably, afferents targeting hair cells of the cristae were positive for myosin VIIA while afferents targeting the utricular and saccular maculae were negative (HH stage 26–28). Although previous studies have reported that myosin VIIA is restricted to sensory hair cells, our data shows that myosin VIIA is also expressed in neurons of the developing chick ear. Our study suggests a possible role for myosin VIIA in axonal migration/pathfinding and/or innervation of vestibular afferents. In addition, myosin VIIA could be used as an early marker for vestibular neurons during the development of the avian AVG.     

Evidence for an Na+-K+-Cl- cotransporter in mammalian type I vestibular hair cells

In amniotes,there are two types of hair cells, designated I and II, that differ intheir morphology, innervation pattern, and ionic membrane properties.Type I cells are unique among hair cells in that their basolateralsurfaces are almost completely enclosed by an afferent calyceal nerveterminal. Recently, several lines of evidence have ascribed a motilefunction to type I hair cells. To investigate this, elevated externalK⁺, which had been used previouslyto induce hair cell shortening, was used to induce shape changes indissociated mammalian type I vestibular hair cells. Morphologicallyidentified type I cells shortened and widened when the externalK⁺ concentration was raisedisotonically from 2 to 125 mM. The shortening did not require externalCa²⁺ but was abolished whenexternal Cl was replacedwith gluconate or sulfate and when externalNa⁺ was replaced withN-methyl-D-glucamine.Bumetanide (10-100 µM), a specific blocker of theNa⁺-K⁺-Cl cotransporter,significantly reduced K⁺-inducedshortening. Hyposmotic solution resulted in type I cell shape changessimilar to those seen with highK⁺, i.e., shortening and widening.Type I cells became more spherical in hyposmotic solution, presumablyas a result of a volume increase due to water influx. In hypertonicsolution, cells became narrower and increased in length. These resultssuggest that shape changes in type I hair cells induced by highK⁺ are due, at least in part, toion and solute entry via anNa⁺-K⁺-Cl cotransporter, whichresults in cell swelling. A scheme is proposed whereby the type I haircell depolarizes and K⁺ leaves thecell via voltage-dependent K⁺channels and accumulates in the synaptic space between the type I haircell and calyx. Excess K⁺ couldthen be removed from the intercellular space by uptake via thecotransporter.