Ion channels in mammalian vestibular afferents may set regularity of firing.

Rodent vestibular afferent neurons offer several advantages as a model system for investigating the significance and origins of regularity in neuronal firing interval. Their regularity has a bimodal distribution that defines regular and irregular afferent classes. Factors likely to be involved in setting firing regularity include the morphology and physiology of the afferents' contacts with hair cells, which may influence the averaging of synaptic noise and the afferents' intrinsic electrical properties. In vitro patch clamp studies on the cell bodies of primary vestibular afferents reveal a rich diversity of ion channels, with indications of at least two neuronal populations. Here we suggest that firing patterns of isolated vestibular ganglion somata reflect intrinsic ion channel properties, which in vivo combine with hair cell synaptic drive to produce regular and irregular firing.

Vestibular efferents contain peripherin.

Vestibular efferents have a common origin with the motoneurons of the facial nerve. In adults they share a number of common features, such as the same transmitter. Here we show using retrograde transport and immunohistochemistry, that the vestibular efferents, like facial motoneurons, contain peripherin. This supports the suggestion that peripherin-positive fibers at the apex of the cristae ampullaris are efferents.

The anatomy of the vestibular nuclei.

The vestibular portion of the eighth cranial nerve informs the brain about the linear and angular movements of the head in space and the position of the head with respect to gravity. The termination sites of these eighth nerve afferents define the territory of the vestibular nuclei in the brainstem. (There is also a subset of afferents that project directly to the cerebellum.) This chapter reviews the anatomical organization of the vestibular nuclei, and the anatomy of the pathways from the nuclei to various target areas in the brain. The cytoarchitectonics of the vestibular brainstem are discussed, since these features have been used to distinguish the individual nuclei. The neurochemical phenotype of vestibular neurons and pathways are also summarized because the chemical anatomy of the system contributes to its signal-processing capabilities. Similarly, the morphologic features of short-axon local circuit neurons and long-axon cells with extrinsic projections are described in detail, since these structural attributes of the neurons are critical to their functional potential. Finally, the composition and hodology of the afferent and efferent pathways of the vestibular nuclei are discussed. In sum, this chapter reviews the morphology, chemoanatomy, connectivity, and synaptology of the vestibular nuclei.

Expression of a voltage-dependent potassium channel protein (Kv3.1) in the embryonic development of the auditory system.

The present study traces the development of a voltage-dependent potassium channel protein (Kv3.1) in the avian homologue of the cochlear nucleus, in the cochleovestibular ganglion, and in the otic epithelium from early developmental stages until near hatching. Immunohistochemistry with antibodies to the carboxy terminus (recognizing the Kv3.1b splice variant) and to the amino terminus (recognizing either form of Kv3.1) was used on Hamburger-Hamilton-staged chicken embryos. There were three periods in the relative levels of immunostaining in these regions. Early (E2-6), when precursor cells proliferate, migrate, and form axons, there was staining when using either antibody. In the middle period (E6-11), marked by hair cell differentiation, dendritic growth, and early synapse formation, staining levels decreased. In the late period (E11-19), when auditory function begins, staining increased rapidly, especially for Kv3.1b. Early Kv3.1 expression occurs in neuronal and hair cell precursors before they differentiate or function. Later, in the otic epithelium, a high level of Kv3.1 in cilia may precede or coincide with the onset of hair cell function. In neurons, some features of its localization correlate with axon outgrowth and synapse formation, others with the onset of neural activity and function.

[Detection of GABAA alpha 2 mRNA in rat vestibular end organ with in-situ hybridization].

OBJECTIVE: To investigate the effect of the gamma-aminobutyric acid (GABA) on rat vestibular end organ. METHODS: In the rat inner ear paraffin slides, GABAA receptor alpha 2 subunit mRNA in vestibular end organ has been detected with in-situ hybridization. Digoxigenin-GABAA alpha 2 cDNA prob (549 base pair), Anti-Digoxigenin-AP (Fab fragments) and BM Purple AP Substrate (precipitating) have been used. RESULTS: GABAA receptor alpha 2 subunit mRNA has been found in all vestibular ganglion cells and nerve ending surrounding type I hair cells in rat's cristae ampullaris. GABAA alpha 2 has not been found in hair cells and supporting cells. As a positive control, GABAA alpha 2 mRNA has been found in Purkinje cells and granule cells in rat's cerebellum. GABAA alpha 2 has not been found in OMP negative control, non-prob negative control and non-anti-digoxingenin control in inner ear and cerebellum slides. CONCLUSIONS: GABAA alpha 2 receptor has been found in vestibular afferent nerve endings and vestibular ganglion cells. It strongly indicates that GABA is an afferent nerve transmitter in the vestibular end organ and plays an important role in the vestibular afferent nerve transmission.

Neurochemical characterization of the vestibular nerves in women with vulvar vestibulitis syndrome.

Women with vulvar vestibulitis syndrome (VVS) have a distinct burning pain provoked by almost any stimuli in the area around the vaginal introitus. In a previous study we observed an increased number of intraepithelial free nerve endings in women with VVS. The aim of the present study was to neurochemically characterize the superficial nerves in the vulvar vestibular mucosa of women with VVS. Immunohistochemical methods were used to detect neuropeptides normally found in various types of nerve fibers. Calcitonin gene-related peptide, which is known to exist in nociceptive afferent nerves, was the only neuropeptide detected in the superficial nerves of the vestibular mucosa. These findings confirm our previous theory that the free nerve endings within the epithelium are nociceptors.

Expression of BDNF and TrkB mRNAs in the crista neurosensory epithelium and vestibular ganglia following ototoxic damage.

Following ototoxic lesion with the aminoglycoside gentamicin, the vestibular neurosensory epithelia undergo degeneration and then limited spontaneous regeneration. The spatio-temporal expression of brain-derived neurotrophic factor (BDNF) and of its high affinity receptor (trkB) mRNA was investigated in the vestibular end organs and ganglia of chinchillas following gentamicin ototoxicity. In the vestibular ganglia of untreated chinchillas, the level of expression of BDNF mRNA is low. At 1 and 2 weeks after intraotic treatment with gentamicin, BDNF mRNA levels in the vestibular ganglia were elevated significantly compared to untreated chinchillas and chinchillas 4 weeks after treatment. At 4 weeks after gentamicin treatment, BDNF mRNA levels were at intact levels of expression. In the crista ampullaris, high levels of BDNF transcripts were found in the untreated chinchillas. At 1 and 2 weeks after treatment, when only supporting cells are present in the crista, BDNF mRNA was undetectable. Four weeks after aminoglycoside treatment BDNF mRNA was present in the epithelium but at lower levels than in the intact epithelium. In contrast to its ligand, high levels of trkB mRNA hybridization were present in the vestibular ganglia of untreated chinchillas and trkB mRNA levels did not change following gentamicin treatment. In the vestibular epithelia, trkB mRNA was not detected either in the intact epithelium or after gentamicin ototoxicity. These data suggest that BDNF may be involved in the maintenance of the vestibular ganglia and contribute to neurite outgrowth to new and repaired hair cells following ototoxic damage.

Differential subcellular immunolocalization of voltage-gated calcium channel alpha1 subunits in the chinchilla cristae ampullaris.

The immunohistochemical localization of alpha1A, alpha1B, alpha1C, alpha1D and alpha1E voltage-gated calcium channel subunits was investigated in the chinchilla cristae ampullaris and Scarpa's ganglia at the light and electron microscopy level with the use of specific antipeptide antibodies directed against these subunits. The stereocilia membrane of type I and type II hair cells was immunoreactive for alpha1B along its entire length. The basolateral membrane of both types of hair cells was alpha1B, alpha1C and alpha1D immunoreactive. Neurons in the Scarpa's ganglia and afferent nerve terminals in the cristae were immunoreactive for alpha1C and alpha1B. No specific immunoreactivity to alpha1A or alpha1E was seen in the sensory epithelia or ganglia. These findings are consistent with the presence of alpha1B (N-type channel), alpha1C and alpha1D (L-type channels) in the vestibular hair cells, and alpha1B (N-type channel) and alpha1C (L-type channel) in primary vestibular neurons.

[Immunocytochemical study of gamma-aminobutyric acid-ergic innervation in the end-organs of human vestibule].

OBJECTIVE: To investigate gamma-aminobutyric acid-ergic (GABAergic) innervation in the end-organs of human vestibule. METHODS: A modified pre-embedding immunostaining technique of immunoelectron microscopy were applied to accomplish this study with a polyclonal antibody to gamma-aminobutyric acid. RESULTS: GABA-immunoreactive products were confined to the nerve terminals, which were rich in synaptic vesicles and the non-myelinated fibers. The GABA-immunoreactive nerve fibers synapse with afferent calices surrounding the type I hair cells. CONCLUSION: This study shows that GABAergic fibers of human vestibular end-organs belong to the vestibular efferent system.

Course and targets of the calbindin D-28k subpopulation of primary vestibular afferents.

The subpopulation of primary vestibular afferents (PVA) displaying immunoreactivity for the calcium binding protein Calbindin D-28k (Calb) is constituted of particularly large bipolar neurons in the vestibular ganglion (VG) that innervate the central regions of the vestibular end organs exclusively via calyx endings on type I vestibular hair cells. These large-diameter PVA are characterized by irregular spontaneous discharge patterns and predominantly phasic firing properties with respect to natural vestibular stimulation. The present study describes the complete course and terminations of Calb+ PVA in the cerebellar cortex, the cerebellar (CN) and vestibular nuclei (VN) of the mouse. To eliminate the two sources of Calb+ fibers in the cerebellum, i.e., the Calb+ primary vestibular input and the axons of cerebellar Purkinje cells (PC), in their totality, a unilateral eighth nerve transection was performed in the PC-deficient mutant mice, Purkinje cell degeneration (pcd/pcd) and Lurcher (Lc/+). Neurectomy in these mutants results in a complete ipsilateral loss of Calb+ fibers in the cerebellar cortex, the CN and VN. The Calb+ primary vestibular input on the contralateral side terminates solely in the rostral half of the ventral uvula and in the nodulus of the cerebellar cortex. Calb+ fibers traverse all three subdivisions of the CN, but terminations were found only in the lateral and medial cerebellar nuclei. In the VN, Calb+ PVA terminations were restricted to the superior, the ventral part of the lateral, the lateral portion of the medial, and the inferior vestibular nuclei. Calb + terminals were also present in the small cell group Y and Cajal's interstitial nucleus of the vestibular nerve as well as in defined areas of the reticular formation. All Calb + PVA are strictly unilateral. The results show that the Calb+ subpopulation of VG neurons is the sole source of Calb+ fibers and terminals in the PC-deficient cerebellum and the VN. The central input of this distinct subgroup of PVA is distributed in narrow posterior vermal areas and parts of the CN and VN. The cerebellar mutants, Purkinje cell degeneration and Lurcher, provide excellent tools to selectively investigate the subgroup of Calb+ PVA in the mouse in its entirety.

Select types of supporting cell in the inner ear express aquaporin-4 water channel protein.

Aquaporins (AQPs) confer a high water permeability on cell membranes and play important parts in secretory and absorptive epithelia in kidney and other organs. Here we investigate whether AQPs are expressed in the sensory epithelia of the inner ear, where a precise volume regulation is crucial. By use of specific antibodies it was found that the inner ear contains AQP1 and 4 while being devoid of detectable levels of AQP2, 3 or 5. Immunofluorescence and postembedding immunogold labelling revealed a strictly non-epithelial distribution of AQP1, confirming previous data. In contrast, AQP4 protein and mRNA (visualized by in situ hybridization) were concentrated in select types of supporting cell, including Hensen's cells and inner sulcus cells. Immunogold particles signalling AQP4 were confined to the basolateral plasma membrane of Hensen's cells and to the basal plasma membrane of Claudius cells and inner sulcus cells. AQP4 was also found in supporting cells of the vestibular end organs, but was absent from transitional epithelial cells and dark cells. Strong labelling for AQP4 and AQP4-mRNA was associated with the central part of the cochlear and vestibular nerves. Hair cells were consistently unlabelled. Our findings indicate that AQP4 may facilitate osmotically driven water fluxes in the sensory epithelia of the inner ear and thus contribute to the volume and ion homeostasis at these sites.

Glutamate as a primary afferent neurotransmitter in the medial vestibular nucleus as detected by in vivo microdialysis.

An in vivo microdialysis study using alpha-chloralose-anesthetized cats was performed to elucidate whether glutamate is actually released from the vestibular nerve terminals in the medial vestibular nucleus (MVN) with electrical stimulation of the vestibular nerve. When repetitive stimuli composed of rectangular pulses (200 micros in duration, 0.5 mA, and 0.1-50 Hz) were applied to the vestibular nerve for 10 min, a significant frequency-dependent increase in the release of glutamate was observed in the MVN. However, the levels of other amino acids such as aspartate, glycine and GABA remained unaltered with the stimuli. These findings indicate that glutamate is the primary afferent neurotransmitter from the vestibular nerve to the MVN neurons.

AMPA receptors in cultured vestibular ganglion neurons: detection and activation.

The presence and the activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptors were investigated in mouse cultured vestibular ganglion neurons using immunocytochemistry and measurement of intracellular calcium concentration ([Ca2+]i) by spectrofluorimetry. Cultures of dissociated vestibular ganglia from 18 gestation day mouse embryos were grown in vitro for 3-4 days. Immunocytochemical labelling of AMPA receptor subunits GluR2/R3 and GluR4 was detected in neuron cell bodies and proximal neurites and more lightly in glial cells. There was no clear selective subcellular localization of the different subunits. For the GluR1 subunit a signal was observed only in some neurons and neurites and was weak. Vestibular ganglion neurons responded to fast application of 1 mM glutamate and 10 mM aspartate through unknown receptors by a transient increase in [Ca2+]i. The mean amplitude of this rapid increase was about nine times the resting level and recovery was complete within 30-45 s after the application. If separated by an interval of at least 10 min, consecutive applications produced similar calcium responses. AMPA (1 mM) application induced the same type of responses. Five minutes prior to the AMPA exposure, the application of a specific AMPA antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX, 1.5 mM), in the external medium inhibited the response to AMPA. Chelation of external calcium by EGTA (1.5 mM) abolished the responses to drug applications, indicating that an influx of external calcium is involved in the [Ca2+]i increase. These observations suggest that heteromeric AMPA receptors are expressed in vestibular ganglion neurons in culture and play a functional role in their glutamate-induced depolarization. Experiments are in progress using specific AMPA and NMDA antagonists to characterize the participation of the two types of ionotropic glutamate receptors in the glutamate/aspartate-induced intracellular calcium response.

Differential expression of alpha2-7, alpha9 and beta2-4 nicotinic acetylcholine receptor subunit mRNA in the vestibular end-organs and Scarpa's ganglia of the rat.

To further characterize the pattern of expression of the nicotinic acetylcholine receptor (nAChR) subunits in the peripheral vestibular system, we conducted RT-PCR of all known mammalian nAChR alpha and beta subunits in mRNA extracted from adult rat vestibular primary afferent neurons (Scarpa's ganglia) and vestibular end-organs. Transcripts encoding the alpha2-7 and beta2-4 nAChR subunits were found in the vestibular ganglia, while alpha3, alpha5-7, alpha9 and beta2-4 nAChR subunits were expressed in the vestibular end-organs. These results support previous electrophysiological, immunocytochemical and molecular biological data, and also provide a more complete understanding of the role of nAChRs in the neurochemical transmission subserving the efferent-afferent interaction in the vestibular periphery.

Central and peripheral myelin in the rat cochlear and vestibular nerves.

In the bipolar neurons of vertebrate cochlear and vestibular nerves, the myelin envelopes without interruption the axon, the perikaryon and the dendrite. The perikaryal myelin is thin and partially loose, whereas axon and dendrite are enveloped by compacted myelin. The expression of protein 0 and myelin basic protein, constituents of peripheral and central myelin respectively, has been investigated in the rat by immunohistochemical study at the light microscopic level. Our data indicate that both in the cochlear and vestibular nerves the myelin of the perikaryon and dendrite is composed by specific peripheral myelin proteins. The axon segment between the perikaryon and the transitional zone expresses peripheral myelin proteins in the cochlear nerve, while both types of myelin proteins are present in the vestibular nerve. Between the transitional zone and the brainstem the myelin of the axon is exclusively of the central type. The peripheral-central myelin transitional zone is in close proximity to the axonal pole in the vestibular ganglion cells, while in the cochlear nerve it is near the spiral foramina, at variable distance from the axonal pole of ganglion cells.

Immunohistochemical and electrophysiological demonstration of substance P in human vestibular ganglion cells.

This study was performed in order to determine the immunohistochemical expression and distribution pattern as well as the electrophysiological actions of substance P in human vestibular ganglion cells. Substance P-like immunostaining was mainly found in the cytoplasm of small vestibular ganglion cells. In contrast, nerve fibers were not labeled. Using intracellular recording techniques, substance P produced a slow depolarization of membrane potentials accompanied by an increase in membrane resistance. Furthermore, an enhanced firing response to depolarization occurred.

Expression of laminin in vestibular ganglia and nerves in herpes simplex virus infectious chick embryo.

The expression of laminin in the vestibular ganglia and nerves in herpes simplex virus I (HSV-I) infectious chick embryos was examined by immuno-histochemistry. In 9-day-old normal chick embryos, the surface of the vestibular ganglion cells and the vestibular nerves were labelled with laminin, while, in HSV-I infectious chick embryos, the staining of laminin in the vestibular ganglia and nerves was very weak. These results suggest that HSV-I injures vestibular ganglia and nerves, and that laminin is a good marker of the degeneration and innervation of developing vestibular nerves.

Ultrastructural organization of calcitonin gene-related peptide immunoreactive efferent axons and terminals in the vestibular periphery.

The ultrastructural distribution of calcitonin gene-related peptide immunoreactivity (CGRPi) was examined in sections of decalcified temporal bones in order to study the complex peptidergic innervation patterns of this efferent neuromodulator in the peripheral vestibular system of the rat. A new method of preembedding immunoelectron microscopy was developed to accomplish this study. Unmyelinated CGRPi axons, measuring 1 to 3 microns in diameter, passed among the primary afferent fibers in Scarpa's ganglion, and these fibers continued through the subepithelial regions of the vestibular end-organs. Within the neurosensory epithelia of the maculae and cristae, the CGRPi axons ramified to produce numerous CGRPi terminals. Immunoelectron microscopic localization of CGRPi terminals in the maculae and cristae revealed an extensive innervation pattern on the afferent vestibular pathway. Calcitonin gene-related peptide immunoreactive terminals made synaptic contacts with the unmyelinated portions of the primary afferent vestibular fibers innervating both type I and type II hair cells. Abundant synaptic contact between CGRPi terminals and the chalices surrounding type I hair cells was observed. Rare direct contact between CGRPi terminals and type I or type II hair cells was observed. In addition, vesiculated efferent terminals without CGRPi were seen contacting type II hair cells. These data suggest that the efferent vestibular system has a much more complex innervation pattern on the afferent vestibular pathway than previously believed.

Immunocytochemical localization of intermediate filaments in the guinea pig vestibular periphery with special reference to their alteration after ototoxic drug administration.

The present study examined the immunocytochemical localization of various intermediate filaments (IFs), 68 kDa, 160 kDa and 200 kDa neurofilament protein (NFP), cytokeratin (CK) 1, 8, 10 and 19, vimentin, and glial fibrillary acidic protein (GFAP) in the vestibular end-organs and ganglia of normal and streptomycin-treated guinea pigs. In normal animals, 68 kDa, 160 kDa and 200 kDa NFP were found in afferent nerve fibers and nerve terminals (probably nerve chalices). Fine nerve fibers (probably efferent and/or sympathetic nerve fibers) were also immunoreactive to NFP. In the vestibular ganglia, 68 kDa and 160 kDa NFP were predominantly distributed in larger cells, whereas 200 kDa NFP was also found in some small ganglion cells. Cytokeratin 8 and 19 were located in supporting cells, transitional cells, dark cells of vestibular end-organs, and the epithelial cell lining of the membranous labyrinth. Vimentin was observed in the hair cells distributed in the central region of the end organs, supporting cells, most connective tissue cells, and Schwann cells of the vestibular ganglion. Although GFAP-like immunoreactivity was evident in glial cells of the proximal vestibular nerve, no immunoreactivity was detected in the distal portion of the vestibular nerve, vestibular ganglion, or vestibular end-organs. These highly distinct staining patterns of IFs indicated that they may play different roles in the different cell types, and that they may serve as a specific marker for each cell type. In streptomycin-treated guinea pigs, immunoreactivities for NFP and vimentin (found in the hair cells) decreased after treatment, whereas immunoreactivities for the other IFs were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)