Suppression of delayed rectifier K+ channels by gentamicin induces membrane hyperexcitability through JNK and PKA signaling pathways in vestibular ganglion neurons.

Aminoglycoside antibiotics, such as gentamicin, are known to have vestibulotoxic effects, including ataxia and disequilibrium. To date, however, the underlying cellular and molecular mechanisms are still unclear. In this study, we determined the role of gentamicin in regulating the sustained delayed rectifier K+ current (IDR) and membrane excitability in vestibular ganglion (VG) neurons in mice. Our results showed that the application of gentamicin to VG neurons decreased the IDR in a concentration-dependent manner, while the transient outward A-type K+ current (IA) remained unaffected. The decrease in IDR induced by gentamicin was independent of G-protein activity and led to a hyperpolarizing shift of the inactivation Vhalf. The analysis of phospho-c-Jun N-terminal kinase (p-JNK) revealed that gentamicin significantly stimulated JNK, while p-ERK and p-p38 remained unaffected. Blocking Kv1 channels with α-dendrotoxin or pretreating VG neurons with the JNK inhibitor II abrogated the gentamicin-induced decrease in IDR. Antagonism of JNK signaling attenuated the gentamicin-induced stimulation of PKA activity, whereas PKA inhibition prevented the IDR response induced by gentamicin. Moreover, gentamicin significantly increased the number of action potentials fired in both phasic and tonic firing type neurons; pretreating VG neurons with the JNK inhibitor II and the blockade of the IDR abolished this effect. Taken together, our results demonstrate that gentamicin decreases the IDR through a G-protein-independent but JNK and PKA-mediated signaling pathways. This gentamicin-induced IDR response mediates VG neuronal hyperexcitability and might contribute to its pharmacological vestibular effects.

Effects of unilateral vestibular ganglionectomy on glutaminase activity in the vestibular nerve root and vestibular nuclear complex of the rat.

The metabolism of glutamate, the most likely neurotransmitter of vestibular ganglion cells, includes synthesis from glutamine by the enzyme glutaminase. We used microdissection combined with a fluorometric assay to measure glutaminase activity in the vestibular nerve root and nuclei of rats with unilateral vestibular ganglionectomy. Glutaminase activity in the lesioned-side vestibular nerve root decreased by 62% at 4 days after ganglionectomy and remained at similar values through 30 days. No change occurred in the contralateral vestibular nerve root. Glutaminase activity changes in the vestibular nuclei were lesser in magnitude and more complex, including contralateral increases as well as ipsilateral decreases. At 4 days after ganglionectomy, glutaminase activity was 10-20% lower in individual lesioned-side nuclei compared with their contralateral counterparts. By 14 and 30 days after ganglionectomy, there were no statistically significant differences between the nuclei on the two sides. This transient asymmetry of glutaminase activities in the vestibular nuclei contrasts with the sustained asymmetry in the vestibular nerve root and suggests that intrinsic, commissural, or descending pathways are involved in the recovery of chemical symmetry. This recovery resembles our previous finding for glutamate concentrations in the vestibular nuclei and may partially underlie central vestibular compensation after peripheral lesions.

Calcitonin gene-related Peptide and choline acetyltransferase colocalization in the human vestibular periphery.

Within the vestibular system, calcitonin gene-related peptide (CGRP) has been localized in the efferent terminals and their brainstem neuronal cell bodies in several animal models. Presently, very few studies have verified these findings in the vestibular system in adult primates or humans. CGRP immunoreactivity (CGRPi) and its colocalization with choline acetyltransferase immunoreactivity (ChATi) in human vestibular end organs and Scarpa's ganglion were studied using polyclonal antibodies against CGRP and ChAT, at the light-microscopic level. The CGRPi axons ramified to produce numerous CGRPi terminals throughout the neurosensory epithelium of the maculae and cristae, primarily in the basal and midbasal areas. Numerous CGRPi efferent terminals made contact with both type II vestibular hair cells and the afferent chalices surrounding type I vestibular hair cells. All CGRP immunoreactive fibers also exhibited ChATi. As in the animal models, no CGRPi was found within Scarpa's ganglion. This study provides evidence for CGRPi in the human vestibular periphery and validates the biomedical relevance of the current animal models.

Immunohistochemical demonstration of inducible nitric oxide and nuclear factor-kappa B with reference to age-related changes in the mouse spiral and vestibular ganglion.

The morphology and immunohistochemistry of inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-kappa B) were studied on the spiral and vestibular ganglion of young and old ddy mice. The significant decrease in the number of the spiral ganglion cells and a significant expression of iNOS and NF-kappa B were observed in old mice. In contrast, in the vestibular ganglion of all animals examined, decrease in the number of the ganglion cells or expression of iNOS and NF-kappa B were not observed. Although the relevance of enzymatic systems for the protection of vestibular ganglion cells in old individuals from harmful oxidative stress increased with aging should be further clarified, lack of harmful stress due to nitric oxide (NO) may be one of the plausible reasons for that the vestibular ganglion cells were not decreased in number with aging, since iNOS was not detected in the vestibular ganglion cells in the animals tested in the present study.

Localization of nitric oxide synthase isoforms (NOS I, II and III) in the vestibular end organs of the guinea pig.

The localization of nitric oxide (NO) synthase (NOS) isoforms was investigated in the vestibular organ of the pigmented guinea pig by indirect immunohistochemistry. The cytoplasm of both type I and type II vestibular sensory cells as well as vestibular ganglion cells showed both NOS I and III immunoreactivity, whereas there was no reactivity in their nuclei and sensory hairs. The afferent nerve chalices were usually not stained. NOS III staining was also observed in the nerve fibers contacting type II cells and in the subepithelial tissue. The endothelial lining of the blood vessels displayed reactivity for NOS III. The cytoplasm of fluid transporting cells showed weak staining for NOS I and moderate staining for NOS III. Immunostaining for NOS II did not display any reactivity in general. These findings may suggest that NO is a mediator of neurotransmission in the vestibular system in sensory cells and ganglia. NO in the fluid transporting cells may play an important role for maintaining the endolymph and ion homeostasis, and NOS III in vascular endothelial cells implies regulatory effects of NO on vascular wall tonus and vestibular blood supply.

Effect of methimazole, an FMO substrate and competitive inhibitor, on the neurotoxicity of 3,3'-iminodipropionitrile in male rats.

This study was designed to examine the role of flavin-containing monooxygenase (FMO) on the auditory and vestibular neurotoxicity of 3,3'-iminodipropionitrile (IDPN) using the FMO substrate and competitive inhibitor methimazole (MMI). Specifically, the purpose was to block the FMO-mediated conversion of IDPN to the putative neurotoxic metabolite N-hydroxy3,3'-iminodipropionitrile (HOIDPN). In three separate experiments, adult male Long-Evans hooded rats were administered (ip) saline (vehicle), MMI, IDPN, or HOIDPN individually, or a combination of IDPN and MMI or HOIDPN and MMI. Animals were observed daily for signs of the ECC syndrome (excitation with choreiform and circling movements) for 10 days. One to 2 weeks after exposure, a battery of behavioral tests was used to examine vestibular and auditory function. MMI completely blocked the neurotoxicity associated with a 600 mg/kg dose of IDPN and partially blocked the effects of a 1000 mg/kg dose of IDPN. In contrast, MMI failed to block, and instead increased, the neurotoxicity associated with HOIDPN. These data suggest that FMO-mediated metabolism of IDPN is necessary for the generation of a metabolite responsible for the vestibular and auditory neurotoxicities.

Acid phosphatase activity in the chick's inner ear.

The cellular localization of distribution of acid phosphatase (AP) in the plastic sections of newly hatched chick's inner ear were investigated utilizing an azo-coupling method. AP activity as evidenced by azo dye deposits were well defined and localized in various cells of membranous labyrinth. Intense AP activity was detectable in the supranuclear area of hair cells in the basilar papilla and vestibular sensory hair cells. As in the case of the other sites of AP activity, marked AP activity was seen in the supranuclear area of the transitional epithelia of crista ampullaris and in the supranuclear area, or diffusely in the cytoplasm of the dark cells at the base of crista ampullaris. The columnar cells and the cells of tegmentum vasculosum showed moderate to strong AP activity. The statoacoustic and vestibular ganglion cells showed various degrees of AP activity. On the AP activity of statoacoustic or vestibular ganglion, in comparison between the sections from JB-4 Plus embedded specimens and those from LR White embedded specimen, the latter could more intensely demonstrate AP activity than the former. Moreover, sections fixed in 2.5% paraformaldehyde demonstrated more intense AP activity than those fixed in 2.5% glutaraldehyde.

Distribution of choline acetyltransferase mRNA in the efferent vestibular neurons of the chinchilla.

The distribution of choline acetyltransferase messenger RNA (mRNA) among efferent vestibular neurons in the chinchilla was investigated. mRNA coding for choline acetyltransferase, the enzyme that synthesizes acetylcholine, was used as a marker for the cholinergic system. In order to retrogradely label the efferent vestibular neurons, Fluoro-gold was injected through the oval window into the inner ear of anesthetized young male chinchillas (6 to 12 months old). The animals were anesthetized and perfused through the heart 2 days post injection with 4% paraformaldehyde in phosphate buffer. Retrogradely labeled efferent vestibular neurons were mapped in brainstem sections prior to processing for in situ hybridization histochemistry using radiolabeled ribonucleic acid probes complementary to the 3' end of the choline acetyltransferase mRNA. At the levels of the ascending facial nerve and the genu of the facial nerve, we found that approximately 90% of the Fluoro-gold labeled cells in group E1 contained choline acetyltransferase mRNA. All of the group E2 cells that were labeled with Fluoro-gold were found to be cholinergic (contain choline acetyltransferase mRNA). Finally, 60% of the Fluoro-gold-labeled cells in the caudal pontine reticular nucleus contained choline acetyltransferase mRNA.

Cytochrome oxidase histochemistry in Scarpa's ganglion after hemilabyrinthectomy.

Cytochrome oxidase histochemistry was studied in neurons in the vestibular ganglion in gerbils two weeks after hemilabyrinthectomy. This study measured the staining density in ganglion cells on both the lesioned and non-lesioned side of the brainstem. Cytochrome oxidase staining was significantly reduced in ganglion cells ipsilateral to the lesion. This decrease may have been related to the concomitant loss of spontaneous discharge and reduced energy demand for oxidative metabolism.

Histochemical evidence that cochlear efferents are carried with the inferior vestibular nerve in guinea pigs.

Histochemical methods were employed to determine the course of the olivocochlear bundle (OCB) within the vestibular nerve of guinea pigs. Following transection of the inferior vestibular nerve, cholinesterase staining in the cochlea was greatly reduced. Transection of the superior vestibular nerve, however, yielded no detectable change in staining. It is concluded that the cochlear efferent innervation in guinea pigs is carried in the inferior vestibular nerve, at the point of entry into the medial bulla.

The demonstration of choline acetyltransferase activity in cultured vestibular ganglion cells from the fetal rat.

Incubation of vestibular ganglion cells from the rat fetus was successfully done demonstrating bipolar and multipolar cells in the cell cultures produced. Further, the presence of choline-acetyltransferase-positive cells was confirmed immunohistochemically.

Neurochemical evidence for afferent GABAergic and efferent cholinergic neurotransmission in the frog vestibule.

Glutamate decarboxylase and choline acetyltransferase activities with magnitudes similar to those of their homologous enzymes in frog nervous tissue were found in homogenates of the frog labyrinth. Transection of the vestibular nerve resulted in a gradual diminution of choline acetyltransferase activity until it reached an 88% decrease 6 weeks after surgery. In contrast, glutamate decarboxylase activity did not suffer any alteration at any time after nerve excision. The presence of their enzymes of synthesis is evidence of the neurotransmitter participation of GABA and acetylcholine in the frog vestibule; the observed decrease of choline acetyltransferase following vestibule nerve excision supports the efferent synaptic bouton localization of choline acetyltransferase. The suggestion that glutamate decarboxylase is located in a cell type (or compartment) that may well be the hair cell is supported by the fact that this enzyme does not suffer any modification after surgery. These results are in accordance with an efferent cholinergic neurotransmission and a putative afferent role of GABA in the frog vestibule.

Onset and development of neuron-specific enolase immunoreactivity in the peripheral vestibular system of the mouse.

The development of neuron-specific enolase (NSE) immunoreactivity in mouse sensory and ganglion vestibular cells was studied from gestation day 14 to adulthood. NSE staining appeared sequentially in these structures with a pattern that closely paralled their maturation sequences. The onset of NSE reactivity, at gestation day 15, in ganglion cells was concomitant with the formation of contacts between the afferent fibers and sensory cells, and was observed in hair cells, at gestation day 17, during the period in which the first synaptic structures form. The development of NSE staining in the cristae revealed an apex-base and axial gradients of maturation.

Fibre population of the vestibulocochlear anastomosis in humans.

The vestibulocochlear anastomosis in the adult human was studied by light and electron microscopy. It was found to contain both myelinated and unmyelinated axons. The number of myelinated axons ranged from 223 to 695, with a mean of 360, while the unmyelinated axons varied from 638 to 1453, with a mean of 1005. The ratio of unmyelinated to myelinated axons varied from 2.1 to 4.4, with a mean of 3.0.

Choline acetyltransferase and acetylcholinesterase in centrifugal labyrinthine bundles of rats.

Activities of choline acetyltransferase and acetylcholinesterase were measured for the acetylcholinesterase-positive fiber bundles containing axons projecting from the brainstem to the labyrinth of the rat. These activities were compared to those of a well-established cholinergic tract: the facial motor root. The choline acetyltransferase activities were roughly similar between the tracts, consistent with a conclusion that the centrifugal labyrinthine fibers are all cholinergic. The acetylcholinesterase activities were much higher in the centrifugal labyrinthine bundle than in the facial motor root, probably relating to the smaller diameters of the labyrinthine fibers. Transection of the centrifugal labyrinthine bundle led to virtually total loss of its choline acetyltransferase activity lateral to the cut, consistent with a centrifugal direction of all the fibers, but loss of only half its acetylcholinesterase activity, even after 34 days. These results agree with those for well-established cholinergic pathways, including the facial motor root in the present study, and with previous suggestions that a component of the acetylcholinesterase in cholinergic tracts might be synthesized by cells other than the neurons in the tract.

Quantitative distribution of choline acetyltransferase and acetylcholinesterase activities in the rat cochlear nucleus.

Within the cochlear nucleus of the rat, as well as some nearby regions, quantitative histochemical mapping procedures were used to construct maps of the distributions of choline acetyltransferase and acetylcholinesterase activities. The results were in some ways consistent with results previously reported for cat, e.g., very low activities of both enzymes were found in the auditory nerve root, and also in the vestibular nerve root, except where acetylcholinesterase-positive centrifugal fibers are located, very high activities were found in the facial nerve system. In many ways, however, the results for the rat cochlear nucleus contrasted with those for the cat. Notably, choline acetyltransferase activities in some regions of the rat cochlear nucleus were as much as 30- to 60-fold higher than for the comparable regions in the cat, and both enzymes had much more uniform distributions in the rat cochlear nucleus than in the cat. The more prominent cholinergic system in the rat cochlear nucleus might relate to a proportionately larger population of cholinergic interneurons, or, more probably, a more significant innervation by cholinergic, centrifugal pathways, or both, as well as, perhaps, generally higher choline acetyltransferase activities in cholinergic neurons of rat.

Alkaline phosphatase activity in the ganglion cells in and near the temporal bone.

The distribution pattern of alkaline phosphatase was studied in the ganglion cells in and near the temporal bone of some mammals. No or extremely weak activity of alkaline phosphatase was observed in or near the spiral and vestibular ganglion cells of the bat, rat, cat, and monkey. However, intense to moderate enzyme activity was noted exclusively in the peripheral areas of the spiral and vestibular ganglion cells of the guinea pig. The enzyme activity was also demonstrated in the nerve fibers at some distance from both axonal and dendritic processes of the spiral and vestibular ganglion cells of the guinea pig. Trigeminal and geniculate ganglion cells of all mammals examined showed intense to moderate enzyme activity in their capsular cells. There is a marked difference in the phosphate metabolism in the capsular tissues of the ganglion cells in or near the temporal bone of various mammals.

On the acetylcholinesterase activity in the vestibular ganglion of the rat.

Acetylcholinesterase was demonstrated in the ganglion cells of the vestibular ganglion of the rat with Karnovsky's method. The acetylcholinesterase content was evaluated after a 24 hours incubation time by means of a three-grade scale. The cell size was determined by a particle size analyser. No correlation between the two values could be established. A comparison with the spiral ganglion showed a slightly lower acetylcholinesterase content in the vestibular ganglion. The content of acetylcholinesterase in the vestibular ganglion cells seems to be lower than in the spinal ganglion cells.