Olivocochlear neuron central anatomy is normal in alpha 9 knockout mice.

Olivocochlear (OC) neurons were studied in a transgenic mouse with deletion of the alpha 9 nicotinic acetylcholine receptor subunit. In this alpha 9 knockout mouse, the peripheral effects of OC stimulation are lacking and the peripheral terminals of OC neurons under outer hair cells have abnormal morphology. To account for this mouse's apparently normal hearing, it has been proposed to have central compensation via collateral branches to the cochlear nucleus. We tested this idea by staining OC neurons for acetylcholinesterase and examining their morphology in knockout mice, wild-type mice of the same background strain, and CBA/CaJ mice. Knockout mice had normal OC systems in terms of numbers of OC neurons, dendritic patterns, and numbers of branches to the cochlear nucleus. The branch terminations were mainly to edge regions and to a lesser extent the core of the cochlear nucleus, and were similar among the strains in terms of the distribution and staining density. These data demonstrate that there are no obvious changes in the central morphology of the OC neurons in alpha 9 knockout mice and make less attractive the idea that there is central compensation for deletion of the peripheral receptor in these mice.

Activation of tyrosine hydroxylase in the lateral efferent terminals by sound conditioning.

Preconditioning to sound is a well-documented strategy to provide protections against a subsequent acoustic trauma. In the present study, preconditioning (1.0 kHz tone at 81 dB sound pressure level (SPL) for 24 h) protected ABR thresholds by 17-28 dB from an acoustic trauma (2.7 kHz, 103 dB SPL, 30 min) that resulted in a temporary threshold shift. The protection afforded by sound conditioning was shown to be blocked by the administration of 6-hydroxydopamine which disrupts tyrosine hydroxylase in the nerve terminals of the lateral efferent fibers. Furthermore, tyrosine hydroxylase immunoreactivity was up-regulated both by sound conditioning alone, and by the combined treatment of sound conditioning and acoustic trauma. In contrast, acoustic trauma alone resulted in a reduction in tyrosine hydroxylase immunoreactivity compared to unexposed controls. These findings are the first demonstration that tyrosine hydroxylase in the lateral efferents are up-regulated during sound conditioning and suggests a role for the lateral efferent system in protecting against acoustic trauma by sound conditioning.

Efferent axons in the avian auditory nerve.

The sensory hair cells of the inner ear receive both afferent and efferent innervation. The efferent supply to the auditory organ has evolved in birds and mammals into a separate complex system, with several types of neurons of largely unknown function. In this study, the efferent axons in four different species of birds (chicken, starling, barn owl and emu) were examined anatomically. Total numbers of efferents supplying the cochlear duct (auditory basilar papilla and the vestibular lagenar macula) were determined; separate estimates of the efferents to the lagenar macula only were also derived and subtracted. The numbers for auditory efferents thus varied between 120 (chicken) and 1068 (barn owl). Considering the much larger numbers of hair cells in the basilar papilla, each efferent is predicted to branch extensively. However, pronounced species-specific differences as well as regional differences along the tonotopic gradient of the basilar papilla were documented. Myelinated and unmyelinated axons were found, with mean diameters of about 1 microm and about 0.5 microm, respectively. This suggests two basic populations of efferents, however, they did not appear to be distinguished sharply. Evidence is presented that some efferents lose their myelination at the transition from central oligodendrocyte to peripheral Schwann cell myelin. Finally, a comparison of the four bird species evaluated suggests that the efferent population with smaller, unmyelinated axons is the phylogenetically more primitive one. A new population probably arose in parallel with the evolution and differentiation of the specialized hair-cell type it innervates, the short hair cell.

The detrimental effects of potassium bromate and thioglycolate on auditory brainstem response of guinea pigs.

Potassium bromate (KBrO3) is known to be an oxidizing agent that is used not only as a food additive, mainly in the bread-making process, but also as a neutralizer in thioglycolate containing hair curling set. Although it has been shown that bromate poisoning could cause severe and irreversible sensorineural hearing loss as well as renal failure, the action mechanism of bromate-induced otoneurotoxicity especially its combination with thioglycolate remains to be studied. In this study, we attempted to investigate the toxic effects of KBrO3 in combination with or without thioglycolate on the auditory brainstem response (ABR) system in the guinea-pigs which was claimed to be very susceptible to the xenobiotics. In a preliminary test, we have found that after consecutive 2 weeks administration, KBrO3 caused a significant prolongation of wave I-III and the interwave latencies of ABR as well as significantly elevated the threshold of hearing, suggesting that the conduction velocity of the peripheral auditory nerve was delayed. By contrast, the absolute latency of wave IV/V and the interwave latency of wave III-V were not significantly prolonged, suggesting that KBrO3 had no effect on the brainstem. This oto-neurotoxic effect of KBrO3 was markedly enhanced by combining with thioglycolate. Our data also indicated that KBrO3 combined with thioglycolate but not KBrO3 alone prominantly caused a decrease of body weight. However, enzymatic activities (including Na+/K+-ATPase and Ca2+-ATPase) and the level of nitric oxide (NO) was significantly affected in the brainstem. Based on these findings, we tentatively conclude that whether KBrO3 alone or KBrO3 combined with thioglycolate induced oto-neurotoxicity majorly through the peripheral auditory nerve rather than via the central brainstem intoxication.

Na,K-ATPase alpha- and beta-isoforms in the developing cochlea of the mouse.

Immunohistochemistry was used to investigate the presence of Na,K-ATPase alpha- and beta-subunits isoforms (alpha 1, alpha 2, alpha 3, beta 1 and beta 2) in the cochlea of the mouse at different ages between embryological day (E) 19 and postnatal day (P) + 30. alpha 1 was mainly found in the stria vascularis and in the spiral ligament; it increased steadily from p+4. These data correlates well with the morphological and electrophysiological maturation of the cochlea. alpha 3 predominated in the spiral ganglia and the cochlear nerve. This finding is well in accordance with reports that alpha 3 seems to be associated with the nervous system. The beta-subunit was found mainly in those tissues where staining of the alpha-subunit also was seen. Both subunits were localized in tissue regions where fluid regulation is expected to play an important role. For some isoforms, the expression pattern of Na,K-ATPase during development in the mouse is different from that in the rat. The expression of Na,K-ATPase and that of glucocorticoid receptors during development in the inner ear of the mouse show a similar pattern, which may indicate that glucocorticoid receptors could be involved in regulating the expression of Na,K-ATPase.

Localization of ornithine decarboxylase (ODC) in the cochlea of the immature rat.

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, is important in cochlear development. Whereas tissue specific differences in cochlear ODC activity have been demonstrated, cellular localization of ODC protein in the inner ear of the immature rat has not. ODC was localized in inner ear structures using an ODC polyclonal antibody and the effects of cycloheximide on ODC immunoreactivity and enzymatic activity were determined. Tissues demonstrating elevated enzymatic activity contained cells with the strong immunoreactivity. ODC activity was highest in the organ of Corti and lateral wall followed by the cochlear nerve. Immunoreactivity was demonstrated throughout the cochlea with intense staining of the hair cells, pillar cells, Deiter's cells, inner sulcus cells, basilar membrane, stria vascularis, spiral ganglion cell bodies and cochlear nerve fibers. Cycloheximide rapidly diminished cochlear ODC activity and expression of ODC protein. The half-life of cochlear ODC was 30 min. Localization of cellular sites of ODC is important in understanding the role of the ODC-polyamine pathway in cochlear development and will be a valuable marker for tissue damage from ototoxic agents.

[Study on Na(+)-K(+)-ATPase of the spiral ganglion neuron and acetylcholinesterase of the cochlea efferent nerve after electric stimulation in the scale media].

In this study, it was found the concentration of Na(+)-K(+)-ATPase of the spiral ganglion neuron (SGN) increased 3 months after electric stimulation (0.1 mA) for 3 hours. However, current at 1.0 mA would destroy the enzyme production mechanism of SGN. Therefore 0.4 mA might be thought as a critical level because the concentration of the enzyme decreased with just above this level of stimulation. We suggest that the appropriate intensity range of the current stimulation of SGN should be controlled from 0.1 mA-0.4 mA. Acetylcholinesterase of the cochlea efferent nerve would increase with weak current stimulation (0.1 mA) which might play an important role in the protection of SGN during ototoxicity. This experimental results led to the conclusion that Na(+)-K(+)-ATPase of the SGN could be taken as an objective indicator presenting the functional changes of SGN quantitatively.

Protein gene product 9.5 in the developing cochlea of the rat: cellular distribution and relation to the cochlear cytoskeleton.

Protein gene product 9.5 was immunolocalized in the adult and early postnatal (P2-P15) rat cochlea, and its distribution compared with a 200 kDa highly phosphorylated neurofilament subunit (neurofilament 200) and alpha-tubulin. In the adult, Protein gene product 9.5 was expressed exclusively in cochlear nerve fibres and ganglion cells, a small percentage of these (Type II ganglion cells and olivocochlear bundle fibres) being intensely positive for both protein gene product and neurofilament 200. In postnatal development, pillar and Deiters' cells were at first (P2-P15) strongly positive for protein gene product 9.5, and hair cells moderately so. At P2, all nerve fibres and ganglion cells showed co-expression of protein gene product 9.5 and neurofilament 200, but at later stages, the subset of intensely co-labelled neurons appeared, nerve fibres at P7 onwards and ganglion cells from P12. There was no overt correlation between the onset of protein gene product 9.5 and alpha-tubulin expression in any cochlear component. Protein gene product 9.5 expression in ganglion cells was at first (P2 and P7) mainly nuclear, and later also cytoplasmic. It is concluded that there is a clear correlation of high levels of protein gene product 9.5 and neurofilament protein expression, and that protein gene product 9.5 is expressed in some non-neuronal cells of the cochlea during its early development, persisting until after hearing has commenced.

GAD- and GABA-immunoreactivity in the ascending auditory pathway of horseshoe and mustached bats.

A comparative study of the immunostain to antibodies directed against glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid (GABA) in the ascending auditory pathway was carried out in horseshoe bats (Rhinolophus rouxi) and mustached bats (Pteronotus parnellii). In both species GAD/GABA-positive puncta (presumed axonal boutons) and GAD/GABA-positive cells were found in the cochlear nucleus, the superior olivary complex, the nuclei of the lateral lemniscus the inferior colliculus, and the medial geniculate body. General features of the immunostaining pattern in the auditory pathway agree with observations in other mammals. Quantitative analysis of puncta distribution shows that many auditory centers are characterized by subregional differences in puncta density and distribution. This indicates local differences in putatively inhibitory input related to connectivity and tonotopic organization. The following species characteristic features were found: 1) The dorsal non-laminated portion of the dorsal cochlear nucleus in horseshoe bats lacks the GAD/GABA-immunoreactive cells typical for the ventral laminated portion and the dorsal cochlear nucleus of other species. Clearly, a cytoarchitectonic specialization is accompanied by a loss of putatively GABAergic local inhibitory circuits. 2) The ventral division of the medial geniculate body of the mustached bat lacks GAD/GABA-immunopositive cells. Such cells are present in the horseshoe bat and other mammals. This finding implies functional differences in the organization of the medial geniculate body within the same mammalian order.

Postnatal developmental changes in inner ear ornithine decarboxylase (ODC).

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, is important in cellular growth, differentiation and development. Although ODC has been quantitated in cochlear tissues of the adult rat, it has not been assessed quantitatively in developing inner-ear tissues. The purpose of the present study was to quantitate ODC in cochlear tissues of the rat during the period of development of hearing. Cochlear ODC was significantly elevated throughout the period of cochlear maturation in that it increased rapidly during the first 10 days, peaked on day 10 and then declined thereafter. ODC in the lateral wall/organ of Corti tissues was significantly higher than in the cochlear nerve in developing, but not in adult rats. Further examination of separate cochlear tissues from 10-day old rats revealed that ODC activity was higher in the organ of Corti than in the lateral wall or cochlear nerve. Postnatal changes in ODC paralleled functional maturation of hearing and the hypersensitive period for aminoglycoside ototoxicity in the rat. Since aminoglycosides have been shown to inhibit ODC in vitro, aminoglycoside inhibition of polyamine synthesis may mediate the hypersensitivity of developing animals to the effects of these drugs.

Contribution of centrifugal innervation to choline acetyltransferase activity in the cat cochlear nucleus.

Using a quantitative microchemical mapping approach combined with surgical cuts of fiber tracts, the contributions of centrifugal pathways to choline acetyltransferase activity were mapped three-dimensionally in the cat cochlear nucleus. Large reductions of choline acetyltransferase activity, averaging 70%, were measured in almost all parts of the lesion-side nucleus following transection of virtually all its centrifugal connections. More superficial cuts, penetrating just through the olivocochlear bundle, also led to significant reductions of enzyme activity, especially most rostrally in the anteroventral cochlear nucleus and superficial granular region, where the reductions were similar to those following the complete cuts. Lesions encroaching upon the superior olivary complex gave bilateral effects. Transverse cuts between rostral and caudal parts of the cochlear nucleus gave some small effects. The results suggest that, as in rats, most choline acetyltransferase activity in the cat cochlear nucleus is associated with its centrifugal innervation. However, unlike the situation in rats, the enzyme activity in cats is related more to olivocochlear branches than to ventral fibers in the trapezoid body region. Also, the choline acetyltransferase activity related to olivocochlear collateral innervation is much less uniformly distributed within the cochlear nucleus in cats than in rats.

Development of aspartate aminotransferase and glutaminase immunoreactivity in the rat auditory nerve.

Aspartate aminotransferase and glutaminase immunoreactive labeling of the auditory nerve has previously been reported. In the present study, the development of these immunoreactivities was examined in the auditory nerve of the rat, at ages ranging from 17 days gestation to four postnatal weeks. Cells and processes were examined in the cochlea, and fibers and terminals in the cochlear nucleus. In the cochlea, immunoreactive labeling with antisera to both enzymes was first seen at 20 gestational days, in spiral ganglion cells. It was not until two postnatal weeks, however, that this immunoreactive labeling was first seen in primary afferent terminals around spherical cells in the anteroventral cochlear nucleus. This correlates with the establishment of mature synaptic connections and function.

Ornithine decarboxylase in the inner ear of the guinea pig.

L-Ornithine decarboxylase, the rate limiting enzyme of polyamine synthesis and a possible marker enzyme for tissue proliferation and maturation, has been found in the developing guinea pig cochlea using the unlabelled horseradish-peroxidase-antiperoxidase technique. Ornithine decarboxylase-like immunoreactive material was detected in the neurons of the Ganglion spirale and in their axonal and/or dendritic fibers. The location of the enzyme and the possible functional role of ornithine decarboxylase plays in the development and maturation of the auditory organ and of the hearing process are discussed.

Impaired morphological development of the dorsal cochlear nucleus in hamsters treated postnatally with alpha-difluoromethylornithine.

The dorsal cochlear nucleus is a highly organized nucleus in the auditory system in which the ramifications of depletion of specific cell types during development can be studied. Granule cells, small interneurons that are located in all layers of the DCN in the adult hamster, proliferate postnatally and are, therefore, potentially vulnerable to anti-mitotic agents that are administered after birth. The present experiments describe the effects of alpha-difluoromethylornithine, a drug that inhibits proliferation of cerebellar granule cells, on the granule cells in the dorsal cochlear nucleus. As in the cerebellum, the density of granule cells in the dorsal cochlear nucleus is reduced after alpha-difluoromethylornithine treatment. In hamsters treated with alpha-difluoromethylornithine (200 or 500 mg/kg subcutaneously (s.c.), twice daily on postnatal days 4-14), the numerical density of granule cells was reduced in the superficial dorsal cochlear nucleus at 15 days; by 40 days this effect was also apparent in the deep layer, suggesting that cells located superficially that would have migrated into the deep dorsal cochlear nucleus had either failed to develop or did not arrive at their final location. This evidence suggests that the cells normally migrate down from the superficial proliferative zone into the deeper layers. In the drug-treated animals, a layer of mixed granule cells and fusiform cells was thinner than in controls probably due to the reduction in interspersed granule cells since the number of fusiform cells was unaffected. There was also a dose-dependent effect on cell growth; fusiform cells were affected at both doses, while giant cells were only affected at the highest dose. Granule cells form a major input to the fusiform cells and their depletion may account for some of the effects on fusiform cell growth. There could also be additional direct actions of alpha-difluoromethylornithine on this population.

Patterns of glutamate decarboxylase immunostaining in the feline cochlear nuclear complex studied with silver enhancement and electron microscopy.

The cochlear nuclear complex of the cat was immunostained with an antiserum to glutamate decarboxylase (GAD), the biosynthetic enzyme for the inhibitory neurotransmitter GABA, and studied with different procedures, including silver intensification, topical colchicine injections, semithin sections, and immunoelectron microscopy. Immunostaining was found in all portions of the nucleus. Relatively few immunostained cell bodies were observed: most of these were in the dorsal cochlear nucleus and included stellate cells, cartwheel cells, Golgi cells, and unidentified cells in the deep layers. An accumulation of immunoreactive cells was also found within the small cell cap and along the medial border of the ventral cochlear nucleus. Immunostained cells were sparse in magnocellular portions of the ventral nucleus. Most staining within the nucleus was of nerve terminals. These included small boutons that were prominent in the neuropil of the dorsal cochlear nucleus, the granule cell domain, in a region beneath the superficial granule cell layer within the small cell cap region, and along the medial border of the ventral nucleus. Octopus cells showed small, GAD-positive terminals distributed at moderate density on both cell bodies and dendrites. Larger, more distinctive terminals were identified on the large cells in the ventral nucleus, in particular on spherical cells and globular cells. There was a striking positive correlation of the size, location, and complexity of GAD-positive terminals with the size, location, and complexity of primary fiber endings on the same cells. This correlation did not hold in the dorsal nucleus, where pyramidal cells receive many large GAD-positive somatic terminals despite the paucity of primary endings on their cell bodies. The GAD-positive terminals contained pleomorphic synaptic vesicles and formed symmetric synaptic junctions that occupied a substantial portion of the appositional surface to cell bodies, dendrites, axon hillocks, and the beginning portion of the initial axon segments. Thus, the cells provided with large terminals can be subjected to considerable inhibition that may be activated indirectly through primary fibers and interneurons or by descending inputs from the auditory brainstem.

GABAergic neurons and axon terminals in the brainstem auditory nuclei of the gerbil.

The anatomical localization of glutamic acid decarboxylase (GAD), the synthesizing enzyme for GABA, was analyzed in the brainstem auditory nuclei of the adult gerbil. GAD-positive terminals and somata were present in the cochlear nucleus, superior olivary complex, lateral lemniscus, and inferior colliculus in varying concentrations and patterns. One of the highest densities of GAD-positive terminals is found in the superficial layers of the dorsal cochlear nucleus (DCN), whereas the ventral cochlear nucleus (VCN) has somewhat fewer terminals that are arranged in pericellular plexuses. GAD-positive neurons occur mainly in the superficial and fusiform layers of the DCN and are scattered throughout the VCN. Within the superior olivary complex, the highest concentration of immunoreactive terminals and neurons occurs in the ventral and lateral nuclei of the trapezoid body. In contrast, the medial nucleus of the trapezoid body and the medial superior olive contain fewer GAD-positive puncta and probably no immunoreactive somata. The lateral superior olive and superior periolivary nucleus contain a few immunoreactive puncta but a large number of immunoreactive somata. In the midbrain, the nuclei of the lateral lemniscus contain a moderate number of GAD-positive puncta and a large number of different types of GAD-positive neurons. The inferior colliculus also contains a heterogeneous population of labeled somata, most of which are multipolar neurons. In addition, a high concentration of immunoreactive puncta occurs in this region. These data demonstrate a diverse distribution of GAD-positive neurons and puncta throughout the brainstem auditory nuclei and suggest that GABA might be an important neurotransmitter in the processing of auditory information.

Effect of olivocochlear bundle transection on choline acetyltransferase activity in the rat cochlear nucleus.

Using a microdissection and quantitative microassay approach, choline acetyltransferase activities were mapped in the cochlear nuclei of rats having complete transections of the olivocochlear bundle on one side in the brain stem. In rats in which the trapezoid body was not significantly damaged by the lesion, consistent reductions of choline acetyltransferase activity in subregions of the lesion-side cochlear nucleus, as compared to the control side, averaged about 20%. Nevertheless, a profound lesion-side reduction of choline acetyltransferase activity was found in a branch connection from the olivocochlear bundle to the cochlear nucleus. The results suggest that branches from the olivocochlear bundle are cholinergic, but contribute a relatively minor proportion of the cholinergic synapses in all regions of the rat cochlear nucleus. In the light of previous results with more extensive lesions, it can be proposed that synapses in all regions of the rat cochlear nucleus. In the light of previous results with more extensive lesions, it can be proposed that most cholinergic input into the rat cochlear nucleus enters by a ventral route along the trapezoid body. It is noted that this represents a quantitatively somewhat different situation from that in the cat.

Effects of trapezoid body and superior olive lesions on choline acetyltransferase activity in the rat cochlear nucleus.

Using a microdissection and quantitative microassay approach, choline acetyltransferase activities were mapped in the cochlear nuclei of rats having either transection of the trapezoid body or destruction of the superior olivary complex on one side in the brain stem. Lateral trapezoid body transection resulted in dramatic loss of choline acetyltransferase activity in all parts of the ipsilateral cochlear nucleus, while more medial transection had little effect. Destruction of the superior olivary complex resulted in dramatic loss of choline acetyltransferase activity in the ipsilateral cochlear nucleus, and detectable loss also contralaterally. The results suggest that most of the centrifugal cholinergic projections to the rat cochlear nucleus derive from or traverse the vicinity of the superior olivary complex bilaterally and enter the cochlear nucleus ventrally from the region of the trapezoid body.

Distribution and fine structure of neuronal elements containing glutamate decarboxylase in the rat cochlear nucleus.

Distribution and fine structure of gamma-aminobutyric acid (GABA)-containing structures were examined in the rat cochlear nuclear complex by means of immunohistochemistry using glutamate decarboxylase (GAD) as a marker. GAD-like immunoreactive (GADI) terminals were diffusely distributed in the dorsal cochlear nucleus, while in the ventral cochlear nucleus numerous immunoreactive fibers were situated around the cell bodies. These light-microscopic observations were confirmed by electron microscopy. Evidence suggesting that many of GADI boutons in the cochlear nucleus are of intrinsic origin was also shown.

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.