Dwayne D. Simmons

Olivocochlear innervation of inner and outer hair cells during postnatal maturation: Evidence for a waiting period

Reconstructions of the efferent innervation of the hamster (Mesocricetus auratus) cochlea were done during postnatal development. Efferent neurons were labeled via injections of biocytin and horseradish peroxidase into the crossed olivocochlear (OC) bundles using an in vitro brainstem technique. Such injections retrogradely labeled cell bodies in ventral periolivary regions of the superior olive consistent with their being medial OC neurons. Anterogradely labeled axons were traced to the cochlea, where they terminated on or below inner hair cells (IHCs) prior to postnatal day 5 (P5). After P5, labeled axons terminated on IHCs and outer hair cells (OHCs) and after P10, the majority of labeled axons terminated on the OHCs. In the electron microscope, small labeled terminals containing densely packed synaptic vesicles were found both adjacent to IHCs (axosomatic) as well as apposed to afferent and efferent fibers below IHCs prior to P5. By P10, large labeled terminals were axosomatic to OHCs and no longer found on IHCs. Consistent with previous reports, these data suggest that medial OC axons form part of an early primary innervation on and below IHCs before terminating on OHCs. This raises the possibility that OC neurons demonstrate a period of waiting below an intermediate target similar to that described in the development of thalamocortical projections.

Differential expression of the α9 nicotinic acetylcholine receptor subunit in neonatal and adult cochlear hair cells

The expression of the alpha9 nicotinic acetylcholine receptor (nAChR) subunit was investigated in perinatal and adult rat cochleae using [35S] labeled cRNA in situ hybridization techniques. In the adult, alpha9 expression showed both longitudinal and radial gradients. The highest expression occurs over outer hair cells (OHCs) in basal regions, and particularly, OHCs in row 1. In contrast, expression over IHCs is lowest in basal regions and highest in apical regions. During embryonic and postnatal ages, the pattern of alpha9 expression differs. Expression of alpha9 was nearly equivalent over IHCs and OHCs. Additionally, the greater epithelial ridge, which is adjacent to IHCs before birth, shows a high level of alpha9 expression. These data are consistent with current models of efferent synaptogenesis and suggest that the expression of the alpha9 nAChR may be influenced by the arrival of efferent axons.

Olivocochlear innervation of inner and outer hair cells during postnatal maturation: an immunocytochemical study

Changes in the expression of several neurochemical markers associated with either axonal growth (GAP-43), synaptic vesicles (synaptophysin), or the cholinergic population of lateral olivocochlear (OC) efferents were investigated in the postnatal cochlea of hamsters. Growth-associated protein was expressed in the neonatal cochlea but not in the adult; immunoreactivity was found below inner hair cells (IHCs) from postnatal day (P) 2 through P14 and below outer hair cells (OHCs) from P5 to P14. In contrast, synaptophysin was expressed in both the neonate and adult cochlea; immunoreactivity was found below IHCs around P4 and below OHCs at P5. Both GAP-43 and synaptophysin immunoreactivities occurred first below IHCs in basal regions of the cochlea. Efferent fibers containing calcitonin gene-related peptide (CGRP) immunoreactivity were identified as early as P4 within the cochlear nerve but were not observed underneath IHCs until P7. Similar to GAP-43 and synaptophysin immunoreactivity, CGRP expression followed a basal to apical gradient; however, expression below OHCs appeared restricted to apical regions. These data raise the possibility that efferents expressing growth proteins and efferents expressing synaptic vesicle proteins co-exist during the first postnatal week. Furthermore, it is hypothesized that CGRP-containing lateral OC neurons form part of a later, secondary innervation to the cochlea.

The human olivocochlear system : Organization and development

The goals of the present study were to identify olivocochlear neurons in the human brainstem, to establish the time course of their early development and to compare the organization of the human olivocochlear system to that of other mammals. To accomplish these goals, we used immunohistochemistry for choline acetyltransferase (ChAT) and calcitonin gene-related peptide (CGRP) in postmortem brainstems of human subjects ranging in age from 16 fetal weeks to 17 years. By immunostaining, we identified two classes of cells in the superior olivary complex: both classes were seen to be present from the twenty-first fetal week to the seventeenth year. Neurons which are immunostained only for ChAT are located primarily in the dorsomedial, ventral and ventrolateral sectors of the periolivary region. These neurons are predominantly bipolar or multipolar cells, and are probably homologous to medial olivocochlear neurons in other species. A second population of cells is immunoreactive for both ChAT and CGRP. This population includes a cluster of mostly small oval neurons, located on the dorsal edge of the olivary complex, and a variable number of cells found along the margin of the lateral olivary nucleus. These ChAT- and CGRP-immunoreactive cells are likely to be homologous to the lateral olivocochlear system in other mammals. With increasing age, the dorsal cluster of small cells shifts from its original cap-like position over the lateral olivary nucleus to become an extended column of cells lying among the fibers of the olivocochlear bundle.

Developmental mRNA expression of the α10 nicotinic acetylcholine receptor subunit in the rat cochlea

A recently discovered alpha10 subunit of the nicotinic acetylcholine receptor (nAChR) family is believed to form a heteromeric receptor with the alpha9 nAChR subunit in auditory hair cells. In the present study, the alpha10 nAChR subunit expression in the developing and adult rat inner ear was analyzed by PCR and localized using isotopic in situ hybridization. Unlike the alpha9 subunit, the alpha10 subunit was not detected at embryonic day 18 (E18). From E21 through postnatal day 15 (P15), the alpha10 subunit was localized over both inner hair cell (IHC) and outer hair cell (OHC) regions, but in the mature cochlea detectable levels of alpha10 mRNA were found only over the OHC region. From E21 through adult ages, there was also a small but consistent basal to apical gradient of alpha10 expression; that is, higher levels in basal regions and lower levels in apical regions. Previously, we detected the alpha9 nAChR subunit over IHCs as early as E18 and throughout adult ages with a clear basal-apical gradient of expression. Our studies raise the question of whether the alpha9 and alpha10 subunits are differentially regulated during embryonic and postnatal development.

Development of the Inner Ear Efferent System

Roberts and Meredith (1992) wrote: “For more than forty years, the efferent supply to the mammalian ear provided by the olivocochlea bundle has been an enigma,” and this is still true today, in particular for the development of efferents. The inner ear efferents are so unique in their physiology, axonal course, and distribution that this adds to the mystery of their role in hearing and balance (Christopher Kirk and Smith 2003). However, analyzing the development of the vestibulocochlear efferent system may not only give us new insight into the development of this system but may also help to understand how the distribution and neurochemcial properties of the adult vestibulocochlear efferent system all come about.

Postnatal maturation of spiral ganglion neurons: A horseradish peroxidase study

Using an in vitro cochlear preparation from postnatal hamsters, spiral ganglion cells (SGCs) were labeled retrogradely following extracellular injections of HRP into the cochlear nerve. In 24 cochleae from hamsters between postnatal days (P) 0 and 10, the neuronal morphology of 201 SGCs and their peripheral axons were analyzed. From P 0 to 3, labeled SGCs had few distinguishable features. Although SGCs could be traced separately to inner hair cells (IHCs) and outer hair cells (OHCs), they all had roughly bipolar-shaped cell bodies. Approximately half of the labeled SGCs had peripheral axons that spiraled some distance before entering radial fiber bundles. From P 3 to 7, SGCs increased in size by nearly 30% and the number of SGCs with spiraling peripheral axons decreased to near zero. At P 10, the central axon diameter to peripheral axon diameter ratios distinguished two populations of SGCs. The hair-cell innervation patterns of SGCs also changed morphologically as a function of postnatal age. At P 0, radial fiber (RF) terminals of peripheral axons contacted as many as 8 IHCs; by P 3, RFs contacted typically one or two IHCs. The terminal portions of peripheral axons contacting OHCs did not show any appreciable spiral until P 2. By P 5, individual outer spiral fibers (OSFs) had greater spiral lengths underneath row-3 OHCs and the number of OHC contacts was also greatest for row-3 OSFs. These data suggest that SGCs undergo a systematic maturational process. Furthermore, the morphological differentiation of SGCs occurs after they have established separate inner and outer hair cell innervations.

Oncomodulin identifies different hair cell types in the mammalian inner ear

The tight regulation of Ca2+ is essential for inner ear function, and yet the role of Ca2+ binding proteins (CaBPs) remains elusive. By using immunofluorescence and reverse transcriptase‐polymerase chain reaction (RT‐PCR), we investigated the expression of oncomodulin (Ocm), a member of the parvalbumin family, relative to other EF‐hand CaBPs in cochlear and vestibular organs in the mouse. In the mouse cochlea, Ocm is found only in outer hair cells and is localized preferentially to the basolateral outer hair cell membrane and to the base of the hair bundle. Developmentally, Ocm immunoreactivity begins as early as postnatal day (P) 2 and shows preferential localization to the basolateral membrane and hair bundle after P8. Unlike the cochlea, Ocm expression is substantially reduced in vestibular tissues at older adult ages. In vestibular organs, Ocm is found in type I striolar or central hair cells, and has a more diffuse subcellular localization throughout the hair cell body. Additionally, Ocm immunoreactivity in vestibular hair cells is present as early as E18 and is not obviously affected by mutations that cause a disruption of hair bundle polarity. We also find Ocm expression in striolar hair cells across mammalian species. These data suggest that Ocm may have distinct functional roles in cochlear and vestibular hair cells. J. Comp. Neurol. 518:3785–3802, 2010.

Temporal and genetic influences on protection against noise-induced hearing loss by hypoxic preconditioning in mice

The protective benefits of hypoxic preconditioning (HPC) against permanent noise-induced hearing loss (NIHL) were investigated in mice. Hypoxia induced by exposure to 8% O2 for 4 h conferred significant protection against damaging broadband noise delivered 24-48 h later in male and female CBA/J (CBA) and CBA/CaJ mice. No protection was found in C57BL/6 (B6) mice, their B6.CAST-Cdh23(CAST) (B6.CAST) congenics, or in CBAxB6 F1 hybrid mice over the same interval, suggesting that the potential for HPC depends on one or a few autosomal recessive alleles carried by CBA-related strains, and is not influenced by the Cdh23 locus. Protection against NIHL in CBA mice was associated with significant up-regulation of hypoxia-inducible factor-1alpha (HIF-1alpha) within the organ of Corti, not found in B6.CAST. In both CBA and B6.CAST mice, some hypoxia-noise intervals shorter than 24 h were associated with exacerbation of NIHL. Cellular cascades underlying the early exacerbation of NIHL by hypoxia are therefore common to both strains, and not mechanistically linked to later protection. Elucidation of the events that underlie HPC, and how these are impacted by genetics, may lead to pharmacologic approaches to mimic HPC, and may help identify individuals with elevated risk of NIHL.

Applications of neuronal labeling techniques to the study of the peripheral auditory system

Horseradish peroxidase (HRP) techniques have recently been applied to the study of the peripheral auditory system. These techniques make it possible to trace single, auditory-nerve fibers from their peripheral terminals on sensory cells to their central terminations within the cochlear nucleus. For the large, myelinated axons of type-I neurons contacting inner hair cells, HRP can be injected intracellularly through the same electrode used to record neural activity, allowing correlations to be made between structure and function on a single-cell basis. From these data, a cochlear frequency map has been constructed, and morphological correlates of differences in spontaneous discharge have been identified. For the small, unmyelinated axons of type-II neurons contacting outer hair cells, single-unit recordings have not yet been made; however, these neurons have been labeled by gross extracellular injections of HRP. Extracellular injections have also been used to study the peripheral terminations of single efferent neurons projecting to the outer hair cell region. The afferent fibers to the outer hair cell (OHC) region contact primarily OHCs from the third row, while efferent fibers primarily contact cells from the first row. Single efferent fibers innervate OHCs over a significantly larger cochlear length than single afferent fibers of either type. Possible functional interpretations of these morphological differences are discussed.