Reciprocal Innervation of Outer Hair Cells in a Human Infant

Reciprocal synapses are characterized by the presence of both afferent and efferent types of synaptic specializations between two cells. They have been described at the neural poles of outer hair cells (OHCs) in humans with advanced age and two monkey species. Our objective was to study the innervation of the OHCs and determine if reciprocal synapses were present in a young (8-month-old infant) human subject. We studied the synaptic and cytoplasmic morphology of 162 nerve terminals innervating 29 OHCs using serial section transmission electron microscopy. Seventy-six percent of all OHCs were innervated by terminals with reciprocal synapses. This prevalence increased from the first toward the third row (p < 0.001), and 100% of OHCs in the third row demonstrated at least one reciprocal synapse. The prevalence of terminals with reciprocal synapses was higher in the human infant than in older human subjects and was very similar to what has been reported for the chimpanzee. Reciprocal synapses occur in sufficient numbers to be physiologically significant in primates. The nerve terminals were found to segregate into two groups on the basis of their cytoplasmic morphological characteristics: (1) vesicle-rich/neurofilament-poor (VR/NP) and (2) vesicle-poor/neurofilament-rich (VP/NR). All afferent and reciprocal terminals were of the VP/NR variety. The majority of the efferent terminals originated from VR/NP nerve fibers (classical olivocochlear morphology), but 23.5% of the efferent terminals were VP/NR. The hypothesis that peripheral processes of type II spiral ganglion cells form classical afferent, reciprocal, and a number of purely presynaptic terminals on OHCs is discussed. The presence of different types of synaptic specializations on OHCs formed by nerve fibers of the same type (VP/NR) suggests the existence of reciprocal neuronal circuits between OHCs sharing the dendritic arborization of a type II spiral ganglion cell.     

Distribution of the Na,K-ATPase α Subunit in the Rat Spiral Ganglion and Organ of Corti

Processing of sound in the cochlea involves both afferent and efferent innervation. The Na,K-ATPase (NKA) is essential for cells that maintain hyperpolarized membrane potentials and sodium and potassium concentration gradients. Heterogeneity of NKA subunit expression is one mechanism that tailors physiology to particular cellular demands. Therefore, to provide insight into molecular differences that distinguish the various innervation pathways in the cochlea, we performed a variety of double labeling experiments with antibodies against three of the α isoforms of the NKA (NKAα1–3) and markers identifying particular subsets of neurons or supporting cells in whole mount preparations of the organ of Corti and spiral ganglion. We found that the NKAα3 is abundantly expressed within the membranes of the spiral ganglion somata, the type I afferent terminals contacting the inner hair cells, and the medial efferent terminals contacting the outer hair cells. We also found expression of the NKAα1 in the supporting cells that neighbor the inner hair cells and express the glutamate transporter GLAST. These findings suggest that both the NKAα1 and NKAα3 are poised to play an essential role in the regulation of the type I afferent synapses, the medial efferent synapses, and also glutamate transport from the afferent-inner hair cell synapse.     

A quantitative analysis of the afferent innervation of the organ of corti in guinea pig.

A quantitative analysis of the afferent innervation of the organ of Corti was made on normal and vestibular nerve-sectioned guinea pigs. Section of the vestibular nerve at the internal auditory meatus provided an efficient means of eliminating the efferent innervation to the cochlea without significant loss of afferent fibres. Nerve counts on normal and de-efferented animals revealed that about 10-15 % of the cochlear afferent innervation supplies the outer hair cells. The remaining 85-90% of afferent fibres innervate the inner hair cells. As in cats, all tunnel spiral bundle fibres and upper tunnel crossing fibres were efferent to outer hair cells. Since unmyelinated fibres in the osseous spiral bundle were not counted, quantitative analysis of the efferent innervation to inner hair cells could not be made. However, a significant loss of myelinated fibres in the osseous spiral lamina after vestibular nerve section confirms that many myelinated efferent fibres are present in this region.     

Serial section reconstruction of the neural poles of hair cells in the human organ of corti. II. outer hair cells

Study of the anatomy of the cochlea, and in particular the morphology of synaptic relationships between hair cells and cochlear neurons, is essential for elucidation of the mechanisms of transduction of mechanical acoustic signals into electrical neural events. Because considerable gaps remain in our understanding of the microscopic anatomy of these synapses, particularly in the human, a reconstruction of the neural pole of outer hair cells of the human organ of Corti was performed. The data are based on 577 serial sections from the basal turn and 368 sections from the middle turn. This provided complete data on 11 and partial data on 9 outer hair cells. Terminal size of afferent fibers on outer hair cells was much more uniform than that found at the base of inner hair cells. Only small bouton-like terminals were found. Branching of afferent fibers was also seen at the base of outer hair cells. Each outer hair cell received approximately two to eight afferent nerve terminals. Multiple synaptic contacts between a single afferent terminal and an outer hair cell were common. Junctional membrane specialization consisted of synapses, desmosomes, coated vesicles and arrays of microtubules and membrane cisternae. Specialization at synapses consisted of asymmetrical membrane thickening. At outer hair cells the presynaptic membrane was thicker than the postsynaptic membrane. At inner hair cells the converse was true. At outer hair cells 35% of synapses had presynaptic bodies, compared to 83% of synapses at inner hair cells. Reciprocal synapses, with both hair cell to neuron and neuron to hair cell polarities, were found only on outer hair cells. Vesiculated efferent terminals were common at the base of outer hair cells. Both axosomatic and axodendritic efferent synapses were found. In addition, the same efferent fibers were found to synapse both on an outer hair cell and on an afferent dendrite. One example of a probable dendro-dendritic synapse in the outer spiral bundle is presented.     

Reciprocal Synapses Between Outer Hair Cells and their Afferent Terminals: Evidence for a Local Neural Network in the Mammalian Cochlea

Cochlear outer hair cells (OHCs) serve both as sensory receptors and biological motors. Their sensory function is poorly understood because their afferent innervation, the type-II spiral ganglion cell, has small unmyelinated axons and constitutes only 5% of the cochlear nerve. Reciprocal synapses between OHCs and their type-II terminals, consisting of paired afferent and efferent specialization, have been described in the primate cochlea. Here, we use serial and semi-serial-section transmission electron microscopy to quantify the nature and number of synaptic interactions in the OHC area of adult cats. Reciprocal synapses were found in all OHC rows and all cochlear frequency regions. They were more common among third-row OHCs and in the apical half of the cochlea, where 86% of synapses were reciprocal. The relative frequency of reciprocal synapses was unchanged following surgical transection of the olivocochlear bundle in one cat, confirming that reciprocal synapses were not formed by efferent fibers. In the normal ear, axo-dendritic synapses between olivocochlear terminals and type-II terminals and/or dendrites were as common as synapses between olivocochlear terminals and OHCs, especially in the first row, where, on average, almost 30 such synapses were seen in the region under a single OHC. The results suggest that a complex local neuronal circuitry in the OHC area, formed by the dendrites of type-II neurons and modulated by the olivocochlear system, may be a fundamental property of the mammalian cochlea, rather than a curiosity of the primate ear. This network may mediate local feedback control of, and bidirectional communication among, OHCs throughout the cochlear spiral.     

Anatomy of Cochlear Innervation

In this review of cochlear innervation, the differences in the innervation of outer and inner hair cells are emphasized. Of the afferent neurons, 90 to 95 per cent are large, myelinated type I neurons, exclusively connected in an essentially radial unbranched manner to the inner hair cells; 5 to 10 per cent are small, mostly unmyelinated type II neurons connected to the outer hair cells with considerable spiral extension and branching. The few small type II neurons, with their thin unmyelinated axons, probably have a minor functional importance for centripetal information transfer. The functional emphasis of the outer hair cell system is likely at the level of the receptor cells where the outer hair cells monitor receptor function. The efferent innervation also consists of at least two types of neurons. Small neurons from the lateral superior olivary nucleus project to the inner hair cell area in a predominantly homolateral fashion, making almost exclusively synaptic contacts with the afferent dendrites associated with the inner hair cells. Larger neurons from the medial nucleus of the trapezoid body and periolivary nucleus provide the abundant efferent nerve supply of the outer hair cells, predominantly contralateral. They have mostly large synaptic contacts, and, in some species exclusively, with the receptor cells, indicating again the functional emphasis of the outer hair cell system at the receptor cell level.     

Aspartate aminotransferase immunoreactivity in cochlea of guinea pig

The distribution of aspartate aminotransferase-like immunoreactivity in the cochlea of the guinea pig was studied at the light microscopy level. Indirect immunofluorescence histochemistry using antisera against cytoplasmic aspartate aminotransferase prepared from pig heart was applied to surface preparations of the organ of Corti and cryostat sections of the cochlea. In the modiolus, immunofluorescence was localized to spiral ganglion cells and myelinated fibers of the auditory nerve and intraganglionic spiral bundles. In the organ of Corti, immunofluorescence was seen in upper tunnel crossing fibers and at the base of outer hair cells, following a distribution similar to that of the efferent innervation of the outer hair cells. Weak immunofluorescence was seen in the inner spiral bundle and tunnel spiral bundle, but was not present in all preparations. Immunofluorescence was not seen in inner hair cells, nor at the base of inner hair cells, and may have been absent from outer hair cells.It is concluded that spiral ganglion cells and myelinated auditory nerve axons contain aspartate aminotransferase-like immunoreactivity; such immunoreactivity has previously been determined in auditory nerve endings in the cochlear nucleus. Olivocochlear neurons that innervate outer hair cells also contain such immunoreactivity while other cochlear efferents contain little or none.     

A study of cochlear innervation in the young cat with the Golgi method

Individual afferent and efferent nerve fibers were identified and traced in Golgi-impregnated cochleas of cats from newborn to one month old. Afferent radial fibers project radially without varicoshies to terminate at the base of one or two inner hair cells. Outer spiral fibers have both radial and spiral orientations within the organ of Corti, do not form varicosities while crossing the base of the tunnel, and spiral for long distances in the outer spiral bundles. They contact many outer hair cells of more than one row both en passant and by small terminal branchlets. Two separate groups of efferent fibers are identifiable. Thin efferent fibers with many large varicosities spiral for long distances in the inner and tunnel spiral bundles; varicosities in the inner spiral bundle may contact radial afferent fibers or hair cells, depending on age. Thick radial efferent fibers course radially through the tunnel spiral bundle and across the upper part of the tunnel, often in fascicles. They contact a few outer hair cell bases by large terminals. The spiral expanse of the terminals is limited. These fibers are most common in the more basal turns of the organ.The present results confirm the anatomical separation of radial and spiral afferent fiber systems and identify two separate efferent populations beyond the neonatal period in the cat. The major features of afferent innervation discernible in Golgi-impregnated cochleas are present at birth, although some simplification of afferent fibers probably occurs during the first postnatal week. In contrast, the efferent fiber pattern undergoes important changes during the first few weeks after birth. In mature animals, the fine spiral efferents probably contact only afferent fibers, whereas the thick radial efferents may contact both outer hair cells and spiral afferent fibers. The possibility that some individual efferents branch to both inner and outer hair cell regions in the older cats cannot be ruled out.     

Innervation densities of inner and outer hair cells of the human organ of Corti. Evidence for auditory neural degeneration in a case of Usher's syndrome

The innervation densities, i.e. the number of afferent endings per inner and outer hair cell, number of synaptic specializations per inner hair cell and the number of efferent endings per outer hair cell, were evaluated by serial section electron microscopy in the normal human inner ear and a case of Usher's syndrome (retinitis pigmentosa and sensorineural hearing loss). There was a significant decrease in the innervation density in Usher's syndrome as compared to normative controls, which could not be explained by the decrease in density of spiral ganglion cells. This was interpreted as suggesting a decrease in the number of neural processes and endings within the dendritic arborization of remaining spiral ganglion cells in this disorder.     

Neuroanatomical basis of cochlear coding mechanisms.

The afferent cochlear neurons to outer and inner hair cells differ not only in their relative numbers (95% to inner hair cells and only 5% to outer hair cells) and distribution pattern (great convergence for outer hair cells and divergence for inner hair cells) but also in their degeneration behaviour and metabolism. Some special afferent fibres seem to exist. The afferent neurons present three types of ganglion cells in the spiral ganglion with no morphological evidence for an extensive direct interaction between afferent neurons of the outer and the inner hair cell system at any level in the cochlea and even their efferent nerve supply is essentially separated.     

Horseradish peroxidase injection of physiologically characterized afferent and efferent neurones in the guinea pig spiral ganglion

Single afferent and efferent neurones in the guinea pig spiral ganglion were injected with horseradish peroxidase. They could be recovered in subsequent histological processing and traced from the injection site in the ganglion to their final termination in the organ of Corti. All responsive primary afferents innervated the inner hair cells (58 neurones). One outer spiral fibre innervating the outer hair cells was recovered. This cell was non-spiking and unresponsive to acoustic stimulation. Neurones having properties previously attributed to cochlear efferents, terminated on the outer hair cells in regions of the cochlea consistent with their characteristic frequencies.     

Ultrastructural findings in a case of Menière's disease

The temporal bones of an individual with documented unilateral Meniere's disease were prepared for light and electron microscopy. A morphometric analysis was performed on hair cells, spiral ganglion cells, dendritic fibers in the osseous spiral lamina, afferent and efferent endings, and afferent synaptic contacts. In the ear with Meniere's disease, we found hair cell damage, including disruption of the cuticular bodies and basalward displacement of some outer hair cells. There was no significant difference in the number of hair cells or spiral ganglion cells on the two sides. There was a significant decrease, however, in the number of afferent nerve endings and afferent synapses at the base of both inner and outer hair cells in the ear with Meniere's disease as compared to the contralateral ear.     

Localization of gamma-aminobutyric acid A receptor subunits in the rat spiral ganglion and organ of Corti

Gamma-aminobutyric acid (GABA) is thought to be the major inhibitory neurotransmitter in the central nervous system. Although it is distributed in the olivo-cochlear bundles, which constitute the mammalian cochlear efferent system, its function in the cochlea is still obscure. In this study, we investigated the localization of GABAa receptor subunits (alpha1-6, beta1-3, gamma) in the rat cochlea in order to determine the role of GABA in the cochlea. Most spiral ganglion cells were intensely immunolabeled with all the anti-GABAa receptor subunit antibodies. In the organ of Corti, punctate immunoreactivities were observed in inner hair cell regions corresponding to the distribution of GABA. These data suggest that GABAa receptor was present in afferent nerve terminals in inner hair cell regions, and that GABA regulated afferent nerve transmission contacting efferent nerve endings by means of the axo-dendritic synapse function.     

Reconstructions of efferent fibers in the postnatal hamster cochlea

It is well known that adult-like physiological functioning of the mammalian postnatal cochlea occurs coincidentally with the presence of efferent synapses on outer hair cells (OHCs). This study described the cochlear innervation patterns of thick efferent fibers traveling in the vestibular nerve in postnatal hamsters ranging in age from day zero to day 10. At least three kinds of efferent fibers were labeled via an in vitro horseradish peroxidase (HRP) technique: varicose, thin efferents; nonvaricose, thin efferents; and nonvaricose, thick efferents. Nonvaricose thick efferents were reconstructed from the basal third of the cochlea. Reconstructed efferent fibers traversed in the intraganglionic spiral bundle (IGSB) on the peripheral edge of the spiral ganglion and branched profusely in the osseous spiral lamina (OSL). From day zero to day five, large (greater than 1.0 microns) diameter nonvaricose efferent fibers gave rise to branches that either terminated underneath inner hair cells or appeared to end blindly in the OSL. Efferent fibers also had branches that traveled in the inner spiral bundle (ISB) and tunnel spiral bundle (TSB). In cochleae from hamsters six to eight days old, some thin and thick diameter efferent fibers contacted both inner and outer hair cells. By the tenth day, large diameter fibers traveled radially across the tunnel of Corti to terminate on one to five OHCs. As early as day seven, large diameter fibers also appear to terminate preferentially on OHCs in row one. These observations are consistent with the notion that the end of the first postnatal week represents a critical period in the formation of adult-like synapses on the OHCs. The data also suggest a developmental transition period when efferent fibers contact both hair cell types before contacting OHCs separately.     

Serial Section Reconstruction of the Neural Poles of Hair Cells in the Human Organ Of Corti. I. Inner Hair Cells

Study of the anatomy of the cochlea, and in particular the morphology of synaptic relationships between hair cells and cochlear neurons, is essential for elucidation of the mechanisms of transduction of mechanical acoustic signals into electrical neural events. Because considerable gaps remain in our understanding of the microscopic anatomy of these synapses, particularly in the human, a reconstruction of the neural pole of inner hair cells of the human organ of Corti was performed. The data are based on 526 serial sections from the basal turn (10 mm region) and 356 serial sections from the middle turn (26 mm region). This provided complete data on 3 and partial data on 5 inner hair cells. Afferent terminals on inner hair cells were variable in size, ranging 1 to 20 µm in diameter. Branching of large fibers to produce multiple terminals innervating from 1 to 3 inner hair cells was common. Each inner hair cell received approximately 6 to 8 different nerve terminals. In addition, each terminal possessed a variable number of synaptic contacts. Junctional membrane specialization consisted of synapses, desmosomes, coated vesicles and arrays of microtubules and membrane cisternae. Specialization at synapses consisted of asymmetrical membrane thickening. At inner hair cells the postsynaptic membrane was thicker than the presynaptic membrane. Eighty-three percent of synapses had presynaptic bodies. Vesiculated efferent terminals synapsed on afferent fibers at the base of inner hair cells, but never directly on the inner hair cell. These anatomical data demonstrate distinct differences between the human and animal inner ear, which are important in the interpretation of neurophysiological data in animals and the formulation of hypotheses that involve assumptions crossing species.     

Immunohistochemical distribution and fine localization of the gamma-aminobutyric acid in the organ of Corti of normal guinea pigs]

This paper presents the distribution and fine localization of the gamma-aminobutyric acid (GABA) in the Corti's organ of normal guinea pigs by using immunohistochemical technique (ABC-GDN method) and immunoelectron microscopy. GABA-IR was found in axons of efferent neurons in all turns of the cochlear spiral, but no positive endings may be found in the apical. The positive immunoreactive products were seen in the efferent components, including inner spiral bundle, tunnel spiral bundle, tunnel-crossing fibers, and large nerve endings on outer hair cell bases. Some of the GABA-IR negative outer hair cells could be seen between the positive endings. One GABA-IR positive nerve fiber could form synapse with six or seven outer hair cell bases. The GABA-IR positive efferent endings, negative efferent endings and negative afferent endings could be seen on the out hair cell bases with transmission electronic microscope. The GABA-IR positive efferent endings and negative afferent endings could form the neuraxon-neurodendron synapse on the inner hair cells bases. These morphological distribution suggests that the GABA may be one of the cochlear efferent neurotransmitter or modulator.     

Distribution of frequenin in the mouse inner ear during development, comparison with other calcium-binding proteins and synaptophysin

Frequenin is a calcium-binding protein previously implicated in the regulation of neurotransmission. We report its immunocytochemical detection in the mouse inner ear, in the adult, and during embryonic (E) and postnatal (P) development. The distribution of frequenin was compared with those of other calcium-binding proteins (calbindin, calretinin, parvalbumin) and synaptophysin. In the adult mouse inner ear, frequenin immunostaining was observed in the afferent neuronal systems (vestibular and cochlear neurons, their processes and endings) and in the vestibular and cochlear efferent nerve terminals. Frequenin colocalized with synaptophysin in well characterized presynaptic compartments, such as the vestibular and cochlear efferent endings, and in putative presynaptic compartments, such as the apical part of the vestibular calyces. Frequenin was not found in vestibular hair cells and in cochlear inner and outer hair cells. During development, frequenin immunoreactivity was first detected on E11 in the neurons of the statoacoustic ganglion. On E14, frequenin was detected in the afferent neurites innervating the vestibular sensory epithelium, along with synaptophysin. On E16, frequenin was detected in the afferent neurites below the inner hair cells in the organ of Corti. The timing of frequenin detection in vestibular and cochlear afferent neurites was consistent with their sequences of maturation, and was earlier than synaptogenesis. Thus in the inner ear, frequenin is a very early marker of differentiated and growing neurons and is present in presynaptic and postsynaptic compartments.     

Hair cell innervation in the fetal human cochlea

Twelve cochleas from human fetuses ranging in age from 13 to 22 gestational weeks were investigated by transmission electron microscopy in order to follow the development of hair cell innervation. First, we were able to confirm the two general gradients of cochlear maturation, i.e. 'internal-to-external' and 'base-to-apex'. In the 14th week of gestation, the inner hair cell pattern of innervation was almost mature, with well formed afferent synapses and axo-dendritic efferent contacts. At the outer hair cell level, only afferents, probably both spiral and radial ones, were present up to week 16. At week 22, axo-somatic synapses were observed between the medial efferents and the outer hair cells, but they were not yet completely mature. At this stage the myelination had begun within the spiral lamina fibres. These findings confirmed that the different stages of synaptogenesis in the human cochlea are similar to that described in other mammals, and thus the same kind of functional relationships could be proposed.     

Regenerated Synapses Between Postnatal Hair Cells and Auditory Neurons

Regeneration of synaptic connections between hair cells and spiral ganglion neurons would be required to restore hearing after neural loss. Here we demonstrate by immunohistochemistry the appearance of afferent-like cochlear synapses in vitro after co-culture of de-afferented organ of Corti with spiral ganglion neurons from newborn mice. The glutamatergic synaptic complexes at the ribbon synapse of the inner hair cell contain markers for presynaptic ribbons and postsynaptic densities. We found postsynaptic density protein PSD-95 at the contacts between hair cells and spiral ganglion neurons in newly formed synapses in vitro. The postsynaptic proteins were directly facing the CtBP2-positive presynaptic ribbons of the hair cells. BDNF and NT-3 promoted afferent synaptogenesis in vitro. Direct juxtaposition of the postsynaptic densities with the components of the preexisting ribbon synapse indicated that growing fibers recognized components of the presynaptic sites. Initiation of cochlear synaptogenesis appeared to be influenced by glutamate release from the hair cell ribbons at the presynaptic site since the synaptic regeneration was impaired in glutamate vesicular transporter 3 mutant mice. These insights into cochlear synaptogenesis could be relevant to regenerative approaches for neural loss in the cochlea.