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.     

Cochlear ganglion cells in the alligator lizard

Two types of ganglion cells are present in the cochlear ganglion of the alligator lizard. Myelinated cells are the predominant type but a small population of unmyelinated cells is also present. Ganglion cells are reconstructed and morphometrically analyzed from serial light and electron micrographs. The results are interpreted to indicate that the unmyelinated ganglion cells are degenerating neurons. No normal population of unmyelinated nerve fibers was found. The anatomy of the cochlear nerve of the lizard is described and compared with the mammalian auditory nerve.     

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.     

Innervation of the apical turn of the human cochlea: a light microscopic and transmission electron microscopic investigation.

HYPOTHESIS: A light and transmission electron microscopic investigation of the apical turn of a freshly fixed human cochlea. BACKGROUND: Our knowledge about the human cochlea rests to a large extent on animal species research. An opportunity to obtain tissue from normal-hearing persons occurs during surgery for life-threatening petroclival meningioma. This study presents detail on the morphology and innervation of the apical part of the human cochlea using light microscopic and transmission electron microscopic level sectioning. METHODS: The tissue was histologically processed after removal during petroclival meningioma surgery. The cochlea was serially sectioned perpendicularly to its long axis, and at regular distances semithin sections were reembedded and prepared for transmission electron microscopy. Nerve fibers/fascicles were traced from the area of the spiral ganglion to the level of the inner hair cells, and a cochleotopic "map" of the cochlear nerve supplying the apical portion was constructed. RESULTS: The apical turn was found to be innervated by 3,694 myelinated nerve fibers representing approximately 10% of the total number of fibers innervating the cochlea. The total number of unmyelinated nerve fibers was 513. The majority belonged to the efferent olivocochlear system and the intraganglionic spiral bundle or represented Type II afferent neurons innervating outer hair cells. CONCLUSION: The significance of the anatomic findings in relation to cochlear implantation is discussed.     

Uptake of gentamicin by vestibular efferent neurons and superior olivary complex after transtympanic administration in guinea pigs

Transtympanic administration of gentamicin is a widely accepted and effective approach for treating patients with intractable vertigo. Previous studies have demonstrated the uptake, distribution and effects of gentamicin in peripheral vestibular and cochlear structures after transtympanic injection. However, little is known about whether transtympanically administered gentamicin is trafficked into more central auditory and vestibular structures and its effect on these structures. In this study, we used immunofluorescence to determine the distribution of gentamicin within the auditory and vestibular brainstem. We observed gentamicin immunolabeling bilaterally in the vestibular efferent neurons, and in the superior olivary complex, and ipsilaterally in the cochlear nucleus 24h after transtympanic administration of gentamicin, and that the drug could still be detected in these locations 30 days after injection. In contrast, no gentamicin labeling was detected in the vestibular nuclear complex. In the vestibular efferent neurons and superior olivary complex, gentamicin labeling was detected in the cytoplasm and cell processes, while in the cochlear nucleus gentamicin is mainly localized outside and adjacent to the cell bodies of neurons. Nerve fibers in cochlear nucleus, root of eighth nerve, as well as descending pathways from the superior olivary complex, are also immunolabeled with gentamicin continuously. Based on these data, we hypothesize that retrograde axonal transport of gentamicin is responsible for the distribution of gentamicin in these efferent nuclei including vestibular efferent neurons and superior olivary complex and anterograde axonal transport into the ipsilateral cochlear nucleus.     

Tyrosine Hydroxylase Expression in Type II Cochlear Afferents in Mice

Acoustic information propagates from the ear to the brain via spiral ganglion neurons that innervate hair cells in the cochlea. These afferents include unmyelinated type II fibers that constitute 5 % of the total, the majority being myelinated type I neurons. Lack of specific genetic markers of type II afferents in the cochlea has been a roadblock in studying their functional role. Unexpectedly, type II afferents were visualized by reporter proteins induced by tyrosine hydroxylase (TH)-driven Cre recombinase. The present study was designed to determine whether TH-driven Cre recombinase (TH-2A-CreER) provides a selective and reliable tool for identification and genetic manipulation of type II rather than type I cochlear afferents. The “TH-2A-CreER neurons” radiated from the spiral lamina, crossed the tunnel of Corti, turned towards the base of the cochlea, and traveled beneath the rows of outer hair cells. Neither the processes nor the somata of TH-2A-CreER neurons were labeled by antibodies that specifically labeled type I afferents and medial efferents. TH-2A-CreER-positive processes partially co-labeled with antibodies to peripherin, a known marker of type II afferents. Individual TH-2A-CreER neurons gave off short branches contacting 7–25 outer hair cells (OHCs). Only a fraction of TH-2A-CreER boutons were associated with CtBP2-immunopositive ribbons. These results show that TH-2A-CreER provides a selective marker for type II versus type I afferents and can be used to describe the morphology and arborization pattern of type II cochlear afferents in the mouse cochlea.     

Efferent innervation in the goldfish saccule examined by acetylcholinesterase histochemistry

In contrast to the abundance of information available regarding the anatomy and physiology of afferents within the goldfish saccule, the efferent system of this auditory endorgan has been scarcely studied morphologically. In this study, acetylcholinesterase histochemistry with diaminobenzidine enhancement was used to describe the morphology of efferents. Under light microscopy, labeled fibers appeared in the distal portion of the saccular nerve, penetrated the basement membrane and formed a horizontal mesh-like plexus near the base of hair cells. Many vertical branchlets with terminal swellings protruded upward toward hair cells from the plexus. Under electron microscopy, dense extracellular labeling was present around efferent terminals, which often formed clusters on hair cells. Labeling was also present around unmyelinated fibers of passage within the sensory epithelium and the distal saccular nerve. These fibers contained coarse microtubules and small vesicles, and often ran in a bundle with other similar fibers. Based on their position within the epithelium, histochemistry and ultrastructural characteristics, these fibers were concluded to be efferents. These fibers became myelinated and unlabeled in the proximal saccular nerve. These results suggest that acetylcholinesterase can be a marker of entire distal unmyelinated portions of efferent fibers and demonstrated abundant efferent innervation in the goldfish saccule.     

Neurotoxicity of kainic acid in the rat cochlea during early developmental stages

Summary The neurotoxic effect of kainic acid (KA) was investigated by electron microscopy in rat cochleas at two developmental stages: 17 days of gestation (17 G) and postnatal day 1 (PN 1). In each animal, one cochlea was injected with 1 nmol KA diluted into 2 ml artificial perilymph, while the other cochlea was only injected with artificial perilymph as a control. Ten minutes later, the cochleas were perfused with fixative, removed and processed for electron microscopy. The KA injection resulted in marked swelling of the majority of afferent fibers, i.e. the peripheral processes of spiral ganglion neurons. In the 17 G cochlea, swollen fibers were traced from the perikarya to the undifferentiated otocyst epithelium. Following birth, swollen afferents in the PN 1 cochlea were in contact with both inner (IHCs) and outer hair cells (OHCs), which were now differentiated. At both stages of development, a subclass of small afferent nerves were unaffected. At PN 1, the KA-insensitive afferents only contacted the OHCs. These fibers probably belong to the spiral system of afferents and are related to type II spiral ganglion cells. Conversely, KA-sensitive afferents probably belong to the radial system, related to type I spiral ganglion cells. This system is specific for IHCs in adult cochleas and appears to innervate both IHCs and OHCs at early developmental stages. These findings also indicate that KA neurotoxicity appears very early in the cochlea, at a prenatal time (17 G) before the presynaptic partners of afferent terminals (namely the IHCs) are differentiated. Thus, the neurotoxicity caused seems to be mediated via receptors located on the postsynaptic fiber and is widely distributed, as an immediate effect is visualized along the entire length of the unmyelinated peripheral afferent neurite.     

The Olivocochlear Reflex Strength and Cochlear Sensitivity are Independently Modulated by Auditory Cortex Microstimulation

In mammals, efferent projections to the cochlear receptor are constituted by olivocochlear (OC) fibers that originate in the superior olivary complex. Medial and lateral OC neurons make synapses with outer hair cells and with auditory nerve fibers, respectively. In addition to the OC system, there are also descending projections from the auditory cortex that are directed towards the thalamus, inferior colliculus, cochlear nucleus, and superior olivary complex. Olivocochlear function can be assessed by measuring a brainstem reflex mediated by auditory nerve fibers, cochlear nucleus neurons, and OC fibers. Although it is known that the OC reflex is activated by contralateral acoustic stimulation and produces a suppression of cochlear responses, the influence of cortical descending pathways in the OC reflex is largely unknown. Here, we used auditory cortex electrical microstimulation in chinchillas to study a possible cortical modulation of cochlear and auditory nerve responses to tones in the absence and presence of contralateral noise. We found that cortical microstimulation produces two different peripheral modulations: (i) changes in cochlear sensitivity evidenced by amplitude modulation of cochlear microphonics and auditory nerve compound action potentials and (ii) enhancement or suppression of the OC reflex strength as measured by auditory nerve responses, which depended on the intersubject variability of the OC reflex. Moreover, both corticofugal effects were not correlated, suggesting the presence of two functionally different efferent pathways. These results demonstrate that auditory cortex electrical microstimulation independently modulates the OC reflex strength and cochlear sensitivity.     

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.     

Effects of section of the medial efferent tracts (crossed and uncrossed) on cochlear frequency selectivity

The cochlear innervation of guinea pigs was sectioned midway between the midline and the external side of the cochlear nucleus, in a rostrocaudal direction at the level of the floor of the fourth ventricle, to study the effects of medial efferent pathways on cochlear frequency selectivity estimated with tuning curves of single auditory nerve fibers. Single-unit tuning curves were affected by this type of efferent sectioning. Thus, the ipsi-lateral efferent system seems to be involved, through a tonic action, in the mechanisms responsible for high frequency cochlea selectivity.     

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.     

"Neural" responses to acoustic stimulation after destruction of cochlear hair cells.

Electrophysiological and histological observations in guinea pig's cochleas after amikacin treatment (14 X 450 mg/kg) confirm the results obtained in a former experiment: clear, short-latency, click-evoked responses were recorded in cochleas with only very few hair cells remaining at the extreme apex. Detailed analysis of these responses strongly indicates a neural origin and confirms their low-frequency sensitivity. Careful histological observations confirm the extensive hair cell loss and the preservation of nerve fibers in the remnants of the organ of Corti and of the vestibular sense organs. These results suggest that the acoustical vibrations either stimulate the vestibular receptors or act directly or through some kind of mechano-electrical transduction on the remaining cochlear nerve fibers.     

Physiology, pharmacology and plasticity at the inner hair cell synaptic complex

This report summarizes recent neuropharmacological data at the IHC afferent/efferent synaptic complex: the type of Glu receptors and transporter involved and the modulation of this fast synaptic transmission by the lateral efferents. Neuropharmacological data were obtained by coupling the recording of cochlear potentials and single unit of the auditory nerve with intra-cochlear applications of drugs (multi-barrel pipette). We also describe the IHC afferent/efferent functioning in pathological conditions. After acoustic trauma or ischemia, acute disruption of IHC-auditory dendrite synapses are seen. However, a re-growth of the nerve fibres and a re-afferentation of the IHC were completely done 5 days after injury. During this synaptic repair, multiple presynaptic bodies were commonly found, either linked to the membrane or "floating" in ectopic positions. In the meantime, the lateral efferents directly contact the IHCs. The demonstration that NMDA receptors blockade delayed the re-growth of neurites suggests a neurotrophic role of NMDA receptors in pathological conditions.     

Serotonergic innervation of the inner ear: is it involved in the general physiological control of the auditory receptor?

The auditory pathway of mammals is composed of two complementary ascending afferent and descending efferent independent systems. The brainstem nuclei and cochlear projections for these systems are now well-known. In addition, a highly conspicuous distribution for serotonergic fibers was recently reported. This study focused on these serotonergic fibers and their neurons of origin. We identified several different types of serotonergic brainstem neurons surrounding the superior olivary complex and around the periolivary nuclei. Even though the 5-hydroxytryptamine (5-HT) efferent cochlear innervation originates in the periolivary area of the superior olivary complex system projecting to the cochlea, it is not involved in the transduction of pure tones during auditory processing. However, recent findings, after cochlear blockade of serotonin transporters, strongly suggested that this neuroactive substance has an important turnover within the auditory receptor. The presence of a conspicuous peripheral nerve distribution together with a particular brainstem origin could define a complex role for this innervation. Therefore, 5-HT fibers projecting to the cochlea might be involved, as in other parts of the auditory pathway, in alertness, attention, control of sleep or wakefulness cycles, and state of urgency prior to the transduction processing at the auditory receptor. A lack, or reduction, of the function of these fibers could result in pathological alterations.     

Transient evoked otoacoustic emissions after vestibular nerve section in chinchillas

Transient evoked otoacoustic emissions are believed to be sensitive to the effects of the cochlear efferent system. The most well-known function of this system is inhibitory on cochlear response. It has been demonstrated that crossed medial efferent system section produces inhibitory control of the outer hair cells mechanisms responsible for non-linear transient evoked otoacoustic emissions generation. However, we suppose that the uncrossed medial efferent system plays a role in outer hair cell function too. We recorded the non-linear part of transient evoked otoacoustic emissions in 17 chinchillas before and after section of the vestibular nerve (crossed and uncrossed fibers). Responses at frequencies bands centered on 0.8, 1.6, 2.4, 3.2 and 4.0 kHz, as well as total emission responses, were analyzed. After vestibular nerve section, there were significant increases in the amplitudes of the 2.4- and 4.0 kHz responses and of the total response. These results indicate that the medial efferent system is important to maintain normal cochlear mechanics. Uncrossed medial efferent system and lateral efferent system seem to be not important in maintaining normal cochlear mechanics.     

Regeneration in the VIII nerve.

After transection of the VIII nerve in the inner meatus, 95% of the cochlear neurons and practically all efferent fibres degenerate and disappear. In long-term survivals, different phenomena indicate the regeneration properties of many, probably efferent or vestibular nerve fibres. The few remaining afferent neurons react to the lesion of their central axon with enormous proliferations of their peripheral branches in the organ of Corti.     

The origin of efferent labyrinthine fibres: a comparative study in vertebrates

Parent cells of efferent acoustic and efferent vestibular fibers were determined anatomically in all classes of vertebrates by use of retrograde axonal transport of horseradish peroxidase. These neurons were found in the brainstem, in particular in the reticular formation. In the goldfish, efferent labyrinthine neurons could be demonstrated in a medial position (lateral to the fasciculus longitudinalis medialis). In the frog, efferent neurons appeared more lateral, dorsomedial to the facial motor nucleus. In reptiles and birds, efferent acoustic neurons separate from efferent vestibular neurons. In mammals, efferent vestibular neurons are located more dorsally, lateral to the genu of the facial nerve. Efferent acoustic neurons take their origin from the superior olivary complex.     

Response of the cochlear nucleus neurons of the cat to tone-burst-trains

Although both posteroventral cochlear nucleus (PVCN) and dorsal cochlear nucleus (DCN) are innervated by the descending branch of auditory nerve fibers, their intrinsic morphological organizations are so different that their physiological roles are expected to be different in signal processing. Temporal information coding of acoustic signals in the cochlear nucleus was examined by using stimuli of "tone-burst-trains (TBT)". Responses of cochlear nucleus neurons of anesthetized cats were recorded either intracellularly or extracellularly. Responses of the neurons to TBT stimuli were classified into "adaptive type" and "non-adaptive type". The "adaptive type" neurons were mainly recorded from PVCN. Responses of these neurons to TBT stimuli decayed exponentially, because of short-term adaptation, in the subsequent tone-bursts. These neurons faithfully preserve the adaptative behavior of auditory nerve fibers. On the contrary, the "non-adaptive type" neurons were mainly found in DCN. They showed variety of responses to TBT stimuli including facilitation, disinhibition and inhibition depending on duration and/or interval of tone-bursts. Our results suggest that some "non-adaptive type" neurons, showing facilitative and/or inhibitory responses to TBT stimuli, act as temporal filters that extract temporal information from acoustic signals.     

Transganglionic transport of D-aspartate from cochlear nucleus to cochlea--a quantitative autoradiographic study

This study concerns the connections of the inner and outer hair cells and the different types of ganglion cells of the mammalian cochlea with the central nervous system by making use of their putative neurotransmitters. D-[3H]Aspartate (D-ASP), a putative marker for glutamatergic neurons, was injected into the cochlear nucleus of cats and guinea pigs and the cochleas prepared for light microscopic autoradiography after varying survival times. A quantitative, statistical autoradiographic method is described. Grain counts per unit area were made for each of 14 tissue compartments in the cochlea and normalized to permit comparisons between cases. An operationally defined background labeling level was computed for each case and a statistical test for significance applied to the neuron-containing tissue compartments. With increasing survival times, significant labeling appeared successively in the cochlear nerve root, in each type of spiral ganglion cell, and in the neuron-containing tissue compartments of the organ of Corti. The findings are consistent with uptake of D-ASP and retrograde transport by cochlear nerve axons from the cochlear nucleus to the perikarya and peripheral processes of the spiral ganglion. We conclude that axons of all spiral ganglion cells project to the cochlear nucleus and that this nucleus is directly connected with both the inner and outer hair cells. Transganglionic transport of D-ASP from the cochlear nucleus is consistent with the hypothesis that the cochlear nerve axons use glutamate or aspartate as a neurotransmitter.