Fine structure of cochlear innervation in the cat

Examination of adult and juvenile cat cochleas by electron microscopy and semi-serial sections permitted identification of the cytological features characteristic of the afferent and efferent nerve fiber populations identified in Golgi impregnations of the cochlea. This study demonstrated the distribution of synaptic contacts made by these fiber populations. As in the Golgi findings, radial and outer spiral afferent fibers were identified in well separated zones of the inner spiral bundle. The trunks of the outer spiral fibers, containing many microtubules and few neurofilaments, at first coursed spirally below the inner hair cells on the proximal face of the inner pillar, turned abruptly between adjacent pillar cells and entered the tunnel without branching. Radial afferents, containing many neurofilaments and a few microtubules, coursed through the inner spiral bundle, maintaining a radial or oblique orientation and proceeded directly toward the inner hair cells. Efferent fibers in the region of the inner spiral bundle were distinguishable by size, by orientation, and, to a lesser extent, by cytology. Small (1 micron) efferent fibers, containing few neurofilaments, an occasional microtubule, and mitochondria, occurred in the inner and tunnel spiral bundles and formed large varicosities, which contacted radial afferents. A separate population of much thicker efferents, containing many neurofilaments, mitochondria and dense-cored vesicles, but no microtubules, did not enter the inner spiral bundle but coursed directly to the level of the tunnel spiral bundle on the proximal face of the inner pillar cells. These fibers crossed the tunnel at the level of the tunnel spiral bundle and, upon reaching the outer hair cells, formed large synaptic contacts on outer hair cells and on outer spiral fibers as well. Some of these efferent fibers also synapse on afferent fibers while crossing the tunnel. The findings agree with previous observations with the Golgi method showing that entirely separate populations of spiral ganglion cells innervate the inner and outer hair cells. Likewise, there are efferent fibers innervating only inner or outer hair cells, but the probability of efferent fibers to both inner and outer hair cells cannot be ruled out.     

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.     

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.     

The spiral ganglion and the innervation of the human organ of Corti

Five human cochleas were evaluated using the block surface method. The numbers of hair cells, nerve fibres in the osseous spiral lamina, and spiral ganglion cells were determined and correlated. The ultrastructural organization corresponds essentially to that of the mammalian ear, with the exception of multiple synaptic contacts of afferent nerve fibres with inner hair cells and surprisingly large numbers of outer spiral fibres.     

Comparative anatomy of the cochlea and auditory nerve in mammals

The numbers and structure of hair cells; afferent, efferent, and reciprocal synapses as seen at the base of hair cells; innervation patterns of first order cochlear neurons; and number and morphology of spiral ganglion cells will be discussed and compared in the guinea pig, rat, cat, monkey and man. Despite many similarities both in the organ of Corti and the spiral ganglion in these species, there are a number of differences which may have important physiologic implications. In the organ of Corti, the major differences among species are the length and width of the basilar membrane, the number of inner and outer hair cells, and the length of hairs on both inner and outer hair cells. Significant differences in the innervation pattern of the inner hair cell among these species include the number of afferent nerve terminals per inner hair cell, the degree of branching of afferent fibers, and the number of synapses per afferent nerve terminal. Among outer hair cells, the number of afferent nerve terminals per outer hair cell, presence or absence of a pre-synaptic body, presence or absence of reciprocal synapses, the number of efferent terminals per outer hair cell, and the presence of dendodendritic synapses in outer spiral bundles may be differences important physiologically. In the spiral ganglion, there are significant differences in the number of spiral ganglion cells, the number of cochlear nerve fibers, the percentage of spiral ganglion cells which are myelinated, and the presence of synapses on spiral ganglion cells.     

In vitro morphological study of cochlear epithelium.

1. The mammalian cochlear epithelium was successfully kept alive in long-term tissue cultures. The Na and K concentration of the culture medium was similar to that of perilymph. 2. The most vulnerable cells in the culture epithelium were the outer and inner hair cells followed by the inner zones cells and the interdental cells of the spiral limbus. The difference in disintegration time between the outer and inner hair cells was about 48 hrs under the same culture conditions. The same phenomenon was noted between the rows of outer hair cells. 3. When the outer hair cells were not supported by Deiters' cells or when the cochlear epithelium was separated between Deiters' cells and Hensens cells, the outer hair cells degenerated quickly. 4. The interdental cells appeared to be relatively highly differentiated and showed much activity. No evidence of secretory function of interdental cells could be obtained. 5. The inner sulcus cells, Hensens cells, Claudius' cells and Reissners membrane cells were flattened and arranged like covering epithelium.     

Synaptophysin immunohistochemistry in the human cochlea

Light microscopy and immunohistochemical analyses of a freshly prepared human cochlea, removed at meningioma skull base surgery, were performed with particular emphasis on synaptophysin (SY) reactivity. Synaptophysin, a 38-kDa glycoprotein, is one of the most abundant integral membrane proteins of small presynaptic vesicles and is a useful marker for sites of synaptic transmission of the efferent olivocochlear system in the cochlea. Following fixation and decalcification, cryosections of 30 microm were prepared. To introduce immunostaining, free-floating sections were exposed to monoclonal SY antibody. Positive SY immunostaining was solely restricted to the neural and sensory structures and did not include supporting cells of the organ of Corti. Dense reaction products were noted around the hair cells, especially at the basal portion of the inner and outer hair cells and their neural poles, as well as around the inner spiral bundle, tunnel spiral bundle, outer spiral bundle and upper tunnel crossing fibers. The majority of spiral ganglion cells stained positively. An intermingling network of thin unmyelinated nerve fibers stained densely, especially at the basal portions of the cochlea. The spiral limbus, inner and outer sulcus cells, basilar membrane, myelinated nerve fibers, spiral ligament and the stria vascularis were unstained. Human cochlea obtained during surgery offers excellent conditions for immunohistochemical analysis. In the basal cochlea in the organ of Corti, outer hair cell area, there may be alterations due to noise trauma from the drilling procedure.     

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.     

Structural/audiometric correlations in a human inner ear with noise-induced hearing loss

A morphological analysis was performed on a human cochlea removed during skull base surgery. The patient experienced a noise-induced hearing loss following 30 years of mechanical exposure. The tissue was processed according to the block surface technique and the organ of Corti, osseous spiral lamina and spiral ganglion were analyzed at different levels. There was a circumscribed lesion approx. 10 mm from the round window extending to about 13 mm. At this site, the dominant pathological feature was the loss of outer hair cells that was comprehensive in the centermost area and partial in the peripheral region of the damage. The degradation of inner hair cells was less severe with signs of cell atrophy yet with limited loss. Outer pillar cells were often collapsed leading to deformation of the acoustic ridge. The Deiters cells were often present and physically interactive with remaining nerve fibers. In the reticular lamina, surgical manipulation and dissection resulted in tears which may be attributed to a reduction of intercellular strength between cells. In the damaged area, there was a 45% loss of myelinated nerve fibers measured at the osseous spiral lamina. Pathological changes could not be observed in the spiral ganglion with certainty although the type II cells innervating the outer hair cells were often difficult to discern.     

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.     

Acoustic lesions in the mammalian cochlea: implications for the spatial distribution of the 'active process'.

The spatial contribution of mechanically active hair cells to tuning and sensitivity at a single point in the mammalian cochlea has been investigated in the basal turn of the guinea pig cochlea. Following the destruction of outer hair cells with acoustic overstimulation it was possible to record apparently normal tuning and sensitivity from spiral ganglion neurones innervating inner hair cells located on the apical edges of substantial lesions. The distance between the recording site, where neurones showed normal sensitivity, and areas of the cochlea showing 60-100% of the outer hair cells either damaged or missing varied between 0.2 and 1.3 mm which incorporates approximately 70 to 450 outer hair cells. In one animal neurones that demonstrated normal sensitivity were recorded within 0.2 mm of a lesion where 67% of the outer hair cells were either missing or showed severe damage to their stereocilia and within 0.5 mm of areas of the organ of Corti showing damage to 97% of the outer hair cells. This distance includes approximately 50 inner hair cells or 180 outer hair cells. The location of these neurones, whose sharp tuning presumably mirrors basilar membrane mechanics, suggests that a substantial proportion of point tuning in the cochlea may be derived over a distance of less than 0.5 mm and involve fewer than 200 active outer hair cells.     

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.     

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.     

Patterns of GABA-like immunoreactivity in efferent fibers of the human cochlea

Olivocochlear efferent neurons originate in the superior olivary complex of the brainstem and terminate within sensory cell regions of the organ of Corti. Components of this complex include the lateral olivocochlear bundle whose unmyelinated axons synapse with radial afferent dendrites below inner hair cells and the medial olivocochlear bundle, from which myelinated axons form a direct synaptic contact with outer hair cells. gamma-Aminobutyric acid (GABA), a major neurotransmitter of the central nervous system believed to be responsible for most fast-inhibitory transmissions, has been demonstrated with interspecies variation between mammal and primate auditory efferents. In the present study, we evaluate the immunocytochemical presence of GABA in 10 human cochleae using light and electron microscopy. GABA-like immunostaining could be observed in inner spiral fibers, tunnel spiral fibers, tunnel-crossing fibers, and at efferent endings synapsing with outer hair cells. To approximate medial efferent fiber quantifications, we counted labeled terminals at the base of each outer hair cell and then compared this sum with the number of tunnel crossing fibers. We found a 'branching ratio' of 1:2 implicating a doubling in quantifiable efferent fibers at the level of the outer hair cell. In human, the distribution of GABA-like immunoreactivity showed a consistent presence throughout all turns of the cochlea. A new method for application of immunoelectron microscopy on human cochleae using a pre-embedding technique is also presented and discussed.     

Glutamate is the Afferent Neurotransmitter in the Human Cochlea

Glutamate, the most important afferent neurotransmitter in the auditory system, is thought to be the afferent transmitter between the cochlear inner hair cells and afferent neurons, hitherto visualized only in the cochlea of animal species. It has been identified for the first time in sections from the human inner ear. L-glutamate, NMDAR2B and the enzyme glutamine synthetase were identified by using monoclonal antibodies. The distribution pattern of the transmitter L-glutamate in the human cochlea is similar to that observed in other mammals. L-glutamate was identified adjacent to outer and inner hair cells and in the spiral ganglion. Similar distributions were found for glutamine synthetase and the ionotropic NMDA receptor subunit NMDAR2. The identification of neurotransmitters and their receptors in the human cochlea has implications for the pharmacotherapy of inner ear diseases.     

Glutamate is the afferent neurotransmitter in the human cochlea

Glutamate, the most important afferent neurotransmitter in the auditory system, is thought to be the afferent transmitter between the cochlear inner hair cells and afferent neurons, hitherto visualized only in the cochlea of animal species. It has been identified for the first time in sections from the human inner ear. L-glutamate, NMDAR2B and the enzyme glutamine synthetase were identified by using monoclonal antibodies. The distribution pattern of the transmitter L-glutamate in the human cochlea is similar to that observed in other mammals. L-glutamate was identified adjacent to outer and inner hair cells and in the spiral ganglion. Similar distributions were found for glutamine synthetase and the ionotropic NMDA receptor subunit NMDAR2. The identification of neurotransmitters and their receptors in the human cochlea has implications for the pharmacotherapy of inner ear diseases.     

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.     

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.     

The receptor and neuron distribution in the cochlea of the bat, Taphozous kachhensis

Cochlear receptors and spiral ganglion neurons in the inner ear of the bat, Taphozous kachhensis of the family Emballonuridae, were quantitatively analyzed on the basis of surface specimen technique, histological sections, and three-dimensional computer reconstruction. The length of the basilar membrane was 12.1 (+/- 0.4) mm. There were about 23,000 neurons in the spiral ganglion and about 1430 inner hair cells and 4635 outer hair cells in the organ of Corti. Cochlear neurons had two density maxima, one basal and one apical. Inner hair cells had basically the same distribution but less pronounced. Outer hair cells had only one density maximum in the apical region corresponding to the respective maxima of inner hair cells and cochlear neurons. Basal density maxima of inner hair cells and neurons are most probably involved in the analysis of echolocation signals, and presumably correlated with maximum sensitivity of the auditory system. A comparison with the bat, Nyctalus noctula of the family Vespertilionidae revealed a conspicuous similarity in some quantitative aspects of the cochlear structure. These particular similarities may be looked upon as convergent adaptations given by similar flight, hunting, and echolocation strategies in both bat species.     

Acetylcholinesterase in the cochlea on normal guinea pigs by light and electron microscopy

The distribution of AchE activity in the cochleae of 42 normal guinea pigs was studied with thiocholine Cu-ferrocyanide method. Light microscopy revealed the AchE activity in the following areas: 1. the inner spiral bundle, 2. the spiral tunnel bundle, 3. the upper tunnel radial fibers, and 4. the large nerve endings at the base of the outer hair cells. Activity of AchE was most abundant in the basal turn and decreased gradually towards the apex. There was also a decreasing AchE from the first to the third row of outer hair cells. In the fourth turn, AchE activity was demonstrated only in the large nerve endings under the first row of outer hair cells. Under electron microscopy, the reaction was found to be localized to the following structures: 1. the axolemma of the small efferent fibers and their presynaptic enlargements in the inner spiral bundle, 2. the outer surface of the upper tunnel radial fibers and the spiral tunnel bundle, and 3. the large efferent nerve endings in contact with the base of the outer hair cells.