Immunocytochemical Localization of a High-affinity Glutamate-Aspartate Transporter, GLAST, in the Rat and Guinea-pig Cochlea

Glutamate transporters play an important role in the reuptake of glutamate after its release from glutamatergic synapses. Four such transporters have so far been cloned from the rat brain. One, the glutamate-aspartate transporter GLAST, has been detected in the mammalian cochlea, in which the principal afferent synapse of the auditory nerve, between the inner hair cells and neurites of type I spiral ganglion neurons, has been suggested to be glutamatergic. The distribution of GLAST was therefore investigated to provide clues to the handling of glutamate in the cochlea. This was studied using light and electron microscopic immunocytochemistry in rats and guinea pigs with antibodies raised against synthetic peptides based on the sequence for GLAST. Significant immunoreactivity was found in the myelin sheath formed by satellite cells surrounding the type I spiral ganglion neurons, and along the plasma membranes of supporting cells around the inner hair cells; other cells in both locations were only weakly labelled, if at all. The absence of substantial numbers of synapses in the spiral ganglion suggests that GLAST is unlikely to be associated with the uptake of synaptic glutamate after release in this region. lmmunoreactivity associated with the inner hair cells is consistent with the utilization of glutamate at the afferent synapse.     

Hair cell innervation by spiral ganglion neurons in the mouse

Horseradish peroxidase (HRP) was injected extracellularly into the auditory nerve of adult mice so that the enzyme could infuse individual spiral ganglion neurons. Forty-two well-stained neurons were reconstructed through serial sections from their cell bodies to peripheral terminations in the organ of Corti with the aid of a light microscope and drawing tube. No neuron was observed to innervate both inner and outer hair cells (IHCs and OHCs). Previous observations from neonatal mammals that reported that IHCs and OHCs were innervated by the same neuron are thus presumed to describe a transient developmental phenomenon. Two populations of spiral ganglion neurons were determined on the basis of the differences in receptor innervation. The type I neurons innervated exclusively IHCs by way of thick (1-2 microns) radial fibers, whereas the type II neurons innervated only OHCs by way of thin (approximately 0.5 micron) outer spiral fibers. Certain features of the peripheral process in the vicinity of the cell body were highly correlated with fiber type. This pattern of separate innervation of IHCs and OHCs by type I and type II neurons, respectively, may represent the general plan of afferent organization for the adult mammalian cochlea.     

Neuropeptide Y in Rat Spiral Ganglion Neurons and Inner Hair Cells of Organ of Corti and Effects of a Nontraumatic Acoustic Stimulation

Neuropeptide Y (NPY) is an important neuromodulator found in central and peripheral neurons. NPY was investigated in the peripheral auditory pathway of conventional housed rats and after nontraumatic sound stimulation in order to localize the molecule and also to describe its response to sound stimulus. Rats from the stimulation experiment were housed in monitored sound-proofed rooms. Stimulated animals received sound stimuli (pure tone bursts of 8 kHz, 50 ms duration presented at a rate of 2 per second) at an intensity of 80 dB sound pressure level for 1 hr per day during 7 days. After euthanizing, rat cochleae were processed for one-color immunohistochemistry. The NPY immunoreactivity was detected in inner hair cells (IHC) and also in pillar and Deiters’ cells of organ of Corti, and in the spiral ganglion putative type I (≥1,009 μm³) and type II (≤225 μm³) neurons. Outer hair cells (OHC) showed light immunoreaction product. Quantitative microdensitometry showed strong and moderate immunoreactions in IHC and spiral ganglion neurons, respectively, without differences among cochlear turns. One week of acoustic stimulation was not able to induce changes in the NPY immunoreactivity intensity in the IHC of cochlea. However, stimulated rats showed an overall increase in the number of putative type I and type II NPY immunoreactive spiral ganglion neurons with strong, moderate, and weak immunolabeling. Localization and responses of NPY to acoustic stimulus suggest an involvement of the neuropeptide in the neuromodulation of afferent transmission in the rat peripheral auditory pathway.     

Opposing expression gradients of calcitonin‐related polypeptide alpha (Calca/Cgrpα) and tyrosine hydroxylase (Th) in type II afferent neurons of the mouse cochlea

Type II spiral ganglion neurons (SGNs) are small caliber, unmyelinated afferents that extend dendritic arbors hundreds of microns along the cochlear spiral, contacting many outer hair cells (OHCs). Despite these many contacts, type II afferents are insensitive to sound and only weakly depolarized by glutamate release from OHCs. Recent studies suggest that type II afferents may be cochlear nociceptors, and can be excited by ATP released during tissue damage, by analogy to somatic pain‐sensing C‐fibers. The present work compares the expression patterns among cochlear type II afferents of two genes found in C‐fibers: calcitonin‐related polypeptide alpha (Calca/Cgrpα), specific to pain‐sensing C‐fibers, and tyrosine hydroxylase (Th), specific to low‐threshold mechanoreceptive C‐fibers, which was shown previously to be a selective biomarker of type II versus type I cochlear afferents (Vyas et al., 2016 ). Whole‐mount cochlear preparations from 3‐week‐ to 2‐month‐old CGRPα‐EGFP (GENSAT) mice showed expression of Cgrpα in a subset of SGNs with type II‐like peripheral dendrites extending beneath OHCs. Double labeling with other molecular markers confirmed that the labeled SGNs were neither type I SGNs nor olivocochlear efferents. Cgrpα starts to express in type II SGNs before hearing onset, but the expression level declines in the adult. The expression patterns of Cgrpα and Th formed opposing gradients, with Th being preferentially expressed in apical and Cgrpα in basal type II afferent neurons, indicating heterogeneity among type II afferent neurons. The expression of Th and Cgrpα was not mutually exclusive and co‐expression could be observed, most abundantly in the middle cochlear turn.     

Single unit recordings in the auditory nerve of congenitally deaf white cats: Morphological correlates in the cochlea and cochlear nucleus

It is well known that experimentally induced cochlear damage produces structural, physiological, and biochemical alterations in neurons of the cochlear nucleus. In contrast, much less is known with respect to the naturally occurring cochlear pathology presented by congenital deafness. The present study attempts to relate organ of Corti structure and auditory nerve activity to the morphology of primary synaptic endings in the cochlear nucleus of congenitally deaf white cats. Our observations reveal that the amount of sound-evoked spike activity in auditory nerve fibers influences terminal morphology and synaptic structure in the anteroventral cochlear nucleus. Some white cats had no hearing. They exhibited severely reduced spontaneous activity and no sound-evoked activity in auditory nerve fibers. They had no recognizable organ of Corti, presented >90% loss of spiral ganglion cells, and displayed marked structural abnormalities of endbulbs of Held and their synapses. Other white cats had partial hearing and possessed auditory nerve fibers with a wide range of spontaneous activity but elevated sound-evoked thresholds (60–70 dB SPL). They also exhibited obvious abnormalities in the tectorial membrane, supporting cells, and Reissner's membrane throughout the cochlear duct and had complete inner and outer hair cell loss in the base. The spatial distribution of spiral ganglion cell loss correlated with the pattern of hair cell loss. Primary neurons of hearing-impaired cats displayed structural abnormalities of their endbulbs and synapses in the cochlear nucleus which were intermediate in form compared to normal and totally deaf cats. Changes in endbulb structure appear to correspond to relative levels of deafness. These data suggest that endbulb structure is significantly influenced by sound-evoked auditory nerve activity. J. Comp. Neurol. 397:532–548, 1998. © 1998 Wiley-Liss, Inc.     

Unmyelinated axons of the auditory nerve in cats.

This paper describes some central terminations of type II spiral ganglion neurons as labeled by extracellular injections of horseradish peroxidase (HRP) into the auditory nerve of cats. After histological processing with diaminobenzidine, both thick (2-4 microns) and thin (0.5 microns) fibers of the auditory nerve were stained. Whenever traced, thick fibers always originated from type I spiral ganglion neurons and thin fibers always from type II ganglion neurons. Because the labeling of type II axons faded as fibers projected into the cochlear nucleus, this report is limited to regions of the ventral cochlear nucleus near the auditory nerve root. The central axons of type II neurons are unmyelinated, have simple yet variable branching patterns in the cochlear nucleus, and form both en passant and terminal swellings. Under the light microscope, most swellings are located in the neuropil but they are also found in the vicinity of cell bodies, nodes of Ranvier of type I axons, and blood vessels. Eighteen en passant swellings in the neuropil were located by light microscopy and resectioned for electron microscopy; two of these swellings exhibited ultrastructural features characteristic of chemical synapses. The data indicate that inputs from outer hair cells might be able to influence auditory processing in the cochlear nucleus through type II primary neurons.     

Synaptic transfer from outer hair cells to type II afferent fibers in the rat cochlea

Type II cochlear afferents receive glutamatergic synaptic excitation from outer hair cells (OHCs) in the rat cochlea. However, it remains uncertain whether this connection is capable of providing auditory information to the brain. The functional efficacy of this connection depends in part on the number of presynaptic OHCs, their probability of transmitter release, and the effective electrical distance for spatial summation in the type II fiber. The present work addresses these questions using whole-cell recordings from the spiral process of type II afferents that run below OHCs in the apical turn of young (5-9 d postnatal) rat cochlea. A "high potassium puffer" was used to elicit calcium action potentials from individual OHCs and thereby show that the average probability of transmitter release was 0.26 (range 0.02-0.73). Electron microscopy showed relatively few vesicles tethered to ribbons in equivalent OHCs. A "receptive field" map for individual type II fibers was constructed by successively puffing onto OHCs along the cochlear spiral, up to 180 μm from the recording pipette. These revealed a conservative estimate of 7 presynaptic OHCs per type II fiber (range 1-11). EPSCs evoked from presynaptic OHCs separated by >100 μm did not differ in amplitude or waveform, implying that the type II fiber's length constant exceeded the length of the synaptic input zone. Together these data suggest that type II fibers could communicate centrally by maximal activation of their entire pool of presynaptic OHCs.     

Morphology of labeled afferent fibers in the guinea pig cochlea

Cochlear afferent and efferent fibers in the guinea pig were labeled by focal extracellular injections of horseradish peroxidase into the spiral ganglion of the basal turn. The morphology and pattern of termination of these fibers were studied by light microscopy. Fibers labeled by injections into the peripheral side of the ganglion could be grouped on the basis of their courses and terminations in the cochlea into two classes of afferent fibers, two classes of efferent (olivocochlear) fibers, and other presumably autonomic fibers. This paper describes the characteristics of labeled afferent fibers and their parent ganglion cells. Peripheral afferent fibers were grouped into two major classes: thick (mean diameter 1.7 micron) radial fibers projecting in a primarily radial fashion from the spiral ganglion and terminating on single inner hair cells and thin (mean diameter 0.5 micron) outer spiral fibers that spiral basalward in the organ of Corti to terminate on outer hair cells, usually in one row. For outer spiral fibers, the number of outer hair cells contacted and the length of the terminal region depend on the row of outer hair cells contacted, with third-row fibers forming, on the average, the most extensive region of termination. Within the spiral ganglion, two types of ganglion cells could be distinguished: type-I ganglion cells of large size (mean soma area = 216 microns 2) with a ratio of central process diameter to peripheral process diameter greater than one and type-II ganglion cells of smaller size (mean soma area = 100 microns 2) and a central to peripheral process ratio near one. In three cochleae in which injections were made central to the ganglion, 11 type-I ganglion cells have been traced to radial fibers contacting inner hair cells and eight type-II ganglion cells have been traced to outer spiral fibers contacting outer hair cells. Thus the afferent innervation of the guinea pig cochlea is similar to the pattern described in other mammals, in which there is separate innervation of the inner and outer hair cells by the two types of ganglion cells. The central axons of both types of ganglion cells were traced individually through serial sections of a block of tissue containing the cochlea, the auditory nerve, and the cochlear nucleus. They followed similar courses in the auditory nerve, and the axons followed into the cochlear nucleus bifurcated in similar regions of the interstitial portion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization and developmental ontogeny of the pro-apoptotic Bnip3 mRNA in the postnatal rat cortex and hippocampus

Stimuli such as noise or hypoxia can induce a release of ATP into the cochlear fluid spaces. At nanomolar concentrations, ATP affects neurotransmission and electrochemical regulation of sound transduction. At higher concentrations, ATP may exert cytotoxicity acting on specific P2X(7) receptor subunits, thus contributing to the pathophysiology of noise-induced cochlear injury. Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) are pivotal to regulation of extracellular nucleotide concentrations and therefore P2 receptor signaling in the cochlea. Here, we characterize the distribution of NTPDase3 ectonucleotidase (preferentially hydrolyzes ATP over ADP) in cochlear tissues and investigate the effect of noise exposure on NTPDase3 expression. Marked NTPDase3 immunoreactivity in the primary afferent neurones of the spiral ganglion, extending in the distal neurite processes to the synapses beneath the inner and outer hair cells, suggests involvement in auditory neurotransmission. Immunolabeling in the lateral wall and epithelial cells lining the cochlear partition was also evident. Semi-quantitative immunohistochemistry revealed increased NTPDase3 immunolabeling in the synaptic regions of the inner and outer hair cells at sound intensities that induce temporary threshold shift. The results suggest a role for NTPDase3 in regulating ATP signaling associated primarily with auditory neurotransmission, and the potential neuroprotective nature of noise-induced up-regulation of this ectonucleotidase in the cochlea.     

Occurrence and distribution of non-NMDA glutamate receptor mRNAs in the cochlea.

The expression of mRNAs encoding five putative non-NMDA glutamate receptors was investigated using in situ hybridization with radiolabeled riboprobes. Hybridization was observed in spiral ganglion neurons with probes complementary to mRNA products of the glutamate receptor genes GluR2 and GluR3. No specific hybridization was observed with probes for GluR1, GluR4 or GluR5. The results support the hypothesis that glutamate is the transmitter between cochlear inner hair cells and spiral ganglion neurons, and that it acts via non-NMDA glutamate receptors.     

Current concepts in cochlear ribbon synapse formation

In mammals, hair cells and spiral ganglion neurons (SGNs) in the cochlea together are sophisticated “sensorineural” structures that transduce auditory information from the outside world into the brain. Hair cells and SGNs are joined by glutamatergic ribbon‐type synapses composed of a molecular machinery rivaling in complexity the mechanoelectric transduction components found at the apical side of the hair cell. The cochlear hair cell ribbon synapse has received much attention lately because of recent and important findings related to its damage (sometimes termed “synaptopathy”) as a result of noise overexposure. During development, ribbon synapses between type I SGNs and inner hair cells form in the time window between birth and hearing onset and is a process coordinated with type I SGN myelination, spontaneous activity, synaptic pruning, and innervation by efferents. In this review, we highlight new findings regarding the diversity of type I SGNs and inner hair cell synapses, and the molecular mechanisms of selective hair cell targeting. Also discussed are cell adhesion molecules and protein constituents of the ribbon synapse, and how these factors participate in ribbon synapse formation. We also note interesting new insights into the morphological development of type II SGNs, and the potential for cochlear macrophages as important players in protecting SGNs. We also address recent studies demonstrating that the structural and physiological profiles of the type I SGNs do not reach full maturity until weeks after hearing onset, suggesting a protracted development that is likely modulated by activity.     

The development of vestibulocochlear efferents and cochlear afferents in mice

We have reinvestigated the embryonic development of the vestibulocochlear system in mice using anterograde and retrograde tracing techniques. Our studies reveal that rhombomeres 4 and 5 include five motor neuron populations. One of these, the abducens nucleus, will not be dealt with here. Rhombomere 4 gives rise to three of the remaining populations: the facial branchial motor neurons; the vestibular efferents; and the cochlear efferents. The migration of the facial branchial motor neurons away from the otic efferents is completed by 13.5 days post coitum (dpc). Subsequently the otic efferents separate into the vestibular and cochlear efferents, and complete their migration by 14.5 dpc. In addition to their common origin, all three populations have perikarya that migrate via translocation through secondary processes, form a continuous column upon completion of their migrations, and form axonal tracts that run in the internal facial germ. Some otic efferent axons travel with the facial branchial motor nerve from the internal facial genu and exit the brain with that nerve. These data suggest that facial branchial motor neurons and otic efferents are derived from a common precursor population and use similar cues for pathway recognition within the brain. In contrast, rhombomere 5 gives rise to the fourth population to be considered here, the superior salivatory nucleus, a visceral motor neuron group. Other differences between this group and those derived from rhombomere 4 include perikaryal migration as a result of translocation first through primary processes and only then through secondary processes, a final location lateral to the branchial motor/otic efferent column, and axonal tracts that are completely segregated from those of the facial branchial and otic efferents throughout their course inside the brain. Analysis of the peripheral distribution of the cochlear efferents and afferents show that efferents reach the spiral ganglion at 12.5 dpc when postmitotic ganglion cells are migrating away from the cochlear anlage. The efferents begin to form the intraganglionic spiral bundle by 14.5 dpc and the inner spiral bundle by 16.5 dpc in the basal turn. They have extensive collaterals among supporting cells of the greater epithelial ridge from 16.5 dpc onwards. Afferents and efferents in the basal turn of the cochlea extend through all three rows of outer hair cells by 18.5 dpc. Selective labeling of afferent fibers at 20.5 dpc (postnatal day 1) shows that although some afferents are still in early developmental stages, some type II spiral ganglion cells already extend for long distances along the outer hair cells, and some type I spiral ganglion cells end on a single inner hair cell. These data support previous evidence that in mice the early outgrowth of afferent and efferent fibers is essentially achieved by birth.     

Longitudinal gradients of KCNQ4 expression in spiral ganglion and cochlear hair cells correlate with progressive hearing loss in DFNA2

Mutations in the human KCNQ4 gene were recently found by Kubisch et al. [Cell 96 (1999) 437-446] to cause a non-syndromic, autosomal dominant, progressive hearing loss, DFNA2. The mouse Kcnq4 orthologue was previously localized to the outer hair cells (OHCs) of the inner ear, suggesting the pathophysiological effects were due to dysfunctional OHCs. Yet, OHC dysfunction does not provide a plausible explanation for the progressive nature of the frequency specific hearing loss. We have re-examined and extended the expression analyses of KCNQ4 in the murine inner ear using RT-PCR and whole mount in situ hybridization. Our results confirmed that the rat KCNQ4 orthologue is expressed in both inner and outer hair cells. Reciprocal longitudinal gradients were found in inner hair cells (IHCs) and OHCs. The strongest expression of KCNQ4 in IHCc was in the base of the cochlea and in the apex for OHCs. Similar to the IHCs, a basal to apical gradient was present in the spiral sensory neurons. IHCs mediate hearing via their afferent sensory neurons, whereas OHCs function as active cochlear amplifiers. The complete absence of OHCs leads only to severe sensitivity reduction, but not complete hearing loss. Our data suggest that the primary defect leading to initial high frequency loss and subsequent progressive hearing loss for all frequencies may be due to spiral ganglion and/or IHC dysfunction, rather than an OHC aberration.     

Cadmium-Induced Ototoxicity in Rat Cochlear Organotypic Cultures

Cadmium (Cd), a widely used industrial metal, is extremely nephrotoxic and neurotoxic; however, its effects on the peripheral auditory system are poorly understood. To evaluate the ototoxicity of Cd, we treated cochlear organotypic cultures from postnatal day 3 rats with Cd concentrations from 10 to 500 μM for 24 or 48 h. Afterward, we evaluated the degree of damage to hair cells, auditory nerve fibers, and spiral ganglion neurons. Damage to the hair cells, auditory nerve fibers, and spiral ganglion neurons systematically increased in a dose and time-dependent manner. Exposure to Cd concentrations of 10 μM for 24 and 48 h resulted in minor inner and outer hair cell loss in the basal third of the cochlea. As Cd concentrations increased, toxicity spread toward the apex, also in a time-dependent manner. Treatment with 100 μM Cd for 48 h resulted in substantial (>30 %) hair cell loss over the entire cochlea. Cd was also toxic to auditory nerve fibers and spiral ganglion neurons; 100 μM of Cd for 24 h or 10 μM of Cd for 48 h resulted in considerable damage to auditory nerve fibers and spiral ganglion neurons. These findings are the first to demonstrate that Cd can cause significant lesions to peripheral auditory nerve fibers, spiral ganglion neurons, and sensory hair cells in organotypic cultures from postnatal cochleae.     

Peripherin-like immunoreactivity in type II spiral ganglion cell body and projections

Peripherin, an intermediate filament protein, is present in neuronal subpopulations of both peripheral and central nervous systems. The distribution of peripherin was studied in the adult rat cochlea using immunohistochemistry on whole mount material, in cryostat sections and sections of plastic embedded tissue. In the spiral ganglion, peripherin labeling was restricted to the perikarya of a subpopulation of neurons and their peripheral and central processes. Peripherin positive neurons had the following features: (i) they have a large eccentric nucleus, they were often found in a cluster of 2 or 3 cells, (ii) they were often located near the intraganglionic spiral bundle fibers, (iii) they represented roughly 8% of the whole ganglion population and (iv) on the average they had smaller perikarya than non-immunoreactive cells. Immunostaining on semithin plastic sections revealed positive reactivity on Type II ganglion cells, while Type I neurons were negative. Double labeling using peripherin and three neurofilament (NF) subunit antibodies confirmed the presence of both markers within the same spiral ganglion cell type. Type II neurons have been previously documented as the only subpopulation of the spiral ganglion that presents a strong positive NF immunoreactivity within their perikarya. In the organ of Corti, peripherin-positive fibers formed bundles that course beneath the outer hair cells and send branches that end as boutons contacting the outer hair cells. All these characteristics suggest that peripherin-positive cells are Type II neurons, and that peripherin constitutes a reliable marker for this spiral ganglion subpopulation, as well as their peripheral and central processes.     

Collaterals from lateral and medial olivocochlear efferent neurons innervate different regions of the cochlear nucleus and adjacent brainstem.

Two populations of superior olivary neurons which project to different sensory cell regions in the cochlea also give off collateral projections to the ventral cochlear nucleus (VCN) and adjacent brainstem. To determine whether these VCN projections also have different targets they were characterized by selective retrograde amino acid transport. Retrograde transport of 3H-d-aspartate (D-ASP) selectively labeled the unmyelinated fibers and neurons of the lateral olivocochlear (OC) system including a dense collateral projection to the central VCN. Retrograde transport of 3H-nipecotic acid (NIP) labeled the myelinated fibers and neurons of the medial OC system, including collateral projections to the peripheral VCN, subpeduncular granule cells, and nucleus Y. Medial and lateral OC efferent collaterals thus innervate different regions of the CN. Lateral system collaterals overlap extensively with Type I spiral ganglion cell afferent input. They are well positioned to play a role in modulating afferent input to the central auditory system, as is the primary projection of these efferents to the cochlea. The medial system collaterals project near the recently described afferent projections of Type II spiral ganglion cells. The medial system collaterals may therefore be related to the function of outer hair cells, as the medial system primary axons appear to be in the cochlea.     

Short-term synaptic plasticity regulates the level of olivocochlear inhibition to auditory hair cells

In the mammalian inner ear, the gain control of auditory inputs is exerted by medial olivocochlear (MOC) neurons that innervate cochlear outer hair cells (OHCs). OHCs mechanically amplify the incoming sound waves by virtue of their electromotile properties while the MOC system reduces the gain of auditory inputs by inhibiting OHC function. How this process is orchestrated at the synaptic level remains unknown. In the present study, MOC firing was evoked by electrical stimulation in an isolated mouse cochlear preparation, while OHCs postsynaptic responses were monitored by whole-cell recordings. These recordings confirmed that electrically evoked IPSCs (eIPSCs) are mediated solely by α9α10 nAChRs functionally coupled to calcium-activated SK2 channels. Synaptic release occurred with low probability when MOC-OHC synapses were stimulated at 1 Hz. However, as the stimulation frequency was raised, the reliability of release increased due to presynaptic facilitation. In addition, the relatively slow decay of eIPSCs gave rise to temporal summation at stimulation frequencies >10 Hz. The combined effect of facilitation and summation resulted in a frequency-dependent increase in the average amplitude of inhibitory currents in OHCs. Thus, we have demonstrated that short-term plasticity is responsible for shaping MOC inhibition and, therefore, encodes the transfer function from efferent firing frequency to the gain of the cochlear amplifier.     

Identification and ultrastructural localization of a calretinin-like calcium-binding protein (protein 10) in the guinea pig and rat inner ear

Calretinin has been identified as a brain specific calcium-binding protein which appears as a prominent protein in the cochlear nucleus. We identified and localized calretinin in the guinea pig and rat inner ear using polyclonal antibodies. Immunoblot analyses of guinea pig and rat auditory nerve homogenates revealed an immunoreactive band migrating with the same molecular weight as the purified protein, atM_r = 29k. Immunocytochemistry was carried out at the light and electron microscope levels. In the guinea pig cochlea, inner hair cells, Deiters' cells, Hensen's cells and interdental cells of the spiral limbus were stained. Most of the cochlear ganglion cells were immunostained. In the guinea pig vestibular organs, the staining was exclusively neuronal and localized in large nerve fibers and nerve calices of the apex of the cristae. Only some vestibular ganglion cells were stained. In the rat cochlea, inner hair cells and most of the ganglion neurons were immunoreactive. In the rat vestibule, large nerve fibers and calices were stained as were some type II hairs cells. Only some vestibular ganglion cells were reactive. Electron microscopic observations of immunostained guinea pig cochlea and vestibule showed that the staining was cytosolic. In addition, specific sub-localization was also found in the apical portion of the nerve calices in association with microvesicles. These results describe the discrete localization of calretinin in the cochlea and in the vestibular receptors and suggest a function associated with biochemical regulations at the level of microvesicles in vestibular afferent neurons.