Evolution of inner ear neuroanatomy of bats and implications for echolocation.

Phylogenomics of bats suggests that their echolocation either evolved separately in the bat suborders Yinpterochiroptera and Yangochiroptera, or had a single origin in bat ancestors and was later lost in some yinpterochiropterans1-6. Hearing for echolocation behaviour depends on the inner ear, of which the spiral ganglion is an essential structure. Here we report the observation of highly derived structures of the spiral ganglion in yangochiropteran bats: a trans-otic ganglion with a wall-less Rosenthal's canal. This neuroanatomical arrangement permits a larger ganglion with more neurons, higher innervation density of neurons and denser clustering of cochlear nerve fascicles7-13. This differs from the plesiomorphic neuroanatomy of Yinpterochiroptera and non-chiropteran mammals. The osteological correlates of these derived ganglion features can now be traced into bat phylogeny, providing direct evidence of how Yangochiroptera differentiated from Yinpterochiroptera in spiral ganglion neuroanatomy. These features are highly variable across major clades and between species of Yangochiroptera, and in morphospace, exhibit much greater disparity in Yangochiroptera than Yinpterochiroptera. These highly variable ganglion features may be a neuroanatomical evolutionary driver for their diverse echolocating strategies4,14-17 and are associated with the explosive diversification of yangochiropterans, which include most bat families, genera and species.

Vascular Supply of the Human Spiral Ganglion: Novel Three-Dimensional Analysis Using Synchrotron Phase-Contrast Imaging and Histology.

Human spiral ganglion (HSG) cell bodies located in the bony cochlea depend on a rich vascular supply to maintain excitability. These neurons are targeted by cochlear implantation (CI) to treat deafness, and their viability is critical to ensure successful clinical outcomes. The blood supply of the HSG is difficult to study due to its helical structure and encasement in hard bone. The objective of this study was to present the first three-dimensional (3D) reconstruction and analysis of the HSG blood supply using synchrotron radiation phase-contrast imaging (SR-PCI) in combination with histological analyses of archival human cochlear sections. Twenty-six human temporal bones underwent SR-PCI. Data were processed using volume-rendering software, and a representative three-dimensional (3D) model was created to allow visualization of the vascular anatomy. Histologic analysis was used to verify the segmentations. Results revealed that the HSG is supplied by radial vascular twigs which are separate from the rest of the inner ear and encased in bone. Unlike with most organs, the arteries and veins in the human cochlea do not follow the same conduits. There is a dual venous outflow and a modiolar arterial supply. This organization may explain why the HSG may endure even in cases of advanced cochlear pathology.

The Cochlear Spiral Ganglion Neurons: The Auditory Portion of the VIII Nerve.

The VIII nerve is formed by sensory neurons that innervate the inner ear, i.e., the vestibular and the auditory receptors. Neurons of the auditory portion, the cochlear afferent fibers that innervate the sensory hair cells of the organ of Corti, have their somas in the cochlear spiral ganglion where two types of neurons can be distinguished. Afferent Type-I neurons are the 95% of the total population. Bipolar and myelinated fibers, each one innervates only one cochlear inner hair cell (IHC). In contrast, afferent Type-II neurons are only the 5% of the spiral ganglion population. They are pseudounipolar and unmyelinated fibers and innervate the cochlear outer hair cells (OHC) so that one afferent Type-II fiber contacts with multiple OHCs, but each OHC only receives one contact from one Type-II neuron. Both types of VIII nerve fibers are glutamatergic, but these asymmetric innervations of the cochlear sensory cells could suggest that the IHC codifies the truly auditory message but the OHC only informs about mechanical aspects of the state of the organ of Corti. In fact, the central nervous system (CNS) has control over the information transmitted by the Type-I neuron by means of axons from the superior olivary complex that innervate them to modulate, filter and/or inhibit the entry of auditory message to CNS. The aim of this paper is to review the current knowledge about the anatomy and physiology of the auditory portion of the VIII nerve. Anat Rec, 302:463-471, 2019. © 2018 Wiley Periodicals, Inc.

The Human Cochlear Aqueduct and Accessory Canals: a Micro-CT Analysis Using a 3D Reconstruction Paradigm.

OBJECTIVE: We sought to study the anatomic variations of the cochlear aqueduct and its accessory canals in human temporal bones using micro-CT and a 3D reconstruction paradigm. More knowledge about the anatomic variations of these structures, particularly at the basal turn of the cochlea and round window niche, may be important to better preserve residual hearing as well as the neural supply during cochlear implant surgery. METHODS: An archival collection of 30 human temporal bones underwent micro-CT and 3D reconstruction. A surface enhancement paradigm was applied. The application displays reconstructed slices as a 3D object with realistic 3D visualization of scanned objects. Virtual sectioning or "cropping" of the petrous bone presented subsequent areas. Thereby, the bony canals could be followed from inside the basal turn of cochlea and middle ear to the jugular foramen. RESULTS: The cochlear aqueduct was always paralleled by an accessory canal containing the inferior cochlear vein. It ran from the basal turn of the cochlea and exited laterally in the jugular foramen. In 70% of the cases, a secondary accessory canal was observed and it derived mostly from a depression or infundibulum located in the floor of the round window niche. This canal also exited in the jugular foramen. The secondary accessory canal occasionally anastomosed with the primary accessory canal suggesting that it contains a vein that drains middle ear blood to the cranial sinus. CONCLUSION: Micro-CT with 3D surface reconstruction paradigm offers new possibilities to study the topographic anatomy of minor details in the human inner ear. The technique creates simulated transparent "castings" of the labyrinth with a coinciding surface view through enhancement of contrast between boundaries. Accessory canals that drain blood from the cochlea, spiral ganglion, and middle ear could be characterized three-dimensionally.

Spiral Form of the Human Cochlea Results from Spatial Constraints.

The human inner ear has an intricate spiral shape often compared to shells of mollusks, particularly to the nautilus shell. It has inspired many functional hearing theories. The reasons for this complex geometry remain unresolved. We digitized 138 human cochleae at microscopic resolution and observed an astonishing interindividual variability in the shape. A 3D analytical cochlear model was developed that fits the analyzed data with high precision. The cochlear geometry neither matched a proposed function, namely sound focusing similar to a whispering gallery, nor did it have the form of a nautilus. Instead, the innate cochlear blueprint and its actual ontogenetic variants were determined by spatial constraints and resulted from an efficient packing of the cochlear duct within the petrous bone. The analytical model predicts well the individual 3D cochlear geometry from few clinical measures and represents a clinical tool for an individualized approach to neurosensory restoration with cochlear implants.

The localization and physiological effects of cannabinoid receptor 1 in the brain stem auditory system of the chick.

Fast, temporally-precise, and consistent synaptic transmission is required to encode features of acoustic stimuli. Neurons of nucleus magnocellularis (NM) in the auditory brain stem of the chick possess numerous adaptations to optimize the coding of temporal information. One potential problem for the system is the depression of synaptic transmission during a prolonged stimulus. The present study tested the hypothesis that cannabinoid receptor 1 (CB1) signaling may limit synaptic depression at the auditory nerve-NM synapse. In situ hybridization was used to confirm that CB1 mRNA is expressed in the cochlear ganglion; immunohistochemistry was used to confirm the presence of CB1 protein in NM. These findings are consistent with the common presynaptic locus of CB1 in the brain. Rate-dependent synaptic depression was then examined in a brain slice preparation before and after administration of WIN 55,212-2 (WIN), a potent CB1 agonist. WIN decreased the amplitude of excitatory postsynaptic currents (EPSCs) and also reduced depression across a train of stimuli. The effect was most obvious late in the pulse train and during high rates of stimulation. This CB1-mediated influence could allow for lower, but more consistent activation of NM neurons, which could be of importance for optimizing the coding of prolonged, temporally-locked acoustic stimuli.

Differences in cochlear nerve cross-sectional area between normal hearing and postlingually deafened patients on MRI.

OBJECTIVES: To demonstrate that parasagittal constructive interference in steady state (CISS) magnetic resonance imaging (MRI) can be used to accurately measure cochlear nerve cross-sectional area and thereby evaluate for statistically significant differences in the cochlear nerve cross-sectional areas of postlingually deafened and normal-hearing adults. STUDY DESIGN: Cross-sectional study. SETTING: Tertiary care medical center. SUBJECTS AND METHODS: Parasagittal CISS MRIs of postlingually profoundly deafened cochlear implant candidates and normal-hearing patients at a tertiary care academic medical center between 2006 and 2009 were retrospectively identified. Two independent and blinded investigators measured the cochlear nerve height and width and calculated the cross-sectional area [π(H/2)(W/2)] at the fundus of the internal auditory canals. Measurements of both investigators were analyzed for reliability and agreement with an Altman plot, and deafened patient measurements were compared with results of the normal-hearing patients via Wilcoxon rank sum tests. RESULTS: The cochlear nerve cross-sectional area of postlingually deafened patients (mean ± SD = 0.61 ± 0.16 mm(2)) was less than normal-hearing patients (0.94 ± 0.28 mm(2)). The difference was statistically significant (P = .002). There was good agreement between independent observer measurements. CONCLUSION: Parasagittal CISS MRI can be used to measure the cochlear nerve with good interobserver agreement, and there is a significant difference between the cross-sectional area of postlingually deafened and normal-hearing adults. The cross-sectional area may correlate with residual spiral ganglion cells and provide a prognostic indicator for post-cochlear implant performance, which is the focus of our ongoing research.

TSLIM imaging and a morphometric analysis of the mouse spiral ganglion.

Thin-sheet laser imaging microscopy (TSLIM) was used to serially section five whole cochleas from 4-wk-old CBA/JCr mice. Three-dimensional reconstructions of Rosenthal's canal (RC) were produced in order to measure canal length and volume, to generate orthogonal cross sections for area measurements, and to determine spiral ganglion neuron (SGN) number. RC length averaged 2.0 mm ± 0.04 (SEM) as measured along the centroid of the canal compared to an average basilar membrane (BM) length of 5.9 ± 0.05 (SEM). RC volume averaged 0.036 mm(3) ± 0.009 (SEM). Significant increases in the radial area of RC were observed at the base (13%), middle (62%), and apex (90%) of its length. The total number of spiral ganglion neurons (SGNs) in RC in each of the five animals averaged 8626 ± 96 (SEM). SGN number increased at the expanded regions of RC. Increased area and cell number at the base and apex are likely related to extensions of the organ of Corti past the length of RC in these areas. The increase in area and cell number in the middle of the RC appears to be related to the most sensitive frequency region of the organ of Corti. Volume imaging or tomography of the cochlea as provided by TSLIM has the potential to be an efficient and accurate semi-automated method for the quantitative assessment of the number of SGNs and hair cells of the organ of Corti.

Signaling through erbB receptors is a critical functional regulator in the mature cochlea.

Noise, ototoxic substances and various genetic factors are common causes of profound hearing loss. Cochlear implants can often restore hearing in these cases, but only if a sufficient number of responsive auditory nerve fibers remain. Over time, these nerve fibers degenerate in the damaged ear, and it is therefore important to establish factors that control neuronal survival and maintain neural excitability. Recent studies show that neuregulins and their receptors are important for survival and proper targeting of neurons in the developing inner ear. A role for neuregulins as maintainers of the neuronal population in the mature inner ear was therefore hypothesized. Here, this hypothesis was directly tested by chronic local application of substances that block neuregulin receptors. Using auditory brainstem response measurements, we demonstrate that such receptor block leads to a progressive hearing impairment that develops over the course of weeks. This impairment occurs despite a normal number of auditory neurons and preserved outer hair cell function. Real-time quantitative reverse transcriptase-polymerase chain reaction shows alterations in neurotrophin-3 expression, suggesting that this growth factor participates in regulating cochlear sensitivity. The present work demonstrates the critical importance of neuregulin/erbB signaling in long-term functional regulation in the mature guinea pig hearing organ.

Developmental expression of the actin depolymerizing factor ADF in the mouse inner ear and spiral ganglia.

Hair cells, the inner ear's sensory cells, are characterized by tens to hundreds of actin-rich stereocilia that form the hair bundle apparatus necessary for mechanoelectrical transduction. Both the number and length of actin filaments are precisely regulated in stereocilia. Proper cochlear and vestibular function also depends on actin filaments in nonsensory supporting cells. The formation of actin filaments is a dynamic, treadmill-like process in which actin-binding proteins play crucial roles. However, little is known about the presence and function of actin binding molecules in the inner ear, which set up, and maintain, actin-rich structures and regulate actin turnover. Here we examined the expression and subcellular location of the actin filament depolymerizing factor (ADF) in the cochlea and vestibular organs. By means of immunocytochemistry and confocal microscopy, we analyzed whole-mount preparations and cross-sections in fetal and postnatal mice (E15-P26). We found a transient ADF expression in immature hair cells of the organ of Corti, the utricle, and the saccule. Interestingly, the stereocilia were not labeled. By P26, ADF expression was restricted to supporting cells. In addition, we localized ADF in presynaptic terminals of medio-olivocochlear projections after hearing onset. A small population of spiral ganglion neurons strongly expressed ADF. Based on their relative number, peripheral location within the ganglion, smaller soma size, and coexpression of neurofilament 200, we identified these cells as Type II spiral ganglion neurons. The developmentally regulated ADF expression suggests a temporally restricted function in the stereocilia and, thus, a hitherto undescribed role of ADF.

Spiral ganglion neurones: an overview of morphology, firing behaviour, ionic channels and function.

The spiral ganglion cells provide the afferent innervation of the hair cells of the organ of Corti. Ninety-five percent of these cells (termed type I spiral ganglion neurones) are in synaptic contact with the inner hair cells, whereas about 5% of them are type II cells, which are responsible for the sensory innervation of the outer hair cells. To understand the function of the spiral ganglion neurones, it is important to explore their membrane properties, understand their activity patterns and describe the variety of ionic channels determining their behaviour. In this review, a brief description is given of the various experimental methods that allow the investigation of the spiral ganglion cells, followed by the discussion of their action potential firing patterns and ionic conductances. The presence, distribution and significance of the K(+) currents of the spiral ganglion cells are specifically addressed, along with the introduction of the putative subunit compositions of the relevant voltage-gated K(+) channels.

Frequency map for the human cochlear spiral ganglion: implications for cochlear implants.

The goals of this study were to derive a frequency-position function for the human cochlear spiral ganglion (SG) to correlate represented frequency along the organ of Corti (OC) to location along the SG, to determine the range of individual variability, and to calculate an "average" frequency map (based on the trajectories of the dendrites of the SG cells). For both OC and SG frequency maps, a potentially important limitation is that accurate estimates of cochlear place frequency based upon the Greenwood function require knowledge of the total OC or SG length, which cannot be determined in most temporal bone and imaging studies. Therefore, an additional goal of this study was to evaluate a simple metric, basal coil diameter that might be utilized to estimate OC and SG length. Cadaver cochleae (n = 9) were fixed <24 h postmortem, stained with osmium tetroxide, microdissected, decalcified briefly, embedded in epoxy resin, and examined in surface preparations. In digital images, the OC and SG were measured, and the radial nerve fiber trajectories were traced to define a series of frequency-matched coordinates along the two structures. Images of the cochlear turns were reconstructed and measurements of basal turn diameter were made and correlated with OC and SG measurements. The data obtained provide a mathematical function for relating represented frequency along the OC to that of the SG. Results showed that whereas the distance along the OC that corresponds to a critical bandwidth is assumed to be constant throughout the cochlea, estimated critical band distance in the SG varies significantly along the spiral. Additional findings suggest that measurements of basal coil diameter in preoperative images may allow prediction of OC/SG length and estimation of the insertion depth required to reach specific angles of rotation and frequencies. Results also indicate that OC and SG percentage length expressed as a function of rotation angle from the round window is fairly constant across subjects. The implications of these findings for the design and surgical insertion of cochlear implants are discussed.

A frequency-position function for the human cochlear spiral ganglion.

Greenwood's frequency-position function for the organ of Corti (OC) is commonly used to estimate represented frequencies for cochlear implant (CI) electrodes, both in temporal bone studies and in imaging studies of living CI recipients. However, many contemporary CIs position stimulating electrodes near the modiolus, directly targeting the spiral ganglion (SG) cells within Rosenthal's canal. At the extreme base and apex, the SG does not extend as far as the OC, and the radial nerve fibers take a tangential course into the modiolus resulting in a potential offset between the frequency maps of the OC and SG. In this investigation, human cadaveric cochleae (n = 7) were studied in surface preparations after osmium staining. The OC and SG lengths were measured and radial fiber trajectories traced to identify frequency-matched points on each structure. These data allowed derivation of a mathematical function correlating represented frequency along the OC to position along the SG. A cubic function fit the data with a very high intersubject correlation. Better knowledge of the human SG 'neural frequency map' may help to refine electrode design, and to more accurately map CI channel filter bands to the appropriate cochlear place along the SG, which may be advantageous for more sophisticated CI outcomes, such as music appreciation. These data also could be valuable for electroacoustic stimulation, by defining the insertion distance of a CI electrode required to reach specific frequencies (based upon preoperative imaging) in an individual subject, thus helping to avoid trauma to cochlear regions with residual hearing.

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.

Histopathology of human cochlear implants: correlation of psychophysical and anatomical measures.

The cadavaric temporal bones of five subjects who underwent cochlear implantation during life (2 Nucleus and 3 Ineraid) were analyzed using two-dimensional (2D) reconstruction of serial sections to determine the number of surviving spiral ganglion cells (SGCs) in the region of each electrode of the implanted arrays. The last psychophysical threshold and maximum-comfortable sensation level measured for each electrode were compared to their respective SGC count to determine the across-electrode psychophysical variance accounted for by the SGC counts. Significant correlations between psychophysical measures and SGC counts were found in only two of the five subjects: one Nucleus implantee (e.g., r=-0.71; p<0.001 for threshold vs. count) and one Ineraid implantee (e.g., r=-0.86; p<0.05 for threshold vs. count). A three-dimensional (3D) model of the implanted cochlea was formulated using the temporal-bone anatomy of the Nucleus subject for whom the 2D analysis did not result in significant correlations between counts and psychophysical measures. Predictions of the threshold vs. electrode profile were closer to the measured profile for the 3D model than for the 2D analysis. These results lead us to hypothesize that 3D techniques will be required to asses the impact of peripheral anatomy on the benefit patients derive from cochlear implantation.

Cholinergic innervation of the chick basilar papilla.

Antibodies directed against choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine (ACh) and a specific marker of cholinergic neurons, were used to label axons and nerve terminals of efferent fibers that innervate the chick basilar papilla (BP). Two morphologically distinct populations of cholinergic fibers were labeled and classified according to the region of the BP they innervated. The inferior efferent system was composed of thick fibers that coursed radially across the basilar membrane in small fascicles, gave off small branches that innervated short hair cells with large cup-like endings, and continued past the inferior edge of the BP to ramify extensively in the hyaline cell area. The superior efferent system was made up of a group of thin fibers that remained in the superior half of the epithelium and innervated tall hair cells with bouton endings. Both inferior and superior efferent fibers richly innervated the basal two thirds of the BP. However, the apical quarter of the chick BP was virtually devoid of efferent innervation except for a few fibers that gave off bouton endings around the peripheral edges. The distribution of ChAT-positive efferent endings appeared very similar to the population of efferent endings that labeled with synapsin antisera. Double labeling with ChAT and synapsin antibodies showed that the two markers colocalized in all nerve terminals that were identified in BP whole-mounts and frozen sections. These results strongly suggest that all of the efferent fibers that innervate the chick BP are cholinergic.

Immunolocalization of Bone Morphogenetic Protein-2 and -3 and Osteogenic Protein-1 during murine tooth root morphogenesis and in other craniofacial structures.

The distribution of Bone Morphogenetic Protein-2, and -3 (BMP-2 and BMP-3) and Osteogenic Protein-1 (OP-1, also known as BMP-7) during root morphogenesis and in other craniofacial structures was examined in sections of 12- to 18-d-old mouse heads using polyclonal and monoclonal antibodies. BMP-3 and OP-1 were localized in alveolar bone, cementum, and periodontal ligament, whereas BMP-2 was only localized in the alveolar bone of periodontium. All three BMPs were localized in predentine, dentine, odontoblasts, osteoblasts, osteocytes, osteoid, cartilage, chondrocytes and spiral limbus. BMP-2 and OP-1 were also localized in spiral ligament and interdentate cells of the cochlea, whilst BMP-3 was restricted to the spiral ganglion. BMP-3 was also localized in ducts of submandibular and sublingual salivary glands, acini of the lacrimal gland, Purkinje cells in the cerebellum, nerve fibres of the cerebellum and brain, afferent cells of the dorsal root ganglia, inferior alveolar nerve, and peripheral processes of the vestibulocochlear nerve. OP-1 was also localized in hair and whisker follicles, sclera of the eye and in ameloblasts. The demonstration of BMP-3 in the nervous system suggests that this protein may be neurotrophic during development and maintenance of the nervous system. The composite expression of BMPs/OPs during periodontal tissue morphogenesis suggests that optimal therapeutic regeneration may entail the combined use of different BMPs/OPs.

Magnetic resonance imaging of the cochlea, spiral ganglia and eighth nerve of the guinea pig.

The membranous labyrinth of the guinea pig cochlea and retrocochlear neural structures were investigated by magnetic resonance imaging (MRI) using an experimental system with a field strength of 4.7T and a single turn surface coil 25 mm in diameter, or standard resonators of 34 or 70 mm in diameter and gradient field strengths of 950 mTm and 200 mTm. High-resolution 2-D and 3-D images of 0.3-1.0 mm slice thickness were acquired by a rapid acquisition with relaxation enhancement (RARE) sequence and a standard multi-echo technique. Structural and dimensional aspects of the cochlea were resolved in vitro and in vivo down to <50 microm, showing the scala vestibule, scala media, scala tympani, spiral ganglia and the cochlear (eighth) nerve. In vivo perfusions with the gadodiamide (GdDTPA-BMA) chelate-bound paramagnetic gadolinium ion resulted in dynamic temporal enhancement of the scala vestibule and scala tympani, but did not penetrate the scala media.

Intracochlear factors contributing to psychophysical percepts following cochlear implantation.

The performance of cochlear implant patients may be related to intracochlear, histopathological factors. We have performed detailed post-mortem examinations of five human, implanted cochleas and for each electrode correlated the psychophysical threshold, comfortable level and dynamic range with spiral ganglion cell survival, presence of fibrous tissue and/or new bone, and distance between the centers of the electrode bands and Rosenthal's canal. The psychophysical parameters were strongly interrelated. Threshold and comfort levels correlated with the distance between the electrodes and Rosenthal's canal. Threshold levels also correlated with the presence of intracochlear fibrous tissue and new bone, especially with the former. The dynamic range showed a negative correlation with intracochlear pathology, especially with new bone. Comfort levels and dynamic range were related to spiral ganglion cell survival. The distance between the electrodes and the modiolus increased with increasing levels of fibrous tissue and new bone. Spiral ganglion cell survival was decreased with increasing levels of fibrous tissue and new bone.

Image analysis of the human inner ear.

The KS 300 is a multifunctional software image analysis system using an object-oriented programming environment. The possibility of its application for the inner ear was studied by using specimens from humans and squirrel monkeys, immunostained for the brain-derived calcium-binding protein, S-100 protein. Grey images were used for measurements. The cell borders were outlined by hand, using a digitizer. The absolute grey values of the pixels changed when the brightness of the images or other conditions changed. By contrast, the relative grey values, i.e. the absolute grey values correlated to the mean grey values of the histoimage, remained constant. By utilizing these relative grey values, it was possible to compare cells both between different specimens and between different areas within the same specimen. The different grey values of spiral ganglion cells stained for S-100 protein are objective quantitative measurements and are believed to reflect differences in their function. In some regions of both human and squirrel monkey specimens, relatively intensely stained cells predominated, whereas in other regions, relatively weakly stained cells were mainly observed. Thus, our image analysis system using the relative grey values has proved suitable for quantitative analysis of immunostained specimens in order to compare them and to assess cell function.