Hair-cell versus afferent adaptation in the semicircular canals.

The time course and extent of adaptation in semicircular canal hair cells was compared to adaptation in primary afferent neurons for physiological stimuli in vivo to study the origins of the neural code transmitted to the brain. The oyster toadfish, Opsanus tau, was used as the experimental model. Afferent firing-rate adaptation followed a double-exponential time course in response to step cupula displacements. The dominant adaptation time constant varied considerably among afferent fibers and spanned six orders of magnitude for the population ( approximately 1 ms to >1,000 s). For sinusoidal stimuli (0.1-20 Hz), the rapidly adapting afferents exhibited a 90 degrees phase lead and frequency-dependent gain, whereas slowly adapting afferents exhibited a flat gain and no phase lead. Hair-cell voltage and current modulations were similar to the slowly adapting afferents and exhibited a relatively flat gain with very little phase lead over the physiological bandwidth and dynamic range tested. Semicircular canal microphonics also showed responses consistent with the slowly adapting subset of afferents and with hair cells. The relatively broad diversity of afferent adaptation time constants and frequency-dependent discharge modulations relative to hair-cell voltage implicate a subsequent site of adaptation that plays a major role in further shaping the temporal characteristics of semicircular canal afferent neural signals.

Ultrastructural observations of efferent terminals in the crista ampullaris of the toadfish, Opsanus tau.

The present study was conducted to visualize the ultrastructural features of vestibular efferent boutons in the oyster toadfish, Opsanus tau. The crista ampullaris of the horizontal semicircular canal was processed for and examined by routine transmission electron microscopy. The results demonstrate that such boutons vary in size and shape, and contain a heterogeneous population of lucent vesicles with scattered dense core vesicles. Efferent contacts with hair cells are characterized by local vesicle accumulations in the presynaptic terminal and a subsynaptic cistern in the postsynaptic region of the hair cell. Serial efferent to hair cell to afferent synaptic arrangements are common, particularly in the central portion of the crista. However, direct contacts between efferent terminals and afferent neurites were not observed in our specimens. The existence of serial synaptic contacts, often with a row of vesicles in the efferent boutons lining the efferent-afferent membrane apposition, suggests that the efferent influence on the crista may involve both synaptic and nonsynaptic, secretory mechanisms. Further, it is suggested that differences in more subtle aspects of synaptic architecture and/or transmitter and receptor localization and interaction may render the efferent innervation of the peripheral crista less effective in influencing sensory processing.

Memory retention of vestibuloocular reflex motor learning in squirrel monkeys.

The vestibuloocular reflex (VOR) motor learning can be induced chronically by wearing lenses for several weeks to months, or acutely by visual-vestibular mismatch for several hours. Cerebellar long term depression (LTD) has been proposed as a causal mechanism for acute learning. We demonstrate differences in retention of acutely and chronically acquired VOR gains in squirrel monkeys and discuss neuronal correlates and possible roles of cerebellar LTD. Our data is compatible with the idea that cerebellar LTD might be a mechanism responsible for acute VOR adaptation.

Ultrastructural observations of efferent terminals in the crista Ampullaris of the toadfish, opsanus tau.

The present study was conducted to visualize the ultrastructural features of vestibular efferent boutons in the oyster toadfish, Opsanus tau. The crista ampullaris of the horizontal semicircular canal was processed for and examined by routine transmission electron microscopy. The results demonstrate that such boutons vary in size and shape, and contain a heterogeneous population of lucent vesicles with scattered dense core vesicles. Efferent contacts with hair cells are characterized by local vesicle accumulations in the presynaptic terminal and a subsynaptic cistern in the postsynaptic region of the hair cell. Serial efferent to hair cell to afferent synaptic arrangements are common, particularly in the central portion of the crista. However, direct contacts between efferent terminals and afferent neurites were not observed in our specimens. The existence of serial synaptic contacts, often with a row of vesicles in the efferent boutons lining the efferent-afferent membrane apposition, suggests that the efferent influence on the crista may involve both synaptic and nonsynaptic, secretory mechanisms. Further, it is suggested that differences in more subtle aspects of synaptic architecture and/or transmitter and receptor localization and interaction may render the efferent innervation of the peripheral crista less effective in influencing sensory processing.

Capacity of vertical VOR adaptation in squirrel monkey.

Squirrel monkeys were trained using newly developed visual-vestibular mismatch paradigms to test the asymmetrical simultaneous induction of vertical vestibuloocular reflex (VOR) gain changes in opposite directions (high and low) either in the upward and downward directions or in response to high- and low-frequency stimuli. The first paradigm consists of sinusoidal head movement [A sin(omegat)] and a full rectified sinusoidal optokinetic stimulus [+/-|A sin(omegat)|], whereas the second paradigm consists of the sum of two sinusoids with different frequencies [A sin(omega(1)t) + A sin(omega(2)t) for head motion and +/-[A sin(omega(1)t) - A sin(omega(2)t)] for the optokinetic stimulus, omega(1) = 0.1pi, omega(2) = 5pi]. The first paradigm induced a half rectified sinusoidal eye-velocity trace, i.e., suppression of the VOR during upward head motion and enhancement during downward head motion or vise versa, whereas the second paradigm induced suppression of the VOR at the low-frequency omega(1) and enhancement at the high-frequency omega(2) or vise versa. After 4 h of exposure to these paradigms, VOR gains of up and down or high and low frequency were modified in opposite directions. We conclude that the monkey vertical VOR system is capable of up-down directionally differential adaptation as well as high-low frequency differential adaptation. However, experiments also suggest that these gain controls are not completely independent because the magnitudes of the gain changes during simultaneous asymmetrical training were less than those achieved by symmetrical training or training in only one of the two components, indicating an influence of the gain controls on each other. These results confine the adaptive site(s) responsible for vertical VOR motor learning to those that can process up and downward or low- and high-frequency head signal separately but not completely independently.

Monitoring kinetic and frequency-domain properties of eyelid responses in mice with magnetic distance measurement technique.

Classical eye-blink conditioning in mutant mice can be used to study the molecular mechanisms underlying associative learning. To measure the kinetic and frequency domain properties of conditioned (tone - periorbital shock procedure) and unconditioned eyelid responses in freely moving mice, we developed a method that allows adequate, absolute, and continuous determination of their eyelid movements in time and space while using an electrical shock as the unconditioned stimulus. The basic principle is to generate a local magnetic field that moves with the animal and that is picked up by either a field-sensitive chip or coil. With the use of this magnetic distance measurement technique (MDMT), but not with the use of electromyographic recordings, we were able to measure mean latency, peak amplitude, velocity, and acceleration of unconditioned eyelid responses, which equaled 7.9 +/- 0.2 ms, 1.2 +/- 0.02 mm, 28.5 +/- 1 mm/s, and 637 +/- 22 mm/s(2), respectively (means +/- SD). During conditioning, the mice reached an average of 78% of conditioned responses over four training sessions, while animals that were subjected to randomly paired conditioned and unconditioned stimuli showed no significant increases. The mean latency of the conditioned responses decreased from 222 +/- 40 ms in session 2 to 127 +/- 6 ms in session 4, while their mean peak latency increased from 321 +/- 45 to 416 +/- 67 ms. The mean peak amplitudes, peak velocities, and peak acceleration of these responses increased from 0.62 +/- 0.02 to 0.77 +/- 0.02 mm, from 3.9 +/- 0.3 to 7.7 +/- 0.5 mm/s, and from 81 +/- 7 to 139 +/- 10 mm/s(2), respectively. Power spectra of acceleration records illustrated that both the unconditioned and conditioned responses of mice had oscillatory properties with a dominant peak frequency close to 25 Hz that was not dependent on training session, interstimulus interval, or response size. These data show that MDMT can be used to measure the kinetics and frequency domain properties of conditioned eyelid responses in mice and that these properties follow the dynamic characteristics of other mammals.

Relationship between inner-ear fluid pressure and semicircular canal afferent nerve discharge.

The present study was designed to determine (1) the transcupular fluid pressure (deltaP) generated across the semicircular canal cupula in response to sinusoidal head rotation, (2) the translabyrinthine dilational pressure (P0) generated across the membranous labyrinth in response to an increase in endolymph fluid volume (hydrops), (3) afferent nerve discharge patterns generated by these distinct pressure stimuli and, (4) threshold values of deltaP and P0 required to elicit afferent neural responses. The experimental model was the oyster toadfish, Opsanus tau. Micromechanical indentation of the horizontal canal (HC) duct and utricular vestibule was used to simulate sinusoidal head rotation and fluid volume injection. Single-unit neural spike trains and endolymph pressure within the ampulla, on both sides of the cupula, were recorded simultaneously. deltaP averaged 0.013 Pa per 1 degrees/s of sinusoidal angular head velocity and P0 averaged 0.2 Pa per 1 nL of endolymph volume injection. The most responsive afferents had a threshold sensitivity to deltaP of 10(-3) Pa and to P0 of 5 x 10(-2) Pa based on a discharge modulation criterion of 1 impulse/s per cycle for 2 Hz pressure stimuli. Neural sensitivity to AP was expected on the basis of transverse cupular and hair bundle deflections. Analysis of mechanics of the end organ, neuronal projections into the crista, and individual neural firing patterns indicates that P0 sensitivity resulted from pressure-induced distension of the ampulla that led to a nonuniform cupular deformation pattern and hair bundle deflections. This explanation is consistent with predictions of a finite element model of the end organ. Results have implications regarding the role of deltaP in angular motion transduction and the role of P0 under transient hydropic conditions.

Physiology of the semicircular canals after surgical plugging.

Inactivation of individual semicircular canals by surgical occlusion (plugging) of the slender duct has been used in basic studies to elucidate the role of individual canal inputs to vestibular-mediated control systems and in clinical applications to treat certain vestibular disorders. The procedure has been shown to be highly effective in blocking sensitivity of individual canals, at least for moderate angular motion stimuli. Effectiveness does not extend to stimuli involving high accelerations where a residual response persists even after complete occlusion of the duct. The residual can be quite large at high-stimulus frequencies where sensitivity to angular motion approaches that of patent canals. The overall physiological effect of canal plugging is reported here in terms of the frequency-dependent attenuation in gain and phase shift of primary afferents. Plug-canal responses are quantitatively described in terms of biomechanics of the deformable labyrinth.

How endolymph pressure modulates semicircular canal primary afferent discharge.

Histological observation of endolymphatic hydrops in subjects suffering from Meniere's disease/syndrome and the presence of vestibular symptoms in experimentally induced endolymphatic hydrops have led to considerable debate regarding the potential role of inner-ear pressure. Could a condition influencing endolymphatic volume regulation lead to transient changes in translabyrinthine pressure sufficiently large to alter primary afferent discharge rates? To investigate this question we built a highly sensitive laser-based pressure sensor and recorded endolymphatic pressure modulations within the horizontal semicircular canal ampulla in response to mechanical stimuli for simple head rotation and controlled volume injection. The most sensitive primary afferents responded to changes in endolymph pressures as low as 0.005 Pa (re: perilymph)--a pressure considerably lower than that observed in most experimentally induced hydropic conditions. This threshold pressure was generated by a approximately 25 picoliter volume injection stimulus. Semicircular canal afferent responses to endolymphatic pressure can be explained on the basis of pressure-induced hydrops, which causes dilation of the ampulla, deformation of the cupula, hair bundle movement, and afferent discharge. Primary afferent responses to maintained stimuli were transient in nature and recovered to pre-stimulus background discharge rates following a period of adaptation. Results demonstrate that the semicircular canals are indeed sensitive to small changes in translabyrinthine pressure in addition to head-movement-related changes in transcupular pressure.

Neural readaptation to Earth's gravity following return from space.

The consequence of exposure to microgravity on the otolith organs was studied by recording the responses of vestibular nerve afferents supplying the utricular otolith organ to inertial accelerations in four toadfish, Opsanus tau, sequentially for 5 days following two National Aeronautics and Space Administration shuttle orbital flights. Within the first day postflight, the magnitude of response to an applied translation was on average three times greater than for controls. The reduced gravitational acceleration in orbit apparently resulted in an upregulation of the sensitivity of utricular afferents. By 30 h postflight, responses were statistically similar to control. The time course of return to normal afferent sensitivity parallels the reported decrease in vestibular disorientation in astronauts following return from space.

Acute adaptation of the vestibuloocular reflex: signal processing by floccular and ventral parafloccular Purkinje cells.

The gain of the vertical vestibuloocular reflex (VVOR), defined as eye velocity/head velocity was adapted in squirrel monkeys by employing visual-vestibular mismatch stimuli. VVOR gain, measured in the dark, could be trained to values between 0.4 and 1.5. Single-unit activity of vertical zone Purkinje cells was recorded from the flocculus and ventral paraflocculus in alert squirrel monkeys before and during the gain change training. Our goal was to evaluate the site(s) of learning of the gain change. To aid in the evaluation, a model of the vertical optokinetic reflex (VOKR) and VVOR was constructed consisting of floccular and nonfloccular systems divided into subsystems based on the known anatomy and input and output parameters. Three kinds of input to floccular Purkinje cells via mossy fibers were explicitly described, namely vestibular, visual (retinal slip), and efference copy of eye movement. The characteristics of each subsystem (gain and phase) were identified at different VOR gains by reconstructing single-unit activity of Purkinje cells during VOKR and VVOR with multiple linear regression models consisting of sensory input and motor output signals. Model adequacy was checked by evaluating the residual following the regressions and by predicting Purkinje cells' activity during visual-vestibular mismatch paradigms. As a result, parallel changes in identified characteristics with VVOR adaptation were found in the prefloccular/floccular subsystem that conveys vestibular signals and in the nonfloccular subsystem that conveys vestibular signals, while no change was found in other subsystems, namely prefloccular/floccular subsystems conveying efference copy or visual signals, nonfloccular subsystem conveying visual signals, and postfloccular subsystem transforming Purkinje cell activity to eye movements. The result suggests multiple sites for VVOR motor learning including both flocculus and nonflocculus pathways. The gain change in the nonfloccular vestibular subsystem was in the correct direction to cause VOR gain adaptation while the change in the prefloccular/floccular vestibular subsystem was incorrect (anti-compensatory). This apparent incorrect directional change might serve to prevent instability of the VOR caused by positive feedback via the efference copy pathway.

Analysis of the discharge pattern of floccular Purkinje cells in relation to vertical head and eye movement in the squirrel monkey.

Purkinje (P) neurons are highly irregular in their discharge patterns having a high coefficient of variation. We have developed a method to extract the signal from these noisy neurons employing multiple linear regression against the input signals that are causal in P-cell firing, namely retinal slip, head motion parameters, and the efference copy of the oculomotor commands. This method was shown to extract all of the experimental stimulus-related signal as the residual following extraction had the same amplitude distribution and spectral content as those of the spontaneous discharge of P-cells when no external stimuli were applied. The method enables us to decompose P-cell firing modulation into components mediated by mossy fiber inputs to flocculus, that is, retinal slip, vestibular and oculomotor efference copy signals. By evaluating these components in relation to VOR adaptation, one can argue that one role of the flocculus is in converting these sensory and efference copy input signals into eye movement-related signals in conjunction with VOR motor learning.

Input of anterior and posterior semicircular canal interneurons encoding head-velocity to the dorsal Y group of the vestibular nuclei.

Neurons in the Y group of the vestibular nuclei are activated disynaptically from the ipsilateral VIIIth nerve and polysynaptically from the contralateral nerve. The ipsilateral anterior and posterior semicircular canals project to the Y group via interneurons in the vestibular nuclei. Candidate interneurons located in the rostrolateral corner of the superior (SVN) and in the caudal medial (MVN) vestibular nuclei were retrogradely labeled by the iontophoretic injection of biocytin into the Y group. The physiology of these interneurons named Y-group projecting neurons (YPNs) was studied in the SVN. SVN-YPNs were activated antidromically by electric pulse stimulation in the Y group. The properties of SVN-YPNs are distinct from those of SVN flocculus projecting neurons (FPNs). Namely, YPNs have a lower resting rate than FPNs, have more irregular interspike intervals, show a different phase and gain during the vestibuloocular reflex, and are located differentially within the SVN. After the injection of biocytin into the Y group, the locations of Purkinje cells that project to the Y group were confined to the vertical zones of the flocculus and ventral paraflocculus. However, mossy fibers originating in the Y group terminate in both the vertical and horizontal zones of the flocculus and ventral paraflocculus as well as in the ipsilateral nodulus.

Chronic recording of regenerating VIIIth nerve axons with a sieve electrode.

A micromachined silicon substrate sieve electrode was implanted within transected toadfish (Opsanus tau) otolith nerves. High fidelity, single unit neural activity was recorded from seven alert and unrestrained fish 30 to 60 days after implantation. Fibrous coatings of genetically engineered bioactive protein polymers and nerve guide tubes increased the number of axons regenerating through the electrode pores when compared with controls. Sieve electrodes have potential as permanent interfaces to the nervous system and to bridge missing connections between severed or damaged nerves and muscles. Recorded impulses might also be amplified and used to control prosthetic devices.

Influence of surgical plugging on horizontal semicircular canal mechanics and afferent response dynamics.

Mechanical occlusion of one or more of the semicircular canals is a surgical procedure performed clinically to treat certain vestibular disorders and used experimentally to assess individual contributions of separate canals and/or otoliths to vestibular neural pathways. The present experiments were designed to determine if semicircular canal afferent nerve modulation to angular head acceleration is blocked by occlusion of the endolymphatic duct, and if not, what mechanism(s) might account for a persistent afferent response. The perilymphatic space was opened to gain acute access to the horizontal canal (HC) in the oyster toadfish, Opsanus tau. Firing rate responses of HC afferents to sinusoidal whole-body rotation were recorded in the unoccluded control condition, during the process of duct occlusion, and in the plugged condition. The results show that complete occlusion of the duct did not block horizontal canal sensitivity; individual afferents often exhibited a robust firing rate modulation in response to whole-body rotation in the plugged condition. At high stimulus frequencies (about >8 Hz) the average sensitivity (afferent gain; spikes/s per degrees /s of head velocity) in the plugged condition was nearly equal to that observed for unoccluded controls in the same animals. At low stimulus frequencies (about <0.1 Hz), the average sensitivity in the plugged condition was attenuated by more than two orders of magnitude relative to unoccluded controls. The peak afferent firing rate for sinusoidal stimuli was phase advanced approximately 90 degrees in plugged canals relative to their control counterparts for stimulus frequencies approximately 0.1-2 Hz. Data indicate that afferents normally sensitive to angular velocity in the control condition became sensitive to angular acceleration in the plugged condition, whereas afferents sensitive to angular acceleration in the control condition became sensitive to the derivative of acceleration or angular jerk in the plugged condition. At higher frequencies (>8 Hz), the phase of afferents in the plugged condition became nearly equal, on average, to that observed in controls. A three-dimensional biomechanical model of the HC was developed to interpret the residual response in the plugged condition. Labyrinthine fluids were modeled as incompressible and Newtonian; the membranous duct, osseous canal and temporal bone were modeled as visco-elastic materials. The predicted attenuation and phase shift in cupular responses were in close agreement with the observed changes in afferent response dynamics after canal plugging. The model attributes the response of plugged canals to labyrinthine fluid pressure gradients that lead to membranous duct deformation, a spatial redistribution of labyrinthine fluids and cupular displacement. Validity of the model was established through its ability to predict: the relationship between plugged canal responses and unoccluded controls (present study), the relationship between afferent responses recorded during mechanical indentation of the membranous duct and physiological head rotation, the magnitude and phase of endolymphatic pressure generated during HC duct indentation, and previous model results for cupular gain and phase in the rigid-duct case. The same model was adjusted to conform to the morphology of the squirrel monkey and of the human to investigate the possible influence of canal plugging in primates. Membranous duct stiffness and perilymphatic cavity stiffness were identified as the most salient model parameters. Simulations indicate that canal plugging may be the most effective in relatively small species having small labyrinths, stiff round windows, and stiff bony perilymphatic enclosures.

Characteristics of regenerating horizontal semicircular canal afferent and efferent fibers in the toadfish, Opsanus tau.

The horizontal semicircular canal nerve of the toadfish, Opsanus tau, was transected and allowed to regenerate. The time course, morphometrics, and projection patterns of regenerating afferent and efferent vestibular fibers were determined. Nerve transections were performed both pre- and postganglionically, and regeneration was assessed in afferent and efferent fibers by bulk labeling the peripheral axons of the horizontal semicircular canal nerve with biocytin after nerve regrowth. Afferent fibers regrew through the transection site within 14 days and projected to all vestibular nuclei within 3 weeks. Bouton and branch number, axon length, surface area, volume, fiber diameter, and internodal distance were quantified for afferent fibers from eight sites within the vestibular nuclei, and axon number and soma size was quantified for the efferent fibers. Extensive regeneration was seen within 5 weeks of transection in all nuclei, and most morphometric parameters approached or exceeded control levels within 10 weeks. Regeneration appeared to recapitulate morphogenesis with an initial overproduction of boutons and branch points followed by elimination of presumably superfluous structures. Internodal distance remained significantly shorter in regenerating afferent axons than in control fish throughout the 15-week observation period. Efferent fibers also were observed to regenerate. Efferent axon number, diameter, and soma size were indistinguishable from those in controls from 3 weeks posttransection through week 15. Electrophysiological recordings from the horizontal canal nerve during mechanical stimuli of the canal confirmed that the regenerated axons transmitted normal signals. The return of normal equilibrium and behavior coincided with the projection of afferent fibers into the central vestibular nuclei, indicating that functional connections had been reestablished.

Dendritic arbors and central projections of physiologically characterized auditory fibers from the saccule of the toadfish, Opsanus tau.

Neurobiotin was injected iontophoretically into saccular afferents of toadfish (Opsanus tau) after intracellular recording to examine dendritic arbors and central projections with respect to the physiological and directional response properties of the cells. Dendritic arbors of 36 afferents were examined in detail. Maximum diameter of the arbor and the number of terminal points were positively correlated with each other, but neither was predictive of spontaneous activity or sensitivity. Best azimuths were centered around 30 degrees -40 degrees, which corresponds to the angle of the saccule with respect to the fish's midline. In general, best elevations for afferents corresponded to hair cell orientations in the region innervated; unexpectedly low elevations obtained from afferents innervating the middle saccule may reflect curvature of the sensory epithelium against the otolith. Three efferent cells were filled partially. The location and large size of the efferent projections indicate that activity along the saccule could be modulated by a single efferent. All afferents projected to the dorsal zone of the descending octaval nucleus (dDON); many afferents bifurcated to terminate in the anterior octaval nucleus, and a few of those also had terminal fields in the medial zone of DON. All afferent projections into the dDON consisted of multiple axon collaterals projecting to numerous sites along the rostral-caudal extent of the nucleus. Variation in terminal field sites also was noted in the medial to lateral axis of the dDON; however, there were no consistent correlations between terminal field locations, physiology, and best directions of the saccular afferents.

Anatomy and physiology of the primate interstitial nucleus of Cajal. II. Discharge pattern of single efferent fibers.

Anatomy and physiology of the primate interstitial nucleus of Cajal. II. Discharge pattern of single efferent fibers. J. Neurophysiol. 80: 3100-3111, 1998. Single efferent fibers of the interstitial nucleus of Cajal (NIC) were characterized physiologically and injected with biocytin in alert behaving monkeys. Quantitative analysis demonstrated that their discharge encodes a constellation of oculomotor variables. Tonic and phasic signals were related to vertical (up or down) eye position and saccades, respectively. Depending on how they encoded eye position, saccade velocity, saccade size, saccade duration, and smooth-pursuit eye velocity, fibers were characterized as regular or irregular, bi- or unidirectionally modulated, more or less sensitive, and reliable or unreliable. Further, fibers that did not burst for saccades (tonic) and fibers the eye-position and saccade-related signals of which increased in the same (in-phase) or in the opposite (anti-phase) directions were encountered. A continuum of discharge properties was the rule. We conclude that NIC efferent fibers send a combination of eye-position, saccade-, and smooth-pursuit-related signals, mixed in proportions that differ for different fibers, to targets of the vertical neural integrator such as extraocular motoneurons.

Examination of the cupula and stereocilia of the horizontal semicircular canal in the toadfish Opsanus tau.

We imaged the horizontal semicircular canal (HSCC) crista and cupula of toadfish, Opsanus tau, by using a) confocal light microscopy of isolated vital HSCC; b) serial sections of fixed, trichrome-stained HSCC; and c) scanning electron microscopy of fixed HSCCs. HSCC were dissections which included an ampulla and an attached canal tube (long and slender canal portion), and, in some cases, a small portion of the utricular wall. Cupulae were seen as multipartite mucus connective tissue shells rising from the crista and extending toward the ampullary roof. They were composed of several refractile bands traversing the cupulae perpendicular to longitudinal fibers extending from the cupular base to its apex. Alcian green-stained cupulae showed an asymmetric alcianphilic, dark, X-shaped structure, indicating that the pillar is rich in mucin and carbohydrate, an interpretation supported by images of trichrome-stained sections. The cupular antrum is devoid of prominent refractile fibers. No tubes or channels were observed in the cupula or antrum of vital preparations. Cupular shell fibers cover the surface of the crista, are roughly parallel, and are associated with a translucent material having a refractive index greater than the surrounding endolymph. Stereocilia were thin, 100-microm-long structures, with little longitudinal curvature, which end with no end bulb. No strands extend from stereocilia to the roof or other portions of the cupular antrum. Gross movements of stereocilia were not seen in mechanically quiescent preparations. Within the cupular antrum, stereocilia were parallel to connective tissue fibers, all embedded in an isotropic gel. This fiber-reinforced gel and cupular matrix are sensitive to N-acetlyneuraminidase and beta-N-acetyl glucosaminidase, and minimally sensitive to beta-N-acetyl hexosaminidase. Connective tissue fibers may serve to stiffen the gel, whose matrix would restrict lateral motion of embedded fibers and stereocilia thereby providing mechanical support for stereocilia.