Dynamic Displacement of Normal and Detached Semicircular Canal Cupula
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- Rabbitt, R.D., Breneman, K.D., King, C. et al. JARO (2009) 10: 497. doi:10.1007/s10162-009-0174-y
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The dynamic displacement of the semicircular canal cupula and modulation of afferent nerve discharge were measured simultaneously in response to physiological stimuli in vivo. The adaptation time constant(s) of normal cupulae in response to step stimuli averaged 36 s, corresponding to a mechanical lower corner frequency for sinusoidal stimuli of 0.0044 Hz. For stimuli equivalent to 40–200 deg/s of angular head velocity, the displacement gain of the central region of the cupula averaged 53 nm per deg/s. Afferents adapted more rapidly than the cupula, demonstrating the presence of a relaxation process that contributes significantly to the neural representation of angular head motions by the discharge patterns of canal afferent neurons. We also investigated changes in time constants of the cupula and afferents following detachment of the cupula at its apex—mechanical detachment that occurs in response to excessive transcupular endolymph pressure. Detached cupulae exhibited sharply reduced adaptation time constants (300 ms–3 s, n = 3) and can be explained by endolymph flowing rapidly over the apex of the cupula. Partially detached cupulae reattached and normal afferent discharge patterns were recovered 5–7 h following detachment. This regeneration process may have relevance to the recovery of semicircular canal function following head trauma.
Keywordsvestibularinner ear micromechanicscupula regenerationangular motion sensationafferent response dynamics
Here, we directly measured the adaptation time constant and lower corner frequency of semicircular canal cupulae to physiological stimuli in the living animal while simultaneously recording individual afferent responses. Responses were measured both in normal animals and in animals with damaged cupulae (detached at the apex). The oyster toadfish, Opsanus tau, was used as the experimental model because of the similarity in dimensions to the human labyrinth and to facilitate the experimental approach. Results reveal the mechanical lower corner frequency to align with the lowest corner frequency of the population of velocity-sensitive afferent neurons. In a subset of experiments, we examined the motion of cupula that had become detached from the apex of the ampulla. It has been reported previously that detachment at the apex acts like a relief valve and occurs under high transcupular pressures that may arise during trauma (Hillman 1974; Rabbitt et al. 1999). The present work examined temporal responses of detached cupulae and corresponding afferents as well as the time course of reattachment and recovery of normal afferent responses.
Adult oyster toadfish (O. tau) were obtained from the Marine Biological Laboratory (Woods Hole, MA, USA) and all experiments were carried out under protocols approved at the Marine Biological Laboratory or the University of Utah. The surgical approach followed that described previously (Rabbitt et al. 2005). Briefly, each fish was anesthetized with MS222 (5 mg/L in seawater) and partially immobilized by an intramuscular injection of pancuronium bromide (0.05 mg/kg; Sigma, St. Louis, MO, USA) in the tail. The fish was secured in an acrylic tank, with two thirds of its body immersed in bubbled seawater, while the remainder of the body was covered with moist tissues. A craniotomy was made lateral to the dorsal course of the anterior canal to expose the horizontal canal ampulla and ∼8 mm of the slender membranous labyrinth (Fig. 1B). The orientation of the labyrinth viewed from the dorsal direction (microscope objective axis) is illustrated in Figure 1A (Ghanem et al. 1998). Perilymph within the upper region of the surgical cavity was replaced with fluorocarbon (FC-75, 3 M, Minneapolis, MN, USA) to improve optical clarity and electrosurgical cutting outcomes. The electrical insulating properties of fluorocarbon allowed us to cut a fistula in the horizontal canal ampulla ∼300 μm medial to the cupula using an electrosurgical generator (Valleylab, Denver, CO, USA) set to a ∼5-W cutting waveform. A sharpened 76-μm dia. tungsten wire served as the cutting electrode. This generated ∼50- to 100-μm diameter hole in the membranous wall allowing access for delivery of fluorescent microspheres into the endolymph (Fig. 1B, beads). The microspheres were allowed to diffuse from the fistula to the cupula. It is important to note that fluorocarbon is immiscible with endolymph, and as a result, there was a surface tension barrier preventing flow of endolymph out of the fistula (Rajguru and Rabbitt 2007). Canal afferent responses to controlled stimuli were recorded after generation of the fistula and compared to control recordings to confirm normal sensory transduction and neural coding, indicating that the dynamics are indeed dominated by the long and slender canal segment.
Although great effort was taken to prevent damage to the cupula during this procedure, in a subset of animals, we found afferent modulation to be reduced, and in some cases, we observed a fraction of the fluorescent microspheres to diffuse over the apex of the cupula and into the lateral side of the ampulla. These animals defined the “dislodged cupula” group. Even in these damaged cases, the cupula remained structurally sound, but simply detached from the interior surface of the membranous ampulla at its apex. Present results address cupula responses and reattachment in modestly damaged cases where the cupula was dislodged at the apex but remained located in the normal central region of the ampulla.
Mechanical indentation of the slender limb of the membranous duct was used as the primary stimulus. This idea was first introduced by Ewald in 1892 (Camis 1930) and later refined by Dickman et al. to mimic physiological head movements and produce nearly equivalent afferent discharge patterns (Dickman and Correia 1989; Dickman et al. 1988). We used a version of the approach detailed by Rabbitt et al. (1995) where 1 μm of mechanical indentation of the horizontal canal limb generates mechanical cupula motion and afferent responses nearly equivalent to ∼4 deg/s of angular head velocity stimulus in the present experimental organism. In a subset of experiments, the whole animal was rotated using sinusoidal angular velocity stimuli (Boyle and Highstein 1990). Cupula displacements were recorded for mechanical indentation stimuli, but not for angular velocity stimuli.
Horizontal canal neural recordings used glass microelectrodes (∼100 MΩ) following the approach described previously (Boyle and Highstein 1990; Rabbitt et al. 2005). Electrodes were positioned using a micromanipulator at a location ∼500 μm from the horizontal canal ampulla where the nerve branch is accessible (Fig. 1B). Extracellular potentials were measured using standard amplification (EXT-02F, npi electronic, Tamm, Germany). Afferent responses and mechanical stimuli were amplified, filtered at 2 kHz (LHBF-48X, npi electronic), and sampled at 5 kHz (ITC-18, HEKA Instruments, Inc., Bellmore, NY, USA).
Carboxylate-modified, neutrally buoyant fluorescent microspheres (beads) were obtained (Bangs Labs, Inc. Fishers, IN, USA) and surface-modified to bind wheat germ agglutinin (WGA). Raw beads in suspension were washed twice in MES buffer with 10 mg EDAC and incubated for 15 min. Following incubation, the beads were washed and resuspended in 0.1 M borate buffer (pH 8.5) with 1 mg WGA (Sigma-Aldrich) and incubated with gentle mixing for 4 h. The beads were then washed and resuspended in 0.1 M borate buffer with bovine serum albumin (BSA; 10 mg/mL) and mixed continuously for 15 min prior to a final wash and resuspension in MES buffer with BSA (10 mg/mL) for storage. Microspheres were placed in toadfish artificial endolymph (Ghanem et al. 2008), vortex-mixed, and loaded into a glass pipette pulled and cut to ∼50-μm tip diameter. The fluid level in the pipette was adjusted to just exceed the capillary action of the glass. The filled pipette was then lowered through the fluorocarbon until the tip contacted the endolymph through the fistula in the ampulla membrane. Contact with endolymph caused the surface tension between the endolymph and the fluorocarbon to be broken and placed the interior of the pipette in communication with the endolymph. Microspheres were allowed to diffuse out of the pipette and into the ampulla. Over time, some of the beads migrated to the cupula and adhered to its surface. The microsphere loading pipette was removed prior to collecting any data.
Fluorescent microsphere tracking
Images were collected using an upright microscope (Axioskop Tech, Carl Zeiss, Germany) configured for epifluorescence and placed on a vibration isolation table (TMC, Peabody, MA, USA). Long working distance air 5×, 10×, 20× objectives (Plan Apo, Mitutyo, Japan) were used to view the complete ampulla, fluorescent microbeads, and the neural recording electrode. A CCD camera (Retiga-EXi, QImaging, Surrey, BC, Canada) was used to collect fluorescent images at a frame rate of ∼100 ms and exposure time of ∼50 ms. Shutter times were sampled and recorded. Custom software was written (Igor Pro, Wave Metrics, Lake Oswego, OR, USA) to control the stimuli (NI GPIB, National Instruments, Austin, TX, USA), trigger the camera, and to collect the neural data, image data, and stimuli via computer (Apple G4, Cupertino, CA, USA; ITC-18, HEKA Instruments, Inc.). Images (1392 × 1040) were collected over two to ten cycles of periodic mechanical indentation stimuli with shutter times random relative to the stimulus period. Images were time-stamped relative to the stimulus onset trigger over multiple applications of the stimulus and subsequently combined. Since image sampling was random relative to the periodic stimulus, data collected over several cycles provided temporal resolution of ∼50 ms with respect to the stimulus timing trigger.
Figure 1C shows an example image of fluorescent microspheres adhered to the cupula, imaged in the living animal. It was straightforward to estimate the three-dimensional locations of the beads by adjusting the “Z”-axis focus and manually digitizing the centroids of the microspheres from still images. An example showing the distribution of fluorescent microspheres in one animal is provided as orthographic projections in Figure 1D–F. Specific locations and numbers of microspheres adhered to the cupula varied substantially between individual animals. Typically, ten to 50 microspheres adhered to the cupula, with five to 20 located on the central region of interest (ROI). Results reported in the present study are limited to motion of beads in the ROI located near the center of the cupula (Fig. 1, dotted circular), on the central pillar overlaying the center region of the crista (Silver et al. 1998).
Motion of the cupula was tracked by focusing on a subset of fluorescent microspheres located in the ROI and collecting a sequence of ∼300 images while presenting multiple applications of the mechanical indentation stimulus. Since the animal was alive, there were always slight whole organ movements caused by respiration, heartbeat, or random muscle contractions. To remove these movement artifacts, we manually selected image registration ROIs at the apex of the ampulla and at the crista, being careful that the registration ROIs did not include any microspheres adhered to the surface of the cupula. The first image in the sequence was used as the reference configuration. The registration ROIs of all subsequent images were then aligned with the reference image using the approach of (Thévenaz and Unser 1998), as implemented by Wave Metrics (Igor Pro 6, Lake Oswego, OR, USA). All images in the sequence were translated and rotated to align the registration ROIs with the reference configuration. After registration of all images, the same approach of Thévanaz and Unser was applied to track motion of the microspheres 20 × 20 pixel square ROI around each bead intensity centroid. Beads within the ROI were selected manually and the intensity centroid was found using custom software (Igor Pro 6, Wave Metrics). Analysis of microspheres adhered to the membranous labyrinth (ampulla) resulted in motions <200 nm, thus suggesting a noise floor of <200 nm—a value that could be improved by averaging over multiple stimulus presentations if desired. The present study used stimuli producing motions an order of magnitude above this noise level, and therefore, averaging was not required. To track cupula motion, five to ten microspheres within the central ROI were selected and tracked individually. The procedure resulted in microsphere displacements in the “x–y” coordinate frame of the CCD array. These data were combined to determine the component of motion perpendicular to the surface of the cupula, with the surface tangent identified by the group of fluorescent beads.
Mechanical indentation of the membranous labyrinth is known to lead to large transcupular pressures and, if the stimulus is excessive, can detach the cupula at its apex (Hillman 1974; Rabbitt et al. 1999). Although we did not intend to damage the cupula, in several cases, the cupula became detached during preparation of the animal. Detached cupulae showed a diverse range of motions depending on the extent of damage. Afferent responses, if present, reflected this diversity. In the most severe detachment cases, the cupula would swing toward the utricle, leaving a gaping opening for endolymph to flow over its apex from the canal lumen into the utricular vestibule. The cupula always remained attached to the sensory epithelium with an orientation perpendicular to the sensory epithelium and aligned with the hair bundles, thus showing a much stronger attachment at the base vs. a weak attachment at the apex. Grossly dislodged cupulae did not respond to mechanical indentation of the slender duct, at least within the limits of our experimental setup. Consistent with this, afferents in these damaged organs did not modulate in response to angular rotation of the animal or mechanical indentation of the duct. This is not surprising since the fluid pressure drop across a grossly dislodged cupula would approach zero.
In three animals, we observed reattachment of the cupula and recovery of normal afferent discharge responses to mechanical stimuli ∼7 h after the initial damage. Recovery of function occurred in cases where the cupula was dislodged at its apex but oriented properly within the ampulla. Presumably, more severely damaged cupula would take much longer to recover. In the modestly detached cases reported here, the apex of the cupula was observed to slide along the inside surface of the ampulla during mechanical indentation stimuli. The cupula did not return completely to its initial position after cessation of the stimulus. When detached, motion of the cupula at the surface of sensory epithelium was very small relative to motion at the apex, and hence, afferent modulation was reduced or absent. The displaced shape of the dislodged cupula was similar to a cantilever beam with the base firmly secured to the sensory epithelium and the apex free to swing under the ampullary apex.
The two rapidly adapting records in Figure 6C were recorded from the same animal ∼1 h (red) and ∼4.5 h (blue) after damage to the cupula. Results at these two time points were virtually identical, indicating that the regeneration process had not yet reached the apex of the cupula where reattachment was necessary. After ∼7 h (Fig. 6B, curve “b”), the cupula had reattached at its apex and exhibited an adaptation time constant of ∼27 s, consistent with the time constants in control recordings. The time constant did not change at later time points (curve “c”, 10 h), but a drop in gain sometimes occurred due to loss in preload of the indentation stimulus over time (Rabbitt et al. 1995). We did not observe intermediate stages of cupula recovery, suggesting that reattachment process may be quick once the appropriate extracellular adhesion molecules are delivered to the apex.
The average adaptation time constant of the cupula measured in normal animals was 36 s and the average gain was 53 nm per deg/s of angular head velocity. It has been shown previously that step stimuli (Rabbitt et al. 2005), or more complex waveforms (Paulin et al. 2004), can be used to estimate frequency domain responses of vestibular afferents. This nearly linear behavior allows us to use the time constant for step stimuli measured here to predict the mechanical lower corner frequency for sinusoidal angular motion of the head to be ∼0.0044 Hz (ω = 1/τ rad/s). Physically, the mechanical lower corner occurs at the frequency where the viscous drag force of endolymph balances the elastic restoring force of the cupula (Oman et al. 1987; Rabbitt et al. 2004; Steinhausen 1933; Van Buskirk 1987). Above the lower corner frequency, present data confirm that the biomechanics of the semicircular canals serves to integrate the angular acceleration stimuli to generate cupular displacements that are proportional to and in phase with angular velocity of the head (valid at least up to the 10 Hz Nyquist frequency of the present image acquisition approach). For angular motion stimuli below the lower corner frequency, the gain of the cupula is attenuated and the phase is advanced re: angular velocity. This is illustrated in Figure 4 in the Bode form of gain (D) and phase (E). We also used the mechanical time constant to estimate the elasticity of the cupula. This estimate used the viscosity of endolymph (Steer et al. 1967) and the morphology of the toadfish labyrinth (Ghanem et al. 1998), combined with Eqs. 3, 6, and 11 (Appendix) to estimate a cupula elastic shear modulus of ∼0.12 N/m2 (1.2 dyn/cm2).
Across the experimental population, afferents adapted more rapidly than the cupulae in the same animals, thus showing that adaptation to step stimuli (and the lower corner frequency observed for sinusoidal rotation) does not directly reflect displacement of the cupula but also includes adaptive properties of hair cell/afferent complexes. Although there are differences between species, there is no doubt that the hair cell/afferent complexes contribute additional signal processing beyond mechanical inputs that shape afferent responses (Anastasio et al. 1985; Baird et al. 1988; Boyle and Highstein 1990; Brichta and Goldberg 1996; Ezure et al. 1978; Fernández and Goldberg 1971; O’Leary and Honrubia 1976; Peterka and Tomko 1984; Curthoys 1982). This may explain why morphologically based mechanical models of canal function do not describe responses of all afferents (Hullar 2006). Cupular adaptation time constants reported here in normal control animals were recorded near the center of each cupula, on the surface facing the utricular vestibule. This part of the cupula overlies the region of the sensory epithelium innervated by the most rapidly adapting afferents in this species (Boyle et al. 1991; Boyle and Highstein 1990), thus highlighting the difference between the slowly adapting cupula and more rapidly adapting afferents. The decreased mechanical adaptation time constants reported here for dislodged cupula were pathological and cannot explain diversity of afferent adaptation times observed within individual animals under healthy conditions. We did not investigate regional variability in the present study, and it remains possible that different regions of the cupula might adapt with different time constants. It also remains possible that motion of the sensory hair bundles might not occur in perfect step with displacement of the cupula. These hypothetical mechanical explanations for differences between afferent discharge vs. cupula motion, however, seem unlikely given all the data at hand. Rather, present results for normal intact cupulae are consistent with previous reports attributing the diversity in afferent adaptation properties primarily to non-mechanical factors (Highstein et al. 2005; Lysakowski and Goldberg 2003; Rabbitt et al. 2005).
Normally, the cupula is attached around its entire perimeter, occludes the complete cross-section of the ampulla, and prevents endolymph from flowing past (Hillman and McLaren 1979; McLaren and Hillman 1979). In a subset of experiments reported here, the cupula had become detached at its apex, thus allowing endolymph to flow in the restricted space between the cupula and the apex of the ampulla. These animals did not manifest obvious vestibular symptoms prior to the experiment, suggesting that cupula detachment likely occurred during the surgical procedure. The condition was consistent with earlier reports describing cupula detachment resulting from high transcupular pressures induced by mechanical trauma or rapid deformation of the membranous labyrinth (Hillman 1974; Rabbitt et al. 1999). When detached modestly at the apex, cupulae and afferents still responded but with abnormally reduced low-frequency gains and much faster adaptation time constants. This can be understood by endolymph leakage over the apex (e.g., Figs. 5 and 6 and Appendix). Reattachment was observed to occur abruptly after about 5–7 h, consistent with the hypothesis that extracellular adhesive molecules arrived at the damaged apex of the cupula after this period of time. The molecular cell biology of this process is unknown, but may involve up-regulation of cupula generation by supporting cells in the crista (Anniko and Nordemar 1982) and the subsequent transport time from the crista to the apex.
Financial support was provided by the NIDCD R01 DC06685 (Rabbitt) and NASA GSRP 56000135 & NSF IGERT DGE-9987616 (Breneman).
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