M.M. Henson

Olivocochlear neurons in the brainstem of the mouse

The locations of efferent auditory neurons in the white mouse were determined using retrograde transport of HRP from the cochlea. Labeled neurons were localized in the lateral superior olivary nucleus (LSO) and ventral nucleus of the trapezoid body (VNTB). The total number of efferent neurons was determined to be 475; of these 34.5% are medial group efferents and 65.4% are lateral group efferents. The ipsilateral LSO contains 99% of the lateral group neurons and the contralateral VNTB contains 75% of the medial group neurons.

Tension fibroblasts and the connective tissue matrix of the spiral ligament

Fibroblasts with stress fibers (tension fibroblasts) have previously been described in the marginal region of the spiral ligament of bats and mice (Henson et al., 1984, 1985). The location, structure and attachments of these cells and the fact that they contain contraction associated proteins, have suggested a role in the generation of tension within the basilar membrane-spiral ligament complex. In this study the structure of these fibroblasts and their relationships to different types of connective tissue matrices were examined in representative Marsupalia, Insectivora, Chiroptera, Rodentia, Lagomorpha, Carnivora and Primates. Tension fibroblasts occur in all species but they are remarkably different in their actin filament content, their structure and distribution, and in their association with the extracellular matrix and otic capsule. Six types of matrices are described (dense filamentous, bundled, laminated, honeycombed, trabeculated and loose filamentous). The configurations of the cells and fibers indicate that tension on the basilar membrane may be accomplished in different ways and to different degrees in different mammals. The arrangement of the matrix and cells in some animals is markedly different in the parts of the cochlea that respond to high, middle and low frequencies.

Detection and quantification of endolymphatic hydrops in the guinea pig cochlea by magnetic resonance microscopy

Three-dimensional magnetic resonance microscopy (MRM) was used to study normal and hydropic cochleae of the guinea pig. With this technique consecutive serial slices representing the entire volume of isolated, fixed cochleae were obtained. The voxels (volume elements) making up the contiguous slices were isotropic (25 microns 3) and in each slice the boundaries of scala media, including the position of Reissners membrane, were clearly delineated. Three-dimensional reconstructions of the endolymphatic and perilymphatic scale were generated. Custom software was developed to quantify cross-sectional area (CSA) of all scalae. In the normal cochlea all 3 scalae, including scala media, showed a gradual decrease in CSA from base to apex. Marked differences existed between our findings and previously reported cochlear dimensions, especially for the perilymphatic scalae in the basal turn. In hydropic cochleae the scala media was enlarged to a varying extent in different turns and marked changes in the degree of distension of Reissners membrane occurred along the cochlea. MRM and subsequent computer analysis of the isotropic data provide excellent methods for imaging and quantifying the fluid spaces of normal and hydropic cochleae.

Quantitative anatomy of the round window and cochlear aqueduct in guinea pigs

In order to analyze the entry of solutes through the round window membrane, a quantitative description of round window anatomy in relationship to scala tympani is required. High-resolution magnetic resonance microscopy was used to visualize the fluid spaces and tissues of the inner ear in three dimensions in isolated, fixed specimens from guinea pigs. Each specimen was represented as consecutive serial slices, with a voxel size of approximately 25 microm(3). The round window membrane, and its relationship to the terminal portion of scala tympani in the basal turn, was quantified in six specimens. In each image slice, the round window membrane and scala tympani were identified and segmented. The total surface area of the round window membrane averaged 1.18 mm(2) (S.D. 0.08, n=6). The length and variation of cross-sectional area as a function of distance for the cochlear aqueduct was determined in five specimens. The cochlear aqueduct was shown to enter scala tympani at the medial limit of the round window membrane, which corresponded to a distance of approximately 1 mm from the end of the scala when measured along its mid-point. These data are of value in simulating drug and other solute movements in the cochlear fluids and have been incorporated into a public-domain simulation program available at http://oto.wustl.edu/cochlea/.

Rapid decalcification of temporal bones with preservation of ultrastructure

Decalcification of temporal bones, especially from primates, has routinely required long periods of time and has been a major deterrent to many types of morphological studies. In this investigation, temporal bones from the monkey, Macaca fuscata, were decalcified with ethylene diamine tetraacetic acid (EDTA) in a microwave oven. To isolate effects of microwaves on decalcification, tissue was fixed and embedded using routine methods; only decalcification was carried out in the microwave oven. The procedure is described in detail. Instead of months, decalcification was complete in two working days. Control procedures included decalcification at room temperature and use of a regular oven at a temperature equal to that reached in the microwave. The ultrastructure of cochlear tissue was equal to or better than that obtained with routine decalcification.

Course and distribution of efferent fibers in the cochlea of the mouse

The course, distribution and termination of single efferent fibers to the cochlea has been described in only a few animals and relatively few fibers have been studied with knowledge of their ipsilateral or contralateral origin. In order to examine the efferent fibers in the mouse, the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was iontophoretically injected into one side of the brain stem near the location of known efferent nuclei. Examination of surface preparations of the cochlea revealed detailed information for both the lateral olivocochlear (LOC) and medial olivocochlear (MOC) systems. Many, but not all, fibers entered the cochlea within the intraganglionic spiral bundle (IGSB). The LOC fibers were restricted to the ipsilateral cochlea and rarely branched within the IGSB and osseous spiral lamina (OSL). In the organ of Corti, they traveled either basally or apically in the region of the inner hair cells (IHCs), spanning lengths up to 130 microns (basally) and 890 microns (apically). Terminal swellings of these fibers were ca 3.0 microns in diameter. Numerous en passant swellings were present where the fibers formed a plexus in the area of the IHCs. The MOC fibers followed a similar course in the IGSB and OSL, and within the OSL the fibers had few branches. Within the organ of Corti they traveled apically (up to 70 microns) in the nerve bundles located in the IHC area before they crossed the tunnel of Corti. In the region of the OHCs, 9% of the traceable fibers branched to innervate two to three OHCs while 91% appeared to innervate only one OHC. There was no discernible difference in the distribution of contralateral and ipsilateral MOC projections in terms of cochlear region or outer hair cell rows.

Imaging the cochlea by magnetic resonance microscopy

The isolated, fixed cochlea of the mustached bat was studied with three dimensional magnetic resonance (MR) microscopy. The cochlea of this animal is about 4 mm in diameter and its entire volume was imaged. With the field of view and matrix size used, the volume elements (voxels) making up the volume data set were isotropic 25 x 25 x 25 micron cubes. Three dimensional (3D) MR microscopy based on isotropic voxels has many advantages over commonly used light microscopy: 1) it is non destructive; 2) it is much less time consuming; 3) no dehydration is required and shrinkage is minimized; 4) the data set can be used to create sections in any desired plane; 5) the proper alignment of sections is inherent in the 3D acquisition so that no reference points are required; 6) the entire data set can be viewed from any point of view in a volume rendered image; 7) the data is digital and features can be enhanced by computer image processing; and 8) the isotropic dimensions of the voxels make the data well-suited for structural reconstructions and measurements. Good images of the osseous spiral lamina, spiral ligament, scala tympani, scala vestibuli, and nerve bundles were obtained. The vestibular (Reissners) membrane was easily identified in the mustached bat and it appears to bulge into the scala vestibuli. The visibility of this structure suggests that MR microscopy would be well-suited for studies of endolymphatic hydrops.

Specializations for sharp tuning in the mustached bat: The tectorial membrane and spiral limbus

The sense of hearing in the mustached bat, Pteronotus parnellii, is specialized for fine frequency analysis in three narrow bands that correspond to approx 30, 60 and 90 kHz constant frequency harmonics in the biosonar signals used for Doppler-shift compensation and acoustic imaging of the environment. Previous studies have identified anatomical specializations in and around the area of the cochlea that processes the dominant second harmonic component, but similar features have not been found in areas related to sharp tuning and high sensitivity for the first or third harmonics. In this report we call attention to the large size of the tectorial membrane and spiral limbus in all three areas that appear to process the harmonically related constant frequency components. These structures are especially pronounced in the regions of the cochlea that respond to the approx 61 kHz, second harmonic and 91.5 kHz, third harmonic bands; they correspond specifically to areas where the density of afferent nerve fibers is high and where very sharply tuned neurons occur. These data for cochleae with multiple specializations lend strong support to the idea that the mass of the tectorial membrane can be an important factor in establishing the response properties of the cochlea.

Ultrastructure of the inner ear of NKCC1-deficient mice

The transduction of the auditory signal is dependent on the flow of ions within the inner ear. We have generated mice deficient in NKCC1, an ion cotransporter that is thought to be involved in the secretion of K+ by the strial marginal cells. Inner ear histology revealed partial to almost total absence of the scala media and collapse of Reissners membrane. Ultrastructural analysis showed that Reissners membrane consists of 3-4 cell layers instead of the usual two, and a substance of unknown composition is present between Reissners membrane and underlying structures. Within the tunnel of Corti, hair cells and supporting cells were difficult to identify. The location of the tectorial membrane was altered, and a precipitate was observed surrounding it. Severe structural defects were noted in the interdental cells and Boettcher cells, and mild defects were observed in the stria vascularis and in type II and type IV fibrocytes. The finding that major defects occur predominantly in cells that are not known to express NKCC1 suggests that loss of NKCC1 results in functional defects in cells expressing NKCC1 and a morphological effect on cell populations downstream in the proposed K+ recycling pathway.

Unusual nerve‐fiber distribution in the cochlea of the bat Pteronotus p. parnellii (Gray)

The distribution of nerve fibers in the osseous spiral lamina was examined in whole mount preparations of the cochleae of three species of bats which emit pulses containing both frequency‐modulated (FM) and constant‐frequency (CF) components: Pteronotus p. parnellii, Hipposideros diadema, and Rhinolophus ferrum‐equinum. In Pteronotus the nerve‐fiber population was extremely dense at the beginning and end of the large basal coil and relatively sparse between these areas. The striking contrast in nerve‐fiber density seen in Pteronotus (family Mormoopidae) was not seen in the other two “CF/FM” bats (family Rhinolophidae).