Noise-induced transient microlesions in the cell membranes of auditory hair cells.

Several types of nonauditory cells recover from transitory mechanically induced microlesions in their cell membranes. We report evidence that hair cells in the auditory papilla of the alligator lizard suffered similar membrane wounding when exposed to noise loud enough to induce a temporary threshold shift. Lucifer yellow, a molecular marker that does not normally penetrate through the cell membrane into the cytoplasm, was introduced into the extracellular fluid bathing the basolateral membrane of the hair cells. We assessed the effect of loud noise on the function of the ear by measuring compound action potentials of the auditory nerve before exposure to the noise, immediately after cessation of the noise, and after recovering overnight. Hair cells that were exposed to the noise took up much more Lucifer yellow than hair cells that were not exposed. We propose that the Lucifer yellow entered the hair cells via noise-induced lesions in their cell membranes, and that the cells were able to survive and recover functionally.

Sensorineural hearing loss in the mdx mouse: a model of Duchenne muscular dystrophy.

Sensorineural hearing loss has been identified in several types of muscular dystrophy, but few studies have investigated any relationship between Duchenne muscular dystrophy and hearing. An animal model of Duchenne muscular dystrophy, the mdx mouse, exhibits the same genetic defect as humans. We performed brainstem auditory evoked responses on mdx and control mice in order to assess sensorineural hearing loss. The amplitude and latency of wave I for each animal were measured at increasing sound pressure levels. A significant increase in threshold and a decrease in wave I amplitude were found in the mdx mice. These results indicate that significant sensorineural hearing loss is associated with muscular dystrophy in the mdx mouse. Possible cellular mechanisms contributing to the hearing deficit are presented.

Abnormal cardiac sensory innervation associated with experimentally induced, electrocardiographic long QT intervals in chick embryos.

Prolongation of the QT interval in the ECG can be induced in d 17 chick embryos by ablating the nodose placode on the right side on d 1 of development. The nodose placode contains the precursor cells which form the neurons of the nodose (inferior vagal) ganglion. Neurons in this ganglion provide sensory innervation to the heart and other viscera. In this study, we measured ganglion volume and neuron size and number in the right and left nodose ganglia in d 17 experimental and control embryos from whom electrocardiograms had been obtained. A significant reduction in the number of neurons present in the right nodose ganglion, relative to the left ganglion, was evident in all embryos with abnormally prolonged QT intervals. Embryos with prolonged QT, as well as lesioned embryos who demonstrated normal.QT on d 17, also had abnormally small neurons in both right and left nodose ganglia, indicating an additional nonspecific, perhaps permissive, effect of the lesion. These results suggest that abnormal development of the sensory innervation of the heart may be an important link in the chain of events leading to the developmental long QT syndrome expressed by these embryos.

Afferent synaptic changes in auditory hair cells during noise-induced temporary threshold shift.

This study presents evidence in support of the hypothesis that one of the sites of failure during noise-induced temporary threshold shift (TTS) is the afferent synapse between auditory hair cells and auditory nerve fibers. Our results show clear evidence indicating changes in the quantity of afferent synapses and the morphology of presynaptic structures in the alligator lizard auditory hair cells during TTS. In TTS hair cells there are statistically significant decreases in: 1) the number of afferent synapses, 2) the number of synaptic vesicles at the afferent synapses, 3) the size of synaptic bodies, and 4) the packing density of synaptic vesicles around the synaptic body. These results suggest that the presynaptic components of the afferent synapse reflect the functional state of the synapse, and that the reduction of these synapses, both in number and component size, contributes to TTS.

Developmental study of the long QT with deafness syndrome in the chick embryo: cochlear pathology.

Developmental abnormalities of the peripheral auditory structures in an experimental animal model of the cardio-auditory (long QT with deafness) syndrome are described. Prolonged QT intervals in the electrocardiogram and deafness were induced in chick embryos by removal of tissue in the region of the right nodose and otic placodes on the first day of incubation. Electrocardiographic recordings, cochlear potential and brainstem auditory evoked responses were recorded in late embryonic life (E17), and used to identify embryos with long QTs and deafness. External and middle ears were evaluated under a dissecting microscope. Inner ears were evaluated in histological sections. Anomalies of the external and middle ears, such as the external auditory meatus, tympanic membrane and stapes, were attributed to disturbance of neural crest development. Anomalies of the inner ear, such as a complete absence of the cochlear duct and auditory nerve, or decreased length of the basilar papilla, were attributed to disturbance of otic placode development. The fact that a small lesion in the region of the nodose and otic placodes during early development in the chick animal model can produce a long QT interval in the electrocardiogram and deafness suggests that abnormal development in this region early in development might be the underlying cause of the human syndrome.

Gap junctional connections between hair cells, supporting cells and nerves in a vestibular organ.

The pattern of gap-junctional connections between cells in the vestibular neuroepithelium of the posterior semicircular duct of the alligator lizard are described based upon the study of freeze fracture replicas and ultrathin sections with a transmission electron microscope. Both type I and type II hair cells are coupled to adjacent supporting cells by a series of small macular gap junctions located in a ring around the hair cell at the level of the apical circumferential belt of actin filaments. Adjacent supporting cells are extensively interconnected by gap junctions. A few cases of gap junctions between afferent dendrites and supporting cells, and between afferent dendrites and calyceal nerve endings were seen. These morphological observations together with data from other studies in the literature suggest a possible role for supporting cells in altering the micromechanical properties of the hair cell receptor organs during stimulation.

Changes in size and shape of auditory hair cells in vivo during noise-induced temporary threshold shift.

In this study we describe changes in the size and shape of auditory hair cells of the alligator lizard in vivo during noise-induced temporary threshold shift. These changes consist of a decrease in cell volume, a decrease in cell length and an increase in cell width. We speculate that these changes are due to relaxation of cytoskeletal contractile elements and osmotic loss of intracellular water. We also describe a decrease in the surface area of the hair cell plasmalemma, and speculate that it is related to the endocytosis and intracellular accumulation of cell membrane during synaptic vesicle recycling. Finally we describe an increase in the endolymphatic surface area of the hair cell, and speculate that this could alter the micromechanics of the stereociliary tuft to attenuate the effective stimulus.

Changes in the synaptic region of auditory hair cells during noise-induced temporary threshold shift.

During noise-induced temporary threshold shift cytoplasmic vacuoles increase in the synaptic region of auditory hair cells. This structural change in the presynaptic region suggests that afferent synapses between hair cells and auditory nerve fibers fail during the period of threshold elevation, and that synaptic fatigue may play a major role.

Electrocardiographic QT prolongation after ablation of the nodose placode in the chick embryo: a developmental model of the idiopathic long QT syndrome.

Electrocardiographic abnormalities characteristic of the idiopathic long QT syndrome are thought to be caused by an imbalance of sympathetic activity in the heart. Recent evidence indicates that autonomic and sensory innervation density in the end-organ is modulated by reciprocal interactions. Ablation of one neuronal population allows reciprocal increases in growth of the remaining nerves. To test whether QT prolongation could be produced in chick embryos by altering sensory innervation to the heart, microcautery was used to ablate premigratory areas of the right nodose placode, a coalescence of cells in the embryonic ectoderm that generates neurons providing sensory innervation to the heart via the inferior ganglion of the vagus (nodose ganglion). After functional autonomic innervation was established, three-lead ECG were obtained in embryos with the right nodose placode ablated (experimental) and in sham-operated controls (sham) at incubation days 17-20 in a controlled temperature environment. Electrocardiograms were analyzed for RR and QT intervals. The QT interval was corrected for heart rate using the formula QTc = QT/(RR)1/2 using an average of ten complexes. Compared with shams (n = 8), experimental embryos (n = 7) had significantly longer QTc (0.339 +/- 0.005 versus 0.318 +/- 0.004), and slower heart rates (RR = 0.29 +/- 0.005 versus 0.27 +/- 0.007). These findings mimic those in children with the idiopathic long QT syndrome. Experimental manipulation of the sensory innervation to the heart in the chick embryo via the nodose placode may provide an animal model to improve understanding of the pathogenesis of the idiopathic long QT syndrome.

Auditory stereocilia in the alligator lizard.

The normal anatomy of stereociliary tufts in the basilar papilla of the alligator lizard is described and demonstrated with scanning electron micrographs. Stereociliary tufts in the tectorial region differ from those in the free-standing region in several ways. Tectorial stereociliary tufts are short (less than 10 micron in height), unidirectional in orientation, and covered with a tectorial membrane. Free-standing stereociliary tufts are very tall (up to 38 micron in height), bidirectional in orientation and not covered by a tectorial membrane or any other tectorial substance. The heights of the stereociliary tufts along the length and across the width of the basilar papilla were measured in serial light and transmission electron micrographs. Free-standing stereociliary tufts decrease progressively in height along the length of the basilar membrane, being tallest at the apical end and shortest at the basal end. Tectorial stereociliary tufts do not increase progressively along the length of the basilar membrane but do increase progressively across the width of the basilar membrane, being shortest on the neural side. Free-standing stereociliary tufts are structurally simple being a prominent specialization of the lizard cochlea. Tectorial stereociliary tufts are structurally more complicated conforming more closely to the general anatomical pattern of vertebrate auditory hair cells.

Connections between stereocilia in auditory hair cells of the alligator lizard.

The interconnections between stereocilia within individual tufts of auditory hair cells in the basilar papilla of the alligator lizard were examined with a transmission electron microscope. An elaborate array of fibers near the base of each stereocilium (where it tapers to anchor into the cuticular plate) connected it to each of its neighboring stereocilia. The tips of individual stereocilia, which were slightly larger in diameter than their shaft, contacted adjacent stereocilia. Fibers also connected the tip of the kinocilium to neighboring stereocilia in the first row. The remaining regions of the stereocilia were relatively free of connecting fibers. The integrity of these connecting fibers are likely to be important in maintaining the normal micromechanical tuning and mechanoelectric transduction in these auditory hair cells. The addition of 0.1% ruthenium red to the primary fixative enhanced the preservation of the connecting structures, implying the presence of glycosaminoglycans.

Patterns of afferent synaptic contacts in the alligator lizard's cochlea.

Afferent synapses on both free-standing and tectorial hair cells in the alligator lizard's cochlea are described quantitatively. Semiserial sections were photographed with a transmission electron microscope. Hair cells together with their afferent nerves were reconstructed and morphometrically analyzed with the aid of a computer. Each afferent nerve forms many synapses with its hair cell. Tectorial afferents make more than twice the number of synapses with their hair cells as do free-standing afferents. This suggests a possible neuroanatomical basis for the physiological difference in synchrony reported in these two types of auditory nerve fibers; namely, the greater the number of synapses the better a fiber is able to follow faithfully the response of its hair cell.

Permanent noise-induced damage to stereocilia: a scanning electron microscopic study of the lizard's cochlea.

Alligator lizards were exposed to broadband noise ranging in intensity from 106 to 132 dB SPL for two hours and permitted to recover from 19 to 62 days. Hearing loss was assessed by comparing the auditory nerve component of the cochlear potential recorded at the end of the recovery period with that recorded before the noise exposure. The stereocilia in these ears were examined with a scanning electron microscope. These sensory hairs showed pathological changes similar to those described in mammalian cochleas with noise-induced damage. In decreasing order of severity the damage included completely missing auditory papillas, missing hair cells, missing hairs, hairs fallen over, and hairs that were only moderately splayed apart compared with their normal appearance. Long lasting hearing loss seems to be associated with all of these sensory hair pathologies.

Cochlear nerve of the alligator lizard.

The innervation of the auditory organ of the alligator lizard is described. Patterns of distribution of the nerve fibers were studied at the light microscopic level with the horseradish peroxidase technique, and the types of synaptic contacts with hair cells were studied at the transmission electron microscopic level with standard techniques. The innervation of the two regions of the basilar papilla differs in the following ways. In the apical region, some fibers send branches along the length of the basilar papilla, and both afferent (non-vesiculated) and efferent (vesiculated) nerve endings are present. In the basal region, all fibers terminate in the immediate area where they enter the papilla without sending branches along the length of the papilla; efferent endings are lacking, and nerve fibers are of a smaller average diameter. The punctate nature of the innervation of hair cells in the basal region is consistent with the hypothesis that the systematic organization according to frequency sensitivity observed in electrophysiological recordings from basal nerve fibers may be related to the length of the stereocilia on the hair cells with which the nerve synapses.

Structural changes in auditory hairs during temporary deafness.

The stereocilia of auditory hair cells were examined with a scanning electron microscope during periods of noise-induced temporary threshold shift. The stereocilia in the tallest row of each tuft of the noise-exposed ears were clumped together compared with control ears. This suggests to us that clumping of the longest stereocilia might be a reversible mechanism of the hair cell to protect itself from overstimulation. The rate of recovery following exposure to noise was monitored by measuring the first neural component of the cochlear potential response. The duration of the noise was adjusted to ensure sufficient time to fix the ears during the period of temporary deafness. Alcian blue was added to the fixative to preserve the glycocalyx of the stereocilia. The experiments were done in the basal region of the auditory organ of the alligator lizard. This region is characterized by hair cells with long free-standing stereocilia.

Cochlear ganglion cells in the alligator lizard.

Two types of ganglion cells are present in the cochlear ganglion of the alligator lizard. Myelinated cells are the predominant type but a small population of unmyelinated cells is also present. Ganglion cells are reconstructed and morphometrically analyzed from serial light and electron micrographs. The results are interpreted to indicate that the unmyelinated ganglion cells are degenerating neurons. No normal population of unmyelinated nerve fibers was found. The anatomy of the cochlear nerve of the lizard is described and compared with the mammalian auditory nerve.

Stereociliary pathology and noise-induced threshold shift: a scanning electron microscopic study.

Cats were exposed unilaterally to narrow-band noise at an intensity of 118-120 dB SPL for two hours. After long survival times thresholds of single auditory units were measured and the organ of Corti examined with a scanning electron microscope (SEM). The purpose of the study was to correlate pathologies of the hair cell stereocilia seen with the SEM with permanent hearing loss measured by elevated thresholds of single auditory nerve units. Pathological changes in inner hair cell (IHC) stereocilia were more severe and more numerous near the lesion focus diminishing progressively with increasing distance from it. Increasingly elevated thresholds correlated with increased damage to stereocilia of IHCs. Thresholds of nerve fibers innervating IHCs with missing or fused stereocilia were greatly elevated. Thresholds of fibers to IHCs with disarrayed stereocilia also seemed to be elevated but to a lesser degree.

The organization of actin filaments in the stereocilia of cochlear hair cells.

Within each tapering stereocilium of the cochlea of the alligator lizard is a bundle of actin filaments with > 3,000 filaments near the tip and only 18-29 filaments at the base where the bundle enters into the cuticular plate; there the filaments splay out as if on the surface of a cone, forming the rootlet. Decoration of the hair cells with subfragment 1 of myosin reveals that all the filaments in the stereocilia, including those that extend into the cuticular plate forming the rootlet, have unidirectional polarity, with the arrowheads pointing towards the cell center. The rest of the cuticular plate is composed of actin filaments that show random polarity, and numerous fine, 30 A filaments that connect the rootlet filaments to each other, to the cuticular plate, and to the membrane. A careful examination of the packing of the actin filaments in the stereocilia by thin sectin and by optical diffraction reveals that the filaments are packed in a paracrystalline array with the crossover points of all the actin helices in hear-perfect register. In transverse sections, the actin filaments are not hexagonally packed but, rather, are arranged in scalloped rows that present a festooned profile. We demonstrated that this profile is a product of the crossbridges by examining serial sections, sections of different thicknesses, and the same stereocilium at two different cutting angles. The filament packing is not altered by fixation in different media, removal of the limiting membrane by detergent extraction, or incubation of extracted hair cells in EGTA, EDTA, and Ca++ and ATP. From our results, we conclude that the stereocilia of the ear, unlike the brush border of intestinal epithelial cells, are not designed to shorten, nor do the filaments appear to slide past one another. In fact, the stereocilium is like a large, rigid structure designed to move as a lever.