Inner Ear Morphology: Advances in Assessment

The study of inner ear morphology has been substantially augmented by the development of the surface preparation technique and the scanning electron microscope. The former represents an old technique revitalized, while the latter is a new method providing dramatic stereoscopic images at the cellular level. More precise knowledge of inner ear sensory cells and their innervation is now being obtained and hence better understanding of function continues to evolve. The early fixation of human postmortem material circumvents many artefacts; however, there are legal and ethical questions to be settled before this is done routinely. The photomicrographs are from specimens prepared by the author.     

Inner Ear Morphology Is Perturbed in Two Novel Mouse Models of Recessive Deafness

Human MYO7A mutations can cause a variety of conditions involving the inner ear. These include dominant and recessive non-syndromic hearing loss and syndromic conditions such as Usher syndrome. Mouse models of deafness allow us to investigate functional pathways involved in normal and abnormal hearing processes. We present two novel mouse models with mutations in the Myo7a gene with distinct phenotypes. The mutation in Myo7a^I487N/I487N ewaso is located within the head motor domain of Myo7a. Mice exhibit a profound hearing loss and manifest behaviour associated with a vestibular defect. A mutation located in the linker region between the coiled-coil and the first MyTH4 domains of the protein is responsible in Myo7a^F947I/F947I dumbo. These mice show a less severe hearing loss than in Myo7a^I487N/I487N ewaso; their hearing loss threshold is elevated at 4 weeks old, and progressively worsens with age. These mice show no obvious signs of vestibular dysfunction, although scanning electron microscopy reveals a mild phenotype in vestibular stereocilia bundles. The Myo7a^F947I/F947I dumbo strain is therefore the first reported Myo7a mouse model without an overt vestibular phenotype; a possible model for human DFNB2 deafness. Understanding the molecular basis of these newly identified mutations will provide knowledge into the complex genetic pathways involved in the maintenance of hearing, and will provide insight into recessively inherited sensorineural hearing loss in humans.     

Hair Cell Polarization in the Flatfish Inner Ear

The hair cell polarization of the various sensory epithelia in the inner ear was examined in two species of flatfish, the Plaice (Pleuronectes platessa) and the Dab (Limanda limanda). The hair cells in the macula utriculi are polarized in the pattern usually seen for this macula in vertebrates. In the macula sacculi and macula lagenae the hair cell polarization is different from that hitherto described from bony fishes and other vertebrates. The polarization seen in these maculae in the flatfish explains their ability to sense movements in all directions, which is necessary if these sensory areas are the most important inner ear organs in the regulation of postural orientation.     

In Search of the Hagfish Thymus

The Pacific hagfish, Eptatretus stoutii, is capable of a varietyof immunologic responses including allograft rejection and serumantibody production to soluble and cellular antigens. Interesthas revived in the morphology of hagfish lymphoid tissues. Thesearch for a thymus in young specimens has resulted in the discoveryof a phagocytic and antigen-receptive cell population associatedwith the pharyngeal velar muscles. We suggest that a protothymusor precursor of the thymus of higher vertebrates may be containedwithin this velar muscle complex.     

The Morphology and Evolution of the Ear in Actinopterygian Fishes

SYNOPSIS. In this paper we consider various aspects of the anatomyand ultrastructure of the actinopterygian ear and make a numberof suggestions on the possible adaptive significance of thestructural specializations. The focus of the arguments is basedupon the substantial inter-specific variation in teleost auditorysystems as measured anatomically, behaviorally, and physiologically.It is potentially of considerable significance that the majorpoints of inter-specific variation in the teleost ear are associatedwith the gross morphology and ultrastructure of the otolithicorgan most often implicated in sound detection, the sacculus.Analysis of patterns of sacculus ultrastructure has led to theconclusion that there are, in effect, only about five differentsaccular ultrastructural patterns but that these patterns arebroadly found throughout the teleost fishes. Based upon patternsof inter-specific variation in the sacculus and in other aspectsof the ear and more peripheral auditory structures (e.g., swimbladder),it is argued that adaptations encountered in the teleost auditorysystem cannot be used as reliable taxonomic indicators amongfishes. Rather, it is proposed that the teleost auditory systemis quite maleable in the evolutionary sense, and that interspecificsimilarities in many features of the auditory system reflectconvergent evoluuon, rather than phylogenetic affinities. Theactual selective pressures operating in the evoluuon of thefish auditory system are still essentially unknown. In addition,we cannot be certain that similar ear patterns in differentspecies reflect convergent evolution (or common ancestry), orthat conversely, different ear patterns among species reflectdifferences in auditory function.     

Dynamics of Mechanically Coupled Hair-Cell Bundles of the Inner Ear

The high sensitivity and effective frequency discrimination of sound detection performed by the auditory system rely on the dynamics of a system of hair cells. In the inner ear, these acoustic receptors are primarily attached to an overlying structure that provides mechanical coupling between the hair bundles. Although the dynamics of individual hair bundles has been extensively investigated, the influence of mechanical coupling on the motility of the system of bundles remains underdetermined. We developed a technique of mechanically coupling two active hair bundles, enabling us to probe the dynamics of the coupled system experimentally. We demonstrated that the coupling could enhance the coherence of hair bundles’ spontaneous oscillation, as well as their phase-locked response to sinusoidal stimuli, at the calcium concentration in the surrounding fluid near the physiological level. The empirical data were consistent with numerical results from a model of two coupled nonisochronous oscillators, each displaying a supercritical Hopf bifurcation. The model revealed that a weak coupling can poise the system of unstable oscillators closer to the bifurcation by a shift in the critical point. In addition, the dynamics of strongly coupled oscillators far from criticality suggested that individual hair bundles may be regarded as nonisochronous oscillators. An optimal degree of nonisochronicity was required for the observed tuning behavior in the coherence of autonomous motion of the coupled system.     

Differential Staining of Inner Ear Fluids by Protargol

Present day techniques for processing temporal bones involve celloidin embedding. With a few modifications in Bodian's silver staining procedure the celloidin of the endolymphatic spaces stains darker than that of the perilymphatic spaces providing there is no break in the anatomical barrier between them. Essentially the routine procedure of Bodian is used except that metallic copper is omitted from the staining solution, impregnation time is reduced to 3 hr, reduction time is extended to 10 min and no oxalic acid is used for gold toning.     

Induced Bactericidal Response in the Hagfish

Hagfish were shown to be capable of synthesizing bactericidins after injection of gram-negative bacteria. The bactericidins could be detected as early as 2 days after injection. The degree of specificity was not as impressive as in mammalian systems.     

Modalities of GABA and Glutamate Neurotransmission in the Vertebrate Inner Ear Vestibule

GABA and glutamate have been postulated as afferent neurotransmitters at the sensory periphery inner ear vestibule in vertebrates. GABA has fulfilled the main criteria to act as afferent neurotransmitter but may also be a putative efferent neurotransmitter, mainly due to cellular localization of its synthesizing enzyme glutamate decarboxylase derived from biochemical, immunocytochemical, in situ hybridization and molecular biological techniques, whereas glutamate afferent neurotransmission role is supported mainly by pharmacological evidences. GABA and Glu could also act as afferent co-neurotransmitters based upon immunocytochemical techniques. This multiplicity was not considered earlier and postulates a peripheral modulation of afferent information being sent to higher vestibular centers. In order to make a definitive cellular assignation to these putative neurotransmitters it is necessary to have evidences derived from immunocytochemical and pharmacological experiments in which both substances are tested simultaneously. Special issue article in honor of Dr. Ricardo Tapia.     

Spatiotemporal Expression of tmie in the Inner Ear of Rats during Postnatal Development

The circling (cir/cir) mouse is a murine model for human nonsyndromic deafness DFNB6. Transmembrane inner ear (tmie) is the causative gene and its mutation through deletion of a 40-kilobase genomic region including tmie leads to deafness. The function of Tmie is unknown. To better understand the function of Tmie, we focused on the spatiotemporal expression of tmie in the rat cochlea by using a Tmie-specific antibody. Results showed that tmie expression was prominent in early postnatal rat cochleas in the stereocilia bundles of hair cells. The Tmie signal spread from the stereocilia to the hair cell body region and on to organ of Corti cells. No Tmie signal was observed in cell nuclei; Tmie was localized to the cytoplasm. Because Tmie is predicted to have 1 or 2 transmembrane domains, we postulate that it is localized to membrane-based organelles or the plasma membrane. Our results imply that Tmie exists in the cytoplasm and may have a key role in the maturation and structure of stereocilia bundles in developing hair cells. After hair cell maturation, Tmie is thought to be involved in the maintenance of organ of Corti cells.Circling is often observed in mouse and rat deafness mutants and is commonly suggested to be a consequence of inner ear defects that impair vestibular systems.^3,12,14 The circling (cir/cir) mouse is a murine model for human nonsyndromic deafness DFNB6; these mice have abnormal circling behavior, suggesting a balance disorder, and profound deafness.^6,7 The most notable pathologic phenotypes of circling mice are the almost completely degenerated cochlea and remarkably reduced cellularity in spiral ganglion neurons. The causative gene for circling is transmembrane inner ear (tmie), with a 40-kilobase genomic deletion including tmie.¹ tmie is also the causative gene of the spinner (sr/sr) mouse, which has phenotypes similar to circling mice, although the mutation patterns are different.⁸ Spinner mice also show circling behavior, hearing loss, imbalance, and swimming inability. In addition, spinner mice have 2 mutations in the tmie gene: the 40-kb genomic deletion including tmie and a point mutation that leads to a truncated protein.⁸In humans, 7 different homozygous recessive mutations in TMIE currently are known to exist in affected members of consanguineous families segregating severe-to-profound prelingual deafness, consistent with linkage to DFNB6.^9,10 Although the functions of murine Tmie and human TMIE are unknown, this protein appears to be important for normal hearing and vestibular function.In a previous study, we produced transgenic mice overexpressing tmie that resulted in phenotypic rescue of circling.¹¹ Normal expression of transgenic tmie induced phenotypic rescue in circling homozygous mutants, although some mice did not show amelioration of abnormal behavior, hearing ability, or tissue morphology in the inner ear. Therefore the Tmie protein is required for normal inner ear function in mouse.¹¹To better understand the function of Tmie, we focused on the spatiotemporal expression of tmie. Knowing when, where, and to what extent this protein is produced in the developing inner ear will provide important clues to protein function. In adult mouse and rat, tmie is expressed in various tissues.^2,13 Whether Tmie plays an important role in those tissues is uncertain, because circling mice that lack the entire tmie gene have no noteworthy problems in any tissues except those of the inner ear systems.⁶In this study, we were interested in the postnatal stages before and after the onset of hearing (around postnatal day [P] 12) in rats; therefore, the postnatal period P0 to19 was studied. Although all the cells that form the mature cochlea are present at birth, important conformational changes occur during this period, including the formation of the tunnel of Corti and the establishment or retraction of neuronal connections. The expression pattern of tmie in the developing inner ear during early postnatal development has not been investigated previously. Here we document our use of a Tmie-specific antibody to elucidate the spatial and temporal expression of tmie in the rat inner ear during postnatal development.     

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing ¹. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth ^2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes ^4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development⁶.The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos⁷. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells^8-10. We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol¹¹. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.     

A Glycol Methacrylate Method for the Routine Histologic Evaluation of Rat Inner Ear

A method has been developed for the histologic evaluation of rat inner ear using glycol methacrylate (GMA) and steel knife sectioning. The necropsy, fixation, and histologic techniques described are not so complex and difficult as to preclude their routine use. The method is particularly useful in studies in which the entire inner ear of a large number of animals must be evaluated histologically.