Morphological, Morphometrical and Histochemical Study of the Lining and Glandular Epithelia of the Tongue of the Bullfrog Rana catesbeiana

The dorsal surface of the tongue of the bullfrog, Rana catesbeiana, has simple columnar epithelium with a few ciliated cells and goblet cells. The entire surface is covered with numerous filiform papillae and few fungiform. Filiform papillae have a simple columnar epithelium with secretory cells, while the fungiform have a sensory disc on their upper surface the lined by a stratified columnar epithelium with basal, peripheral, glandular and receptor cells. Over the dorsal lingual surface there are numerous winding tubular glands, which penetrate deeply into the muscle of the tongue, mingling with the fibers. The gland epithelium is cylindrical with secretory and supporting cells. The first are absolute on the basis of the gland and the latter are rare in the upper third. The ventral surface of the tongue is lined by a stratified epithelium, with the presence of goblet cells, with ciliated cells among them. Morphometrically, lingual glands varies in length, according to their location: shorter in the anterior region of the tongue (330 mu m) than in the posterior region (450 mu m). Secretory cells of the anterior lingual glands are smaller (1457.7 mm(3)) than the posterior ones (2645.9 mu m(3)). The same can be said of the cell nuclei, 130.0 mu m(3) for the anterior glands and 202.3 mu m(3) for the posterior ones. Secretory cells of the lingual glands contain substances rich in protein and neutral mucopolysaccharides, which characterize the seromucous type. Goblet cells of the dorsal and ventral surface epithelia secrete neutral mucopolysaccharides and proteins, and can be characterized as type G1 cells, and the supporting cells of the superficial glands of the fungiform papillae secrete a mucus rich in neutral mucopolysaccharides, sulfomucins and sialomucins.

The expression of fibroblast growth factors and their receptors in the embryonic and neonatal mouse inner ear

Four different fibroblast growth factor receptors (FGFR) are known, three of which have splice variants (known as the b and c variants) in the FGF-binding domain, to give different patterns of sensitivity to the different FGFs. The expression of the b and c variants of the FGF receptors. together with the expression of the ligands FGF1. FGF2, FGF3, FGF7, FGF8b and FGF8c, was determined by quantitative reverse transcription-polymerase chain reaction in developing whole mouse inner ears, and in dissected components of the postnatal mouse inner ear. At embryonic age (E)10.5 days, when the otocyst is a simple closed sac, the receptor most heavily expressed was FGFR2b, relative to the postnatal day 0 level. Over the period E10.5-E12.5. during which the structures of the inner ear start to form, the expression of the different FGF receptors increased 10(2)-10(4) fold per unit of tissue, and there was a gradual switch towards expression of the 'c' splice variants of FGFR2 and FGFR3 rather than the 'b' variants. At E10.5, the ligands most heavily expressed, relative to the postnatal day 0 level, were FGF3, FGF8b and FGF8c. In the postnatal inner eat. the patterns of expression of receptors and ligands tended to be correlated, such that receptor variants were expressed in the same regions as the ligands that are known to activate them effectively. The neural/sensory region expressed high levels of FGFR3c, and high levels of the ligand FGF8b. The same area also expressed high levels of FGFR1b and FGFR2b, and high levels of FGF3. The lateral wall of the cochlea (including the stria vascularis and the spiral ligament) expressed high levels of FGFR1c and FGF1. 11 is suggested that the different FGF receptors and ligands are expressed in a spatially coordinated pattern to selectively program cochlear development. (C) 2001 Elsevier Science B.V. All rights reserved.

Hedgehog signaling governs the development of otic sensory epithelium and its associated innervation in zebrafish

The inner ear is responsible for the perception of motion and sound in vertebrates. Its functional unit, the sensory patch, contains mechanosensory hair cells innervated by sensory neurons from the statoacoustic ganglion (SAG) that project to the corresponding nuclei in the brainstem. How hair cells develop at specific positions, and how otic neurons are sorted to specifically innervate each endorgan and to convey the extracted information to the hindbrain is not completely understood. In this work, we study the generation of macular sensory patches and investigate the role of Hedgehog (Hh) signaling in the production of their neurosensory elements. Using zebrafish transgenic lines to visualize the dynamics of hair cell and neuron production, we show that the development of the anterior and posterior maculae is asynchronic, suggesting they are independently regulated. Tracing experiments demonstrate the SAG is topologically organized in two different neuronal subpopulations, which are spatially segregated and innervate specifically each macula. Functional experiments identify the Hh pathway as crucial in coordinating the production of hair cells in the posterior macula, and the formation of its specific innervation. Finally, gene expression analyses suggest that Hh influences the balance between different SAG neuronal subpopulations. These results lead to a model in which Hh orients functionally the development of inner ear towards an auditory fate in all vertebrate species.

Study of inner ear and lateral line hair cell regeneration

Death of sensory hair cells in the inner ear results in two global health problems that millions of people around the world suffer: hearing loss and balance disorders. Hair cells convert sound vibrations and head movements into electrical signals that are conveyed to the brain, and as a result of aging, exposure to noise, modern drugs or genetic predisposition, hair cells die. In mammals, the great majority of hair cells are produced during embryogenesis, and hair cells that are lost after birth are not replaceable. However, in the last decades, researches have shown some model organisms that retain the ability to regenerate hair cells damaged after embryogenesis, such as Zebrafish and chicken, providing clues as to the cellular and molecular mechanisms that may block hair cell regeneration in mammals. This discovery initiated a search for methods to stimulate regeneration or replacement of hair cells in mammals, a search that, if fruitful, will revolutionize the treatment of hearing loss and balance disorders. One aim of my project is to study the role of retinoic acid in adult Zebrafish and in mice, which is a metabolite of vitamin A known as an essential molecule to activate hair cell regeneration after cells damaged in Zebrafish embryo. We want to study important genes involved in retinoic acid pathway, such as Aldh1a3 and RARs genes, to check what their role is in the inner ear of adult Zebrafish and compare result obtained in the inner ear of mice. On the other hand, Zebrafish lateral line contains neuromast, which are formed by the same structure than the inner ear: hair cells surrounded by supporting cells and neurons. The lateral line is a structure below the skin's surface that makes easier to damage hair cells to study their regeneration. For that reason, another aim of my project is to study how Sox2 and Atoh1, essential genes during the inner ear development, change their expression during hair cell regeneration in the lateral line. In my project, the most important concepts related to Zebrafish world are explained in order to understand why we have studied this animal and these essential genes. Then, techniques that we used are explained, with their protocol attached in the annexes. Finally, results of my project are shown, but many of them were not expected and they would be needed to follow studying.

Id Gene regulation and function in the prosensory domains of the chicken inner ear: a link between Bmp Signaling and Atoh1

Bone morphogenetic proteins (Bmps) regulate the expression of the proneural gene Atoh1 and the generation of hair cells in the developing inner ear. The present work explored the role of Inhibitor of Differentiation genes (Id1-3) in this process. The results show that Id genes are expressed in the prosensory domains of the otic vesicle, along with Bmp4 and Bmp7. Those domains exhibit high levels of the phosphorylated form of Bmp-responding R-Smads (P-Smad1,5,8), and of Bmp-dependent Smad transcriptional activity as shown by the BRE-tk-EGFP reporter. Increased Bmp signaling induces the expression of Id1-3 along with the inhibition of Atoh1. Conversely, the Bmp antagonist Noggin or the Bmp-receptor inhibitor Dorsomorphin elicit opposite effects, indicating that Bmp signaling is necessary for Id expression and Atoh1 regulation in the otocyst. The forced expression of Id3 is sufficient to reduce Atoh1 expression and to prevent the expression of hair cell differentiation markers. Together, these results suggest that Ids are part of the machinery that mediates the regulation of hair cell differentiation exerted by Bmps. In agreement with that, during hair cell differentiation Bmp4 expression, P-Smad1,5,8 levels and Id expression are downregulated from hair cells. However, Ids are also downregulated from the supporting cells which contrarily to hair cells exhibit high levels of Bmp4 expression, P-Smad1,5,8, and BRE-tk-EGFP activity, suggesting that in these cells Ids escape from Bmp/Smad signaling. The differential regulation of Ids in time and space may underlie the multiple functions of Bmp signaling during sensory organ development.

Transplantation and survival of mouse inner ear progenitor/stem cells in the organ of Corti after cochleostomy of hearing-impaired guinea pigs: preliminary results

In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here, we investigated whether postnatal mouse inner ear progenitor/stem cells (mIESCs) are viable after transplantation into the basal turns of neomycin-injured guinea pig cochleas. We also examined the effects of mIESC transplantation on auditory functions. Eight adult female Cavia porcellus guinea pigs (250-350g) were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. The study group (n=4) received transplantation of LacZ-positive mIESCs in culture medium into the scala tympani. The control group (n=4) received culture medium only. At 2 weeks after transplantation, functional analyses were performed by auditory brainstem response measurement, and the animals were sacrificed. The presence of mIESCs was evaluated by immunohistochemistry of sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis of the data. Intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs were observed in all scalae of the basal turns of the injured cochleas, and a proportion of these cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs engrafted in the cochlear basilar membrane. Our study demonstrates that transplanted cells survived and engrafted in the organ of Corti after cochleostomy.

Injection of adult mouse mesenchymal stem cells into the developing chick inner ear

This series of experiments attempted to characterize the abilities of stem cells derived from bone marrow and adipose tissue to integrate into the sensory epithelium of the inner ear and to differentiate into hair cells or neural cell types.

Sensory enhancement, a pilot perceptual study of subdermal magnetic implants

Subdermal magnetic implants originated as an art form in the world of body modification. To date an in depth scientific analysis of the benefits of this implant has yet to be established. This research explores the concept of sensory extension of the tactile sense utilising this form of implantation. This relatively simple procedure enables the tactile sense to respond to static and alternating magnetic fields. This is not to say that the underlying biology of the system has changed; i.e. the concept does not increase our tactile frequency response range or sensitivity to pressure, but now does invoke a perceptual response to a stimulus that is not innately available to humans. Within this research two social surveys have been conducted in order to ascertain one, the social acceptance of the general notion of human enhancement, and two the perceptual experiences of individuals with the magnetic implants themselves. In terms of acceptance to the notion of sensory improvement (via implantation) ~39% of the general population questioned responded positively with a further ~25% of the respondents answering with the indecisive response. Thus with careful dissemination a large proportion of individuals may adopt this technology much like this if it were to become available for consumers. Interestingly of the responses collected from the magnetic implants survey ~60% of the respondents actually underwent the implant for magnetic vision purposes. The main contribution of this research however comes from a series of psychophysical testing. In which 7 subjects with subdermal magnetic implants, were cross compared with 7 subjects that had similar magnets superficially attached to their dermis. The experimentation examined multiple psychometric thresholds of the candidates including intensity, frequency and temporal. Whilst relatively simple, the experimental setup for the perceptual experimentation conducted was novel in that custom hardware and protocols were created in order to determine the subjective thresholds of the individuals. Abstract iv The overall purpose of this research is to utilise this concept in high stress scenarios, such as driving or piloting; whereby alerts and warnings could be relayed to an operator without intruding upon their other (typically overloaded) exterior senses (i.e. the auditory and visual senses). Hence each of the thresholding experiments were designed with the intention of utilising the results in the design of signals for information transfer. The findings from the study show that the implanted group of subjects significantly outperformed the superficial group in the absolute intensity threshold experiment, i.e. the implanted group required significantly less force than the superficial group in order to perceive the stimulus. The results for the frequency difference threshold showed no significant difference in the two groups tested. Interestingly however at low frequencies, i.e. 20 and 50 Hz, the ability of the subjects tested to discriminate frequencies significantly increased with more complex waveforms i.e. square and sawtooth, when compared against the typically used sinewave. Furthermore a novel protocol for establishing the temporal gap detection threshold during a temporal numerosity study has been established in this thesis. This experiment measured the subjects’ capability to correctly determine the number of concatenated signals presented to them whilst the time between the signals, referred to as pulses, tended to zero. A significant finding was that when altering the length of, the frequency of, and the number of cycles of the pulses, the time between pulses for correct recognition altered. This finding will ultimately aid in the design of the tactile alerts for this method of information transfer. Preliminary development work for the use of this method of input to the body, in an automotive scenario, is also presented within this thesis in the form of a driving simulation. The overall goal of which is to present warning alerts to a driver, such as rear-to-end collision, or excessive speeds on roads, in order to prevent incidents and penalties from occurring. Discussion on the broader utility of this implant has been presented, reflecting on its potential use as a basis for vibrotactile, and sensory substitution, devices. This discussion furthers with postulations on its use as a human machine interface, as well as how a similar implant could be used within the ear as a hearing aid device.

Secretion of middle ear in infants - Occurrence, recurrence and related factors

Objective: The present study aimed at evaluating the occurrence and recurrence of middle ear effusion and possible associated factors in the first two years of life of 190 newborns and infants, participants in the interdisciplinary prevention, detection, and intervention program at the Clínica de Educação para Saúde of Universidade do Sagrado Coração, Methods: Newborns and infants were monthly submitted to anamneses, otoscopy, behavioral hearing assessment using sound instruments and pure tones (pediatric audiometry) and tympanometry. Results: The results revealed that 68.4% of infants presented one or more episodes of middle ear effusion during their two first years, with more recurrence among males. Peak occurrence was between four and 12 months of age and, the earlier the first episode, the higher the probability of recurrence. Greatest incidence was during May and August. It was found that, of the variables investigated, the period of exclusive breastfeeding actuated as a protector factor. With respect of risk factors, it was observed that passive smoking, gastro-esophageal reflux and respiratory allergy were related with the recurrences of effusion. Conclusion: Findings revealed the importance of periodic auditory follow-up for infants during their first two years of life, considered to be the critical period of auditory system maturation, during which sensory deprivation can be responsible for damage to the development of speech, language and other auditory abilities. Copyright © 2005 by Sociedade Brasileira de Pediatria.

Sensibility of the Ear After Otoplasty

Changes in skin sensibility occur in various postoperative plastic surgeries, especially when they involve major skin and subcutaneous dissection. There were no studies so far that objectively compared changes of ear sensibility. This prospective study was conducted to compare ear sensibility before and after otoplasty. Patients with prominent ears (n = 15) underwent bilateral otoplasty. Ear tactile sensibility was tested preoperatively and 6 and 12 months after surgery by Pressure Specified Sensory Device, an apparatus that quantifies cutaneous pressure sensation (g/mm(2)). Comparison between preoperative and 6-months postoperative results indicated an increment on mean skin pressure thresholds; however, mean thresholds between pre- and 12 months postoperative period were similar. Vibratory and hot/cold sensibility did not present any difference during this period. This is the first comparative assessment of ear tactile sensibility using quantitative methods. After otoplasty, initially there was reduction in an ear tactile sensibility, followed by a return to levels similar to preoperative sensibility.

Herpesviruses use bidirectional fast-axonal transport to spread in sensory neurons

Alpha herpesviruses infect the vertebrate nervous system resulting in either mild recurrent lesions in mucosal epithelia or fatal encephalitis. Movement of virions within the nervous system is a critical factor in the outcome of infection; however, the dynamics of individual virion transport have never been assessed. Here we visualized and tracked individual viral capsids as they moved in axons away from infected neuronal cell bodies in culture. The observed movement was compatible with fast axonal flow mediated by multiple microtubule motors. Capsids accumulated at axon terminals, suggesting that spread from infected neurons required cell contact.

Notch signaling in the development of the inner ear: Lessons from Drosophila

The sensory patches in the ear of a vertebrate can be compared with the mechanosensory bristles of a fly. This comparison has led to the discovery that lateral inhibition mediated by the Notch cell–cell signaling pathway, first characterized in Drosophila and crucial for bristle development, also has a key role in controlling the pattern of sensory hair cells and supporting cells in the ear. We review the arguments for considering the sensory patches of the vertebrate ear and bristles of the insect to be homologous structures, evolved from a common ancestral mechanosensory organ, and we examine more closely the role of Notch signaling in each system. Using viral vectors to misexpress components of the Notch pathway in the chick ear, we show that a simple lateral-inhibition model based on feedback regulation of the Notch ligand Delta is inadequate for the ear just as it is for the fly bristle. The Notch ligand Serrate1, expressed in supporting cells in the ear, is regulated by lateral induction, not lateral inhibition; commitment to become a hair cell is not simply controlled by levels of expression of the Notch ligands Delta1, Serrate1, and Serrate2 in the neighbors of the nascent hair cell; and at least one factor, Numb, capable of blocking reception of lateral inhibition is concentrated in hair cells. These findings reinforce the parallels between the vertebrate ear and the fly bristle and show how study of the insect system can help us understand the vertebrate.

Molecular genetics of pattern formation in the inner ear: Do compartment boundaries play a role?

The membranous labyrinth of the inner ear establishes a precise geometrical topology so that it may subserve the functions of hearing and balance. How this geometry arises from a simple ectodermal placode is under active investigation. The placode invaginates to form the otic cup, which deepens before pinching off to form the otic vesicle. By the vesicle stage many genes expressed in the developing ear have assumed broad, asymmetrical expression domains. We have been exploring the possibility that these domains may reflect developmental compartments that are instrumental in specifying the location and identity of different parts of the ear. The boundaries between compartments are proposed to be the site of inductive interactions required for this specification. Our work has shown that sensory organs and the endolymphatic duct each arise near the boundaries of broader gene expression domains, lending support to this idea. A further prediction of the model, that the compartment boundaries will also represent lineage-restriction compartments, is supported in part by fate mapping the otic cup. Our data suggest that two lineage-restriction boundaries intersect at the dorsal pole of the otocyst, a convergence that may be critical for the specification of endolymphatic duct outgrowth. We speculate that the patterning information necessary to establish these two orthogonal boundaries may emanate, in part, from the hindbrain. The compartment boundary model of ear development now needs to be tested through a variety of experimental perturbations, such as the removal of boundaries, the generation of ectopic boundaries, and/or changes in compartment identity.