Regeneration and mammalian auditory hair cells.
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Animals↗
Biomedical subjects
R Romand
Publications and source records attributed to R Romand.
Fimbrin expression in the developing rat cochlea.
The expression of fimbrin in the developing rat cochlea was analyzed using an immunohistochemical technique with fimbrin antibody. The cochlea displayed temporal and lateral-longitudinal gradients for fimbrin expression during development. Fimbrin immunoreactivity first appeared in the inner hair cell stereocilia of the basal turn on the first gestational day studied (day 18). At birth, both inner (IHC) and outer hair cell (OHC) stereocilia of the basal turn showed positive labeling with fimbrin antibody. The progression of appearance was always from IHCs to OHCs and fimbrin immunostaining appeared in the apical hair cells by postnatal day 6. Immunostaining was restricted to stereocilia and the cuticular plate, and no immunoreactivity was observed in neighboring structures of the epithelium. Double labeling using both fimbrin antibody and phalloidin binding revealed similar chronological expression from the earliest stage studied. Increasing fimbrin immunoreactivity was observed in hair cells until late postnatal and adult stages. This study suggests that fimbrin is expressed with F-actin during development and fimbrin together with actin may constitute the two basic molecules that participate in stereocilia formation. We speculate that fimbrin may help maintain the parallel growth of actin filaments within the stereocilia. These data additionally support previous findings that hair cell maturation occurs from the base to the apex and from IHCs to OHCs.
Actins↗
Histochemical and scanning electron microscopic studies of supernumerary hair cells in embryonic rat cochlea in vitro.
In the embryonic organ of Corti supernumerary hair cells were observed when developed in organotypic cultures. Hair cells ranging in up to two rows of inner hair cells (IHCs) and up to nine rows of outer hair cells (OHCs), were observed by phalloidin histochemistry. The total number of hair cells may double in some explanted cochleae compared to control ones. Cuticular plates of hair cells displayed an actin-free zone corresponding to the kinocilium location, differently located and indicating different degrees of differentiation and maturation. Moreover, some hair cells had a small apical surface area and a centrally located kinocilium, revealing immaturity. Under scanning electron microscopy, stereocilia appeared to differentiate normally, as compared to the in vivo development. The staircase pattern of the stereociliary bundles was reached on most of the hair cells with a 'V' shape on the OHCs and hemispherical one on the IHCs. Hair cell polarity was not homogeneous along the length of the tissue. Organs of Corti explanted at birth developed a weaker number of supernumerary hair cells showing a decrease of supernumerary hair cells with the developmental stage of the explant. These results provide evidence for supernumerary hair cells in the mammalian cochlea in culture, without loss or injury to preexisting hair cells.
Animals↗
Development of tonotopy in the inferior colliculus II: 2-DG measurements in the kitten.
The development of size and tonotopy in the inferior colliculus of the kitten was studied using the [14C]2-deoxyglucose technique and tone stimulation with 2 and 15 kHz at a maximum 110 dB sound pressure level. At 2 days of age, frequency-specific labelling cannot be detected. Two kilohertz labelling is distinctly visible in the rostral and central inferior colliculus at day 6; 15 kHz labelling occurs first at day 11. In the rostral and central inferior colliculus, 2 kHz labelling starts at a ventral and central position and shifts dorsalwards and to a more lateral location between postnatal days 6 and 21. Such a shift is not seen in the caudal inferior colliculus. There, the focus of 2 kHz labelling remains rather constant; only the extension of the labelling increases in the older animals. In all parts of the inferior colliculus, 15 kHz labelling starts at a ventromedial position and shifts to a more lateral location while extending also more dorsalwards as the age increases. These changes in 15 kHz labelling continue up to 3 months. In addition to the ventromedial-to-dorsolateral shift and expansion of labelling, there is also a rostral-to-caudal gradient of maturation, in that in older animals frequency-specific labelling reaches farther caudalwards. The reported changes in frequency representation in the inferior colliculus can be explained on the basis of a shift in frequency input and input sensitivity to the laminae of the inferior colliculus, mainly due to maturational changes within the cochlea and/or as a consequence of the increasing size of the inferior colliculus.
Acoustic Stimulation↗
Neurotrophins and the development of cochlear innervation.
The rapid progress in the past few years concerning neurotrophic factor research, has greatly stimulated advances in developmental neurobiology of hearing. We have summarized evidence that neurotrophins are expressed by auditory sensory epithelia during the time at which ganglion cells with neurotrophin receptors send their processes to these epithelia. Recent findings have led to the identification of BDNF and NT3 as responsible substances. Since no NGF mRNA nor the NGF high affinity receptor component trkA mRNA were detectable during the development of cochlear structures, this factor is not likely to be an important neurotrophin at this level. By their biological activity, neurotrophins could be responsible for chemotrophic, differentiation, survival, and maintenance functions at the afferent as well as at the efferent level of the inner ear development.
Afferent Pathways↗
N-myc expression in the embryonic cochlea of the mouse.
N-myc expression in the mouse embryo was examined during the late cochlear organogenesis. Tissue distribution of N-myc expression was histologically analyzed by in situ hybridization of the transcript in the cochlea between 15 and 18 days of gestation. At 15 days of gestation, N-myc expression was found very conspicuous in nervous structure of the cochlea such as the auditory nerve and the spiral ganglion. Moreover, N-myc was also present in the Köllikers organ and in the epithelium surrounding the cochlear canal. A few days later, N-myc expression was still clearly present in the Köllikers organ but less so in nervous structures. This study shows that cochlear tissues derived from the otic placode present a significant level of N-myc transcript during late embryogenesis. N-myc expression seems to be related to cell differentiation in the inner ear.
Animals↗
Distribution and metabolism patterns of plasma 7S- and beta-NGF in the adult male rat.
There is a great deal of controversy on the existence of NGF in body fluids and tissues. To date it remains unknown whether this peptide accumulates preferentially at significant levels in different organs. Thus we undertook the evaluation of kinetic parameters of the disappearance of blood of 125I-7S-NGF and 125I-beta-NGF after intravenous injection in male adult rats. Our results indicate that the plasma half-life of 125I-7S-NGF is approximately twice as long as for 125I-beta-NGF (respectively 61.7 +/- 11.7 min and 36.3 +/- 2.2 min) while the distribution volume is not significantly different between both peptides. Furthermore, the uptake of radioactive NGF by different tissues seems very low as shown by 125I-7S-NGF and 125I-beta-NGF content of the sampled organs compared to the plasma concentration at the same time. These results indicate that the tissue uptake of circulating 7S and beta-NGF is very low in the adult rat. Thus in these animals NGF did not cross the blood-brain barrier and did not accumulate in peripheral organs which are known to contain subsequent amounts of this peptide. This lack of deposition might be due to a binding with plasma proteins (probably alpha 2-macroglobulin).
Animals↗
Distribution of cytoskeletal proteins (neurofilaments, peripherin and MAP-tau) in the cochlea of the human fetus.
We report here an immunohistochemical study of the distribution of intermediate filaments (neurofilament, peripherin) and a microtubule-associated protein, tau, in the human fetal cochlea at 27 weeks of gestation. Neurofilament immunoreactivity (160 and 200 KDa) was localized in afferent and efferent fibers of the cochlear innervation and restricted to a few small spiral ganglion neurons. Peripherin immunoreactivity was specifically distributed in some small ganglion neurons and in their central and peripheral extensions, particularly in fibers reaching the lower part of the outer hair cells. Double immuno-labelling studies with these neurofilaments and peripherin antibodies show that only small neuron cell bodies were stained. Morpholometrical data indicate that immunostained neurons could be related to the Type II neuron population in the spiral ganglion. Tau protein was localized in intraganglionic spiral bundle fibers and in fibers that reach the lower part of hair cells. These observations suggest that neurofilament and peripherin antibodies stain a particular population of human spiral ganglion neurons with Type II characteristics. Moreover, the specificity of peripherin labelling in Type II cells and their processes suggest that peripherin could be used as a probe for the developmental study of this system in the human cochlea. On the other hand, tau antibody appeared as a marker for efferent fibers during development and could give information on the ontogenesis of efferent innervation.
Cochlea↗
Hair cell overproduction in the developing mammalian cochlea in culture.
Hair cells transduce acoustics into electrical signals that are conveyed to the brain by auditory nerve fibres. Hair cells loss in mammals due to ageing, ototoxic drugs or noise, leads to irreversible hearing impairment. One objective would be to replace lost cells by regeneration or production of new hair cells. We report an overproduction of hair cells in the developing cochlea of the rat in culture without adding drugs, without previous injury or special manipulations of the explants. The overproduction of hair cells does not depend on the culture medium or on the innervation of the organ of Corti. Younger foetal explants show higher potency for the production of supplementary hair cells than older ones. This is the first report of the generation of extra hair cells in mammals without previous hair cell loss or treatment with drugs.
Aging↗
Expression of alpha-actinin in the stereocilia of hair cells of the inner ear: immunohistochemical localization.
The distribution of alpha-actinin was studied on surface preparations and cryosections from the organ of Corti of the adult rat by using polyclonal and monoclonal antibodies to smooth muscle alpha-actinin. The polyclonal antibody used gave a positive immunoreactivity in the cuticular plate of the hair cells, pillar cells and supporting cells. A specific labelling of the stereocilia of the outer hair cells (OHCs) and inner hair cells (IHCs) on surface preparations was highly visible. The monoclonal antibody used produces similar patterns of labelling in the organ of Corti. The possible roles of this protein in hair cells are discussed.
Actinin↗
Ontogenesis of F-actin in hair cells.
This report describes the ontogenesis of cochlear stereocilia using scanning electron microscopy for analysis of cilia appearance, and fluorescence microscopy of phalloidin, a label for F-actin, to determine the maturation of the cilia framework. Surface and frozen-sectioned preparations of the otic capsule were obtained from several stages of rat pup development beginning at the 16th gestational day and at various stages until adulthood. In the earliest stage investigated, strong fluorescence labeling was visible on the apical part of Kölliker's organ, revealing a reticular outline of cell junctions. Hair cells started to differentiate at the 18th day of gestation from cells within the primordial receptor area. Phalloidin labeling revealed a sequential appearance of F-actin as the hair cells differentiated from the cells with the Kölliker's organ. The differentiation of receptor cells occurred first with the appearance of a junctional complex between the hair cell and the surrounding cells. Then a cuticular plate appeared followed by the progressive emergence of stereocilia. The F-actin labeling also revealed a progressive differentiation of receptor cells from the cochlear base to its apex. There was also an inner to outer hair cell developmental gradient of label. Inner hair cells developed stereocilia before outer hair cells. The third row of outer hair cells was the last to acquire stereocilia. The adult pattern of stereocilia was reached around the 6th postnatal day. We conclude that the appearance of actin filaments in developing receptor cells and the emergence of stereocilia can be regraded as markers for correlating function and other structural differentiation.
Actins↗
An original organotypic culture method to study the organ of Corti of the newborn rat in vitro.
The inner ear growth is related to several factors which are not yet well known. In order to study the effect of growth factors on the development of the auditory receptor cells, we have chosen first to establish an in vitro model of the inner ear. Newborn rats were selected as the source of tissue because the inner ear is immature enough at this stage of development for studying some relations between receptors and their innervation. Until recently, the Maximov slide assembly technique was the only organ culture system available and silver neurofibrillary methods were used to stain the nervous structures. These are difficult and time-consuming techniques. With the use of a collagen gel drop floating in the culture medium, we have developed a simple and reliable method. Furthermore, an immunohistochemical fiber-staining technique with anti-neurofilament and histochemical staining technique with phalloidin allows us to check in a few days the organotypy at the spiral ganglion and hair cell levels. This floating drop method gave us some preliminary information about the spiral neuron cells which survived.
Animals↗
Ontogenesis of type II spiral ganglion neurons during development: peripherin immunohistochemistry.
In this study, we analysed the distribution of the intermediate filament peripherin in the developing cochlea of the rat. At gestational day 16, weak immunolabeling was observed in neuronal somas throughout the spiral ganglion. At gestational day 20, the peripherin labeling increased in intensity throughout the spiral ganglion. At gestational day 20, the peripherin labeling increased in intensity throughout the cochlea but became especially strong in some ganglion neurons of the basal turn. Homogeneous immunolabeling was observed throughout the spiral ganglion of the apical turn. Double immunofluorescence labeling of the prenatal cochlea with peripherin and neurofilament (NF) antibodies revealed colocalization on the same structures. By postnatal day 3, the peripherin labeling intensity had decreased in the majority of spiral ganglion neurons, but remained strong in some cells of the basal turn. Only a few neurons continued to be immunolabeled into adulthood that correspond to Type II spiral ganglion neurons expressing both NF protein and peripherin, two classes of intermediate filament proteins. In the organ of Corti, the first immunolabeling was observed on gestational day 20 as peripheral fibers reaching the receptor cells. Positive fibers were observed below both inner (IHCs) and outer (OHCs) hair cells. At birth and at postnatal day 3, peripherin immunolabeling was still observed below both IHCs and OHCs. By postnatal day 4, peripherin labeling became more dominant in fibers below OHCs, but some immunoreactivity was still present below IHCs. No immunoreactivity was present in the intraganglionic spiral bundle (IGSB) fibers containing the olivary complex efferent fibers before birth. A few days after birth some fibers of the IGSB started to be immunoreactive.
Animals↗
Developmental differentiation of MAP2 expression in the central versus the peripheral and efferent projections of the inner ear.
The goal of this study was to extend our knowledge of MAP2 localization in the peripheral nervous system of mammals, since most results on MAP2 distribution are obtained in the central nervous system (CNS). This study shows the presence of microtubule-associated protein 2b (MAP2b) and MAP2c in the inner ear and describes the immunocytochemical distribution of MAP in adult and developing spiral ganglion of the rat by using a well-characterized antibody for MAP2a and MAP2b. (This antibody does not recognize the immature MAP2c). MAP2 labeling is already present in spiral ganglion neurons at 16 days of gestation. From this stage and up to the first postnatal week, MAP2 labeling was strong in all spiral ganglion neurons and their central processes. Double immunostaining at the 16-day stage with anti-MAP2 and anti-neurofilament (NF) antibodies mainly showed NF labeling in central branches that corresponded to anatomically and functionally described axons of spiral neurons. The peripheral branches lacked MAP2 labeling. In neonatal and postnatal stages, MAP2 reactivity was located in spiral ganglion perikarya and their neurites. The intensity of adult labeling was, however, lower than in younger animals. The antibody used in this study did not label axons originating in the CNS as seen by a negative response in efferent fibers from the intraganglionic spiral bundle of the cochlea. Our results suggest that during ontogenesis, MAP2 is highly expressed in the central projection of spiral ganglion neurons, and then is reduced to lower quantities in the central branch after the first postnatal week and persists into adulthood.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Development of tone response thresholds, latencies and tuning in the mouse inferior colliculus.
The development of tone response thresholds, latencies and tuning of neurons in the inferior colliculus (IC) of the mouse has been investigated between postnatal days 10 (first responses) and 20. As in adults, response thresholds of neurons are lowest in the center of the IC compared with other areas right from the beginning of responsiveness at day 10. Thresholds decrease rapidly until at days 16-20 (depending on the characteristic frequency of the neurons) adult levels are reached. Response latencies decrease rapidly to adult levels at days 16-18. Broad frequency tuning curves can be measured at days 10 and 11. From day 12 onwards, tuning curve shapes differentiate and adult diversity occurs which indicates presence of inhibition and summation in the pathway to or within the IC at that early age. The tip lengths of the tuning curves increase faster than the sharpness of the tips (Q10 dB values). The developmental courses of the measured parameters are expressed by power functions. The time constants of these functions are used in the discussion of processes underlying the functional maturation in the auditory system of the mouse. The general course of the development of all the here investigated response properties of single neurons in the IC, except tuning curve shape, appears to be determined by maturation at or peripheral to the cochlear level.
Acoustic Stimulation↗
Expression of the beta-nerve growth factor gene in male sex organs of the mouse, rat, and guinea pig.
Steady-state nerve growth factor (NGF) mRNA levels were estimated in male sex organs of the mouse, rat, and guinea pig by RNA blot hybridization analysis. The abundance of NGF mRNAs was in the order vas deferens greater than epididymis greater than or equal to seminal vesicles much greater than testis. NGF mRNA levels in these organs were compared with those estimated for other rat peripheral tissues and were found to correlate with the density of their sympathetic innervation, with the exception of guinea pig prostate. Castration had no significant effect on NGF mRNA levels in the guinea pig prostate, suggesting that NGF synthesis in this tissue is not under direct androgen control. NGF-like and proNGF-like immunoreactivities were localized by immunohistochemical techniques in the secretory cells of the glandular epithelium of the guinea pig prostate and in germ cells in the seminiferous tubules of the mouse testis.
Animals↗
Immunohistochemical localization of nerve growth factor receptor in the cochlea and in the brainstem of the perinatal rat.
Nerve growth factor receptor (NGF-R) localization was studied immunohistochemically in the cochlea and in the brainstem of the perinatal rat, using a specific monoclonal antibody directed against the rat NGF-R. In the cochlea, NGF-R immunoreactivity is positive during the whole perinatal period studied, and is located at the hair cell level, in fibers that reach the organ of Corti, in the intraganglionic spiral bundle and in some small bundles of fibers in the auditory nerve. In the brainstem, NGF-R is detected in auditory structures such as the ventral cochlear nucleus, the superior olivary complex, the nuclei of the trapezoid body and the trapezoid body. Many auditory structures labelled by the NGF-R antibody are implicated in the efferent cochlear innervation. These results suggest that NGF could be implicated in interactions between auditory receptors and efferent innervation of the developing cochlea. This coincides with findings on the immunohistochemical localization of NGF-like protein in the organ of Corti of the developing rat. Moreover, these observations could be related to an early prenatal development of auditory efferent innervation.
Animals↗
Basic FGF-like protein in the lower stato-acoustic system of the neonatal and adult rat.
Basic fibroblast growth factor (bFGF)-like localization was studied immunohistochemically in the lower auditory tract of neonatal and adult rats. During the neonatal period, bFGF-like immunoreactivity is present in the cytoplasm of inner hair cells, spiral ganglion cells, Scarpa's ganglion cells, in auditory brain stem nuclei and in vestibular nuclei. At the adult stage, bFGF-like protein is widely distributed in the auditory brain stem but was not found in the cochlea. These results suggest that bFGF could be implicated in the development as well as in the neuronal maintenance and plasticity of the auditory system.
Age Factors↗