Modification of tonotopic representation in the auditory system during development.

During the early development of the bird and the mammalian peripheral auditory system, a restricted range of low--mid frequencies is recorded in immature animals. These early recordings are correlated to the base or mid-basal region of the cochlea which codes high frequencies in the adult. In order to reconcile the functional observations with anatomical ones, two main hypotheses have been put forward: one called the development of the place principle derived from observations of acoustic trauma in chick cochlea and a second derived from auditory nerve fiber recordings in kittens. Whatever the theories, the tonotopic shift during development is a well-established phenomenon in both birds and mammals that could be explained by a synthetic theory including active and passive cochlear processes. The tonotopic shift observed in the central auditory system mimics quite closely the frequency representation of the peripheral auditory system. The same trend is observed in all auditory nuclei including the cortex, except that the frequency representation is more complex because it shows tonotopic maps that can be twisted in three dimensions. From current observations, there is a simultaneous onset of tonotopic maps across auditory nuclei up to the cortex. A hypothesis is presented related to the frequency changes observed in the cochlea that affect the central auditory pathway, along with possible consequences on auditory behavior.

Specific expression of the retinoic acid-synthesizing enzyme RALDH2 during mouse inner ear development.

Retinoid binding proteins and nuclear receptors are expressed in the developing mouse inner ear. Here, we report that the retinaldehyde dehydrogenase 2 (Raldh2) gene, whose product is involved in the enzymatic generation of retinoic acid (RA), exhibits a restricted expression pattern during mouse inner ear ontogenesis. The Raldh2 gene is first expressed at embryonic day (E) 10.5 in a V-shaped medio-dorsal region of the otocyst outer epithelium, which evolves as two separate domains upon otocyst morphogenesis. At E14.5, Raldh2 is expressed in two areas of the utricle epithelium and specific regions of the saccule and cochlear mesenchyme. Later, Raldh2 transcripts are restricted to two cochlear areas, the stria vascularis and Reissner membrane. Raldh2 mesenchymal expression did not correlate with migrating neural crest-derived melanoblasts. These restricted expression domains may correspond to specific sites of RA synthesis during inner ear morphogenesis.

Cochlear innervation in the developing rat: an immunocytochemical study of neurofilament and spectrin proteins.

We have studied the innervation of the developing cochlea by immunocytochemical staining of the cytoskeletal proteins, neurofilament (NF), and spectrin (brain spectrin and erythrocyte spectrin). NF immunoreactivity was seen in spiral ganglion cell bodies and their processes and in fibers of the intraganglionic spiral bundle (IGSB) on gestational day 16. NF immunoreactivity with monoclonal antibodies to NF160 and NF68 was present beneath both inner hair cells (the IHC) and outer hair cells (OHCs) on gestational day 20. NF200 immunostaining was located only in the IGSB and in fibers reaching the IHC. The first NF200 immunoreactivity beneath the OHCs was seen in the basal turn at birth. NF labelling began to decrease on postnatal day 9 and its intensity became more like that of the adult. Brain spectrin immunostaining was first seen in the IGSB of the basal turn on gestational day 18. It reached the fibers between the spiral ganglion and the IHC on gestational day 20. Brain spectrin immunoreactivity was first seen beneath the OHCs in the basal turn at birth. It reached all the OHCs of the cochlea by postnatal day 4, and began to decrease 9 days after birth. Erythrocyte spectrin immunostaining was first observed during the second postnatal week, when it labelled spiral ganglion cells. The distribution of NF200 and brain spectrin immunoreactivity suggested that efferent innervation of OHCs is present at birth in the rat, and confirms previous studies showing the early efferent innervation of the OHCs of the mouse and the rat at birth, and the time lag between the appearance of the two spectrin isoforms during development.

Myelination kinetics of spiral ganglion cells in kitten.

A study of the regions of myelination of spiral ganglion cell bodies at birth in the kitten revealed that the first myelination occurred before birth. A quantitative analysis of myelinated versus unmyelinated cells, and the distribution of myelin lamellae of the ganglion cell sheath reflect the kinetics of the later stages of cochlear maturation. The onset of the final maturation process begins in the basal region of the first turn, which is 20% of the length of the spiral lamina measured from the basal extremity of the cochlea. This maturation proceeds in an orderly manner from the lower half of the first turn to the apex, but also develops to a smaller degree toward the hook region. Results are compared with previous findings.

Spatial distributions of retinoic acid receptor gene transcripts in the prenatal mouse inner ear.

The expression patterns of the three mouse retinoic acid (RA) receptor gene isotypes (RARalpha, RARbeta, and RARgamma) and retinoid X receptor gene isotypes (RXRalpha, RXRbeta, and RXRgamma) have been investigated by in situ hybridization analysis of their RNA transcripts in the inner ear of mouse fetuses at 18.5 days of gestation. Two RARs (RARalpha and RARgamma) and two RXRs (RXRalpha and RXRbeta) presented an almost ubiquitous transcript distribution with overlapping expression in several regions of the cochlea, such as Kölliker's organ, the organ of Corti, the spiral limbus, and nervous structures. The organ of Corti showed an enhanced in situ labeling with RARalpha and RXRbeta. By contrast, RARbeta and RXRgamma displayed more restricted expression patterns. RXRgamma in particular was strongly expressed in Kölliker's organ and in the spiral ganglion. This expression pattern suggests that RA may be involved in the differentiation of several cochlear cell types. Moreover, the colocalization of several RAR and RXR gene transcripts suggests possible heterodimerization between these receptors in several regions of the cochlea.

Regional differences in fiber size in the cochlear nerve.

The topographical variations in fiber size in the cochlear nerve of cat were quantitatively studied by electron microscopy. Measurements of fiber size as they appeared at the outlet of the spiral lamina show that fibers originating from the basal part of the cochlea are larger than those from the apex. When the diameter of apical fibers and their axoplasms at two different levels are compared, a significant variation in size is observed. As they appear in the internal acoustic meatus the apical fibers are larger at the level of the nerve trunk compared with the same fibers near the ganglion cell bodies. There are significant differences in fiber diameter with regard to their length for fibers derived from the apical turn. These results are compared with previous findings.

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)

The structural maturation of the stato-acoustic nerve in the cat.

The maturation of the stato-acoustic nerve in the cat was studied by light and electron microscopy from the fetal stage to the adult. Measurement of the outer diameter of the fibers and the study of the myelination process revealed that myelination begins earlier for the vestibular nerve than for the cochlear nerve: by the fifty-third day of gestation 64% of the vestibular fibres have already passed the promyelin stage whereas for the cochlear nerve this promyelin stage begins for the majority of fibers on the fifty-seventh gestation day. Afterward, maturation proceeds more rapidly for the cochlear nerve. In the case of both nerves, maturation is still incomplete at two months of age. Concerning the relationship between the thickness of the myelin sheath and the axoplasmic diameter, there is already a good correlation by the fifty-seventh day of gestation in the vestibular nerve, whereas it appears several days after birth in the cochlear nerve.

Localization of neurotrophin-3-like immunoreactivity in the rat cochlear nucleus.

Immunohistochemistry as well as immunohistofluorescence were used to investigate the distribution of the neurotrophin-3 (NT3) in the adult rat cochlear nucleus. We found a widespread distribution of NT3 immunolabeled neurons throughout the three divisions of this nucleus. NT3-like immunoreactivity was clearly population-specific, with some cell groups heavily (various small neurons and granule cells) or moderately (large neurons of the ventral cochlear nucleus) stained, while others remained negative (a major fraction of medium and large neurons of the dorsal cochlear nucleus). Double-labeling experiments were performed using antibody against the glial fibrillary acid protein, a classic marker for mature astrocytes. This colocalization study revealed that NT3 immunoreactivity was also present in a subpopulation of astrocytes, particularly in the glia limitans and their projections. Numerous small cells also colocalized NT3 together with the glial marker in the granule cell domain and in the molecular cell layer of the dorsal cochlear nucleus. These results suggest that NT3 may exist in widespread populations of adult cochlear nucleus neurons as well as in glial cells. This abundant distribution of NT3-like immunoreactivity implies that this neurotrophin may have an important role in the continued maintenance of mature cochlear nucleus and makes it an attractive candidate for playing a role in regulation or stabilization of neuronal circuits in this nucleus.

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.

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.

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.

The spontaneous appearance of hair cell-like cells in the mammalian cochlea following aminoglycoside ototoxicity.

Young rats (in vivo) and cochleas from neonatal rats (in vitro) were treated with ototoxic antibiotics. Scanning electron microscope observations of the cicatricial epithelium of the former outer hair cell region revealed cells with a tuft of microvilli at their apical surface that could contain actin filaments, as observed by phalloidin staining. The apical organization of these hair cell-like cells was reminiscent of fetal hair cells topped with a bundle of microvilli. During both in vivo and in vitro observations, and despite the use of several growth factors in vitro, these hair cell-like cells did not differentiate into mature sensory cells. These hair cell-like cells might represent an attempt by the former sensory epithelium to regenerate.

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).

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.

Survey of intracellular recording in the cochlear nucleus of the cat.

Intracellular recordings were made in the cochlear nucleus of anesthetized cats. In anterior passes, one never obtained sustained depolarizations from 'primary-like' units. For 'chopper' units, however, it was possible to record sustained depolarizations accompaneid by spikes that lasted as long as the tone burst. 'Pauser, 'buildup' and 'on' units also had spike responses that could be accompanied by sustained depolarizations. For 'pauser', 'buildup' and 'on' units, hyperpolarization was not seen during the times when no spike discharges appeared so long as the tone bursts were at the characteristic frequency of the units.

Postnatal maturation of the cochlear neclei in the cat: a neurophysiological study.

The study of the postnatal maturation of the ventral cochlear nuclei (VCN) and the dorsal cochlear nuclei (DCN) was carried out on the cat by means of recordings of the extra-cellular neuronal activity. At birth it is already possible to obtain toneburst responses in the VCN and DCN. At this age the responses are characterised by a small number of spikes grouped in three bursts when the tone-bursts lasted 500 msec. Subsequently, the number of bursts increases until, from 9 or 10 days onwards the responses become sustained. These responses do not acquire their adult characteristics until more than a month after birth. During postnatal maturation of the cochlear nuclei, the VCN is distinguishable from the DCN by the greater number of units revealing spontaneous activity. Another criterion, such as latency, differentiates the VCN from the DCN from the point of view of the kinetics of maturation.

Development of the auditory receptors of the rat: a SEM study.

Fetal and postnatal ontogenesis of the rat cochlea, from the 16th gestational day (16DG) until 3 months post partum, were studied using scanning electron microscopy with emphasis on the stereocilia during the earliest stages of development. The epithelium of the cochlear duct in 16DG rat consisted of plygonal cells topped with numerous microvilli and one central kinocilium, which form the so-called Kölliker's organ. Inner hair cells (IHCs) appeared at 18DG in the basal cochlea. They were characterized by tufts of cilia of the same height and with a kinocilium. The first outer hair cells (OHCs) can be seen at 20DG. The earliest stages of ciliary differentiation, at 18DG for IHCs and 20DG for OHCs, were similar on both types of cells and were characterized by the presence of round bundles of cilia arising from the surrounding microvilli. A three-dimensional V-shaped organization for OHCs and the linear arrangement for IHCs appeared by the end of the first postnatal week, accompanied by the disappearance of transient cilia on the modiolar side of the hair cell and the kinocilium on the external side. The apical pole of OHCs reached adult-like morphology before that of IHCs. Various links between stereocilia were detected already at birth. Morphometric analysis showed that auditory cells from the base of the cochlea reached adult size by the end of the first postnatal week while those from the apex increased their size later. A review of the literature including comparative observations across species on the ontogenesis of the stereocilia shows that hair cells of the stato-acoustic system may present the same early ontogenesis.

Effects of growth factors on the hair cells after ototoxic treatment of the neonatal mammalian cochlea in vitro.

The aim of this study was to test the possible regenerative potential of several molecules and growth factors such as retinoic acid (RA), insulin, epidermal growth factor (EGF) and transforming growth factors alpha (TGFalpha) and beta (TGFbeta) on the neonatal cochlea in vitro after neomycin intoxication. Our studies show that cochlear sensory epithelium behaves differently while maintained in various culture conditions, although we did not observe regeneration whatever the molecules or growth factors tested. The ototoxic action of neomycin in vitro produced a specific death of hair cells, except in the apical region. Organ of Corti of rats 3 days after birth always presented two regions that responded differently to the antibiotic: a widespread scar region extending from the basal cochlea up to the beginning of the apical turn, where most hair cells had disappeared, and a second region called the resistance region localized in the apex, and which was more or less developed depending on culture conditions. The length of the resistance region was modulated by molecules or growth factors added to the feeding solution suggesting that some of them could produce a protective action on hair cells against neomycin. Slight protection effects may be found with RA and insulin, however, the most definite protection results from the combination of insulin with TGFalpha as shown by the large increase in the length of the resistance region compared to organ of Corti treated with antibiotic alone. The tested molecules and growth factors did not promote cochlear hair cell regeneration in vitro after neomycin treatment, however some of them may offer a protective action against ototoxicity.