Postnatal structural development of mammalian Basilar Membrane provides anatomical basis for the maturation of tonotopic maps and frequency tuning.

The basilar membrane (BM) of the mammalian cochlea constitutes a spiraling acellular ribbon that is intimately attached to the organ of Corti. Its graded stiffness, increasing from apex to the base of the cochlea provides the mechanical basis for sound frequency analysis. Despite its central role in auditory signal transduction, virtually nothing is known about the BM's structural development. Using polarized light microscopy, the present study characterized the architectural transformations of freshly dissected BM at time points during postnatal development and maturation. The results indicate that the BM structural elements increase progressively in size, becoming radially aligned and more tightly packed with maturation and reach the adult structural signature by postnatal day 20 (P20). The findings provide insight into structural details and developmental changes of the mammalian BM, suggesting that BM is a dynamic structure that changes throughout the life of an animal.

Distortion product otoacoustic emission phase and component analysis in human newborns.

Apical distortion product otoacoustic emissions (DPOAEs) are comprised of at least two components, as evidenced by the interference pattern of alternating maxima and minima known as fine structure. DPOAE fine structure is produced by the shifting phase relationship in the ear canal, between the generator and characteristic frequency (CF) component of the response. Each component arises from a different cochlear region and, according to theory, reflects a distinct generation mechanism. The analysis of DPOAE components and phase in newborns may provide a window into targeted aspects of cochlear physiology during development. 2f(1)-f(2) DPOAE fine structure was recorded from 15 adults and 14 newborns using a swept-tone technique. DPOAE group delay, as well as magnitude and phase of each component, was compared between age groups. Results show narrower fine structure spacing, a longer group delay (steeper phase gradient) in low frequencies, and a stronger relative contribution from the CF component in newborns. The prolonged group delay for low-frequency DPOAEs could indicate immature basilar membrane motion in the apex of the cochlea and warrants further investigation. The enhanced contribution from the CF component may have implications for clinical practice as well as for theories of cochlear maturation.

Developmental changes of mechanics measured in the gerbil cochlea.

This report describes stiffness and best frequency measurements obtained in vitro from the basilar membrane of the gerbil cochlea at the onset of hearing, during hearing maturation, and after hearing has matured. Our stiffness data constitute the first direct experimental evidence of developmental stiffness changes in the basal and middle turns. Stiffness changes by a factor of 5.5 in the basal turn between postnatal day 11 and adult, and the difference from adult is statistically significant for all ages measured up to postnatal day 16. For the middle turn, stiffness changes by a factor of 1.6 between postnatal day 11 and adult. Whereas for postnatal day 12 and beyond there is no statistically significant difference from adult, our data suggest that there may be a significant difference of stiffness between day 11 and adult in the middle turn. For the basal turn, our motion measurements confirm a passive component to the developmental best frequency shift. For the middle turn, changes in best frequency are not statistically significant. Best frequency was determined by stimulating the tissue at audio frequencies with a glass paddle and measuring motion with a computer-based imaging system. Tissue stiffness was measured with a piezoelectric-based sensor system. Tissue stiffness changes have previously been postulated to contribute to the best frequency shift observed in the cochlear base. Incorporating our data into a simple spring-mass resonance model demonstrates that our experimentally measured stiffness change can account for the change of best frequency. These results suggest that a stiffness change is, in fact, a critical component of the best frequency shift observed in the basal turn of the gerbil cochlea after the onset of hearing.

Development of acetylcholine receptors in cultured outer hair cells.

Efferents, originating in the superior olivary complex, preferentially synapse with cochlear outer hair cells (OHCs), with acetylcholine (ACh) as their primary neurotransmitter. The OHC ACh receptors (AChRs), which have unusual pharmacology, have been cloned and identified as a new subunit (alpha9) of the nicotinic AChR family. The expression of alpha9 AChRs is first detected before birth and peaks between 6 and 10 days after birth (DAB) in developing mice and rats, while functional maturation of the receptor, as determined by measuring the ACh-induced currents, takes place between 6 and 12 DAB. In this study we attempted to examine the development of AChRs in OHCs grown in explanted cultures, deprived of efferent innervation. ACh-induced currents were used as an assay. Reverse transcription-PCR analysis was also performed to detect the expression of alpha9 subunit from cultured OHCs. PCR study indicates that mRNA of the alpha9 subunit was expressed in primary cochlear cultures, similar to that seen in the cochleae of developing animals. Measurement of whole-cell currents showed that ACh-induced outward current was first detected around 5 days in a fraction of cultured OHCs. The number of responsive cells increased between 5 and 12 days in culture. The size of ACh-induced currents also increased during this period. These results suggest that the development of AChRs in cultured OHCs is not affected by removal of efferent innervation.

Development of the gerbil inner ear observed in the hemicochlea.

A frequency-dependent change in hearing sensitivity occurs during maturation in the basal gerbil cochlea. This change takes place during the first week after the onset of hearing. It has been argued that the mass of a given cochlear segment decreases during development and thus increases the best frequency. Changes in mass during cochlear maturation have been estimated previously by measuring the changes in cochlear dimensions. Fixed, dehydrated, embedded, or sputter-coated tissues were used in such work. However, dehydration of the tissue, a part of most histological techniques, results in severe distortion of some aspects of cochlear morphology. The present experiments, using a novel preparation, the hemicochlea, show that hydrated structures, such as the tectorial membrane and the basilar membrane hyaline matrix, are up to 100% larger than estimated previous studies. Therefore, the hemicochlea was used to study the development of cochlear morphology in the gerbil between the day of birth and postnatal day 19. We used no protocols that would have resulted in severe distortion of cochlear elements. Consequently, a detailed study of cochlear morphology yields several measures that differ from previously published data. Our experiments confirm growth patterns of the cochlea that include a period of remarkably rapid change between postnatal day 6 and 8. The accelerated growth starts in the middle of the cochlea and progresses toward the base and the apex. In particular, the increase in height of Deiters' cells dominated the change, "pushing" the tectorial membrane toward scala vestibuli. This resulted in a shape change of the tectorial membrane and the organ of Corti. The tectorial membrane was properly extended above the outer hair cells by postnatal day 12. This time coincides with the onset of hearing. The basilar membrane hyaline matrix increased in thickness, whereas the multilayered tympanic cover layer cells decreased to a single band of cells by postnatal day 19. Before and after the period of rapid growth, the observed gross morphological changes are rather small. It is unlikely that dimensional changes of cochlear structures between postnatal days 12 and 19 contribute significantly in the remapping of the frequency-place code in the base of the cochlea. Instead, structural changes affecting the stiffness of the cochlear partition might be responsible for the shift in best frequency.

Postnatal development of the organ of Corti in cats: a light microscopic morphometric study.

This study quantitatively characterizes the development of the major morphological features of the organ of Corti during the first 2 weeks postnatal, the period when the cat auditory system makes the transition from being essentially non-functional to having nearly adult-like responses. Four groups of kittens (n = 3) were studied at one day postnatal (P1), P5, P10, P15, and compared to adults. Measurements were made of the organ of Corti at 3 cochlear locations: 20%, 60% and 85% of basilar membrane length from the base cochlear locations which in the adult correspond to best frequencies of approximately 20 kHz, 2 kHz and 500 Hz, respectively. In addition, measurements of basilar membrane length and opening of the tunnel of Corti were made in 20 cochlear specimens from kittens aged P0-P6. Results indicate that: (i) at P0 the basilar membrane has attained adult length, and the tunnel of Corti is open over approximately the basal one-half of the cochlea; (ii) the initial opening of the tunnel of Corti occurs at a site about 4 mm from the cochlear base (best frequency of approximately 25 kHz in the adult cochlea); (iii) the thickness of the tympanic cell layer decreases markedly at the basal 20-kHz location; (iv) the areas of the tunnel of Corti and space of Nuel and the angulation of the inner hair cells (IHC) relative to the basilar membrane all show marked postnatal increases at both the middle and apical locations; (v) IHC are nearly adult-like in length and shape at birth, whereas the OHC (at 2-kHz and 500-Hz locations) undergo marked postnatal changes; (vi) disappearance of the marginal pillars and maturation of the supporting cells are not yet complete by P15.

Fibronectin-like immunoreactivity of the basilar membrane of young and aged rats.

Dysfunction of cochlear mechanics has been hypothesized to be a source of age-related hearing loss and the basilar membrane mass and stiffness contribute to normal cochlear mechanics. Fibronectin, a large, extracellular matrix protein and a major component of the basilar membrane, may contribute to both the mass and stiffness of the membrane. Mesothelial cells underlying the basilar membrane may produce the fibronectin and also contribute to the mass of the membrane. Changes in either the fibronectin or the mesothelial cells might, therefore, have an effect on cochlear mechanics. In order to assess basilar membrane changes in aged animals, young adult (2-4 months) and aged (24-26 months) Sprague-Dawley rats were evaluated for the presence of fibronectin-like protein and mesothelial cells. The basilar membrane in the young animals had strong fibronectin-like immunoreactivity throughout its length. The old animals, on the other hand, showed normal fibronectin immunoreactivity in the basilar membrane of the basal turn, but little or no reactivity in the apical cochlear turn. The number of mesothelial cells was reduced throughout the length of the membrane in aged animals, with the greatest loss in the basal turn (60% fewer cells). These two degenerative changes, which appear to be independent of each other, may contribute to the observed threshold shifts in aged cochleas.

Postnatal development of the rat organ of Corti. I. General morphology, basilar membrane, tectorial membrane and border cells.

The development of the rat organ of Corti was studied during the first postnatal weeks. The temporal and the spatial patterns of cochlear development were investigated between 4 and 24 days after birth by means of semi-thin sections at approx. ten equidistant positions along the entire cochlear duct. At all examined positions width, thickness and cross sectional area of basilar membrane, cross-sectional area of tectorial membrane, of cells of Hensen, Claudius and Boettcher and of the organ of Corti were quantitatively analyzed. The most conspicuous maturational changes occur between 8 and 12 days after birth. These are the detachment of the tectorial membrane, the first appearance of filaments within the basilar membrane, the formation of the tunnel of Corti and the opening of the inner spiral sulcus. Quantitative analysis revealed that structures of a given position along the cochlear duct do not develop synchronously. Width of the basilar membrane and cross-sectional area of the tectorial membrane are already mature at the onset of hearing (10-12 days after birth). Length, thickness and cross-sectional area of the basilar membrane as well as cross-sectional area of the organ of Corti and of the cells of Hensen, Claudius and Boettcher still develop after the onset of hearing (up to 20-24 days after birth). We suggest that basic cochlear function is established by structures which are mature before the onset of hearing. Cochlear structures which develop after the onset of hearing might be involved in this improvement during this period.

Postnatal development of the organ of Corti in the wild house mouse, laboratory mouse, and their hybrid.

Length of the basilar membrane, number and distribution of cochlear receptors, and the width of the triad of outer hair cells were analyzed in the course of the postnatal development and in adult individuals in wild and laboratory house mice and in hybrids of these species. While in newborn animals the triad of outer hair cells was wide at the base and narrow at the apex, the opposite was true for adult animals. The parameter decreased at the base and increased at the apex during postnatal development. The center of differentiation of (the reticular lamina of) the organ of Corti was localized at 40-50% of the basilar membrane length from the base and corresponded to the region with the maximum density of inner hair cells. The reticular lamina in the apical half of the cochlea matured earlier than in the basal half. Distribution of receptors did not change after birth. The shortest basilar membrane and the slowest rate of maturation were found in wild mice. Hybrids had the longest basilar membrane and the highest rate of maturation. These facts are considered an effect of heterosis.

Development of 2f1-f2 otoacoustic emissions in the rat.

2f1-f2 otoacoustic emissions have been recorded from the rat cochlea during its development. Acoustic responses were recorded at 3, 5 and 7 kHz using a fixed value of the f2/f1 ratio (= 1.17). The first 2f1-f2 acoustic responses were obtained at 12 days after birth for 2f1-f2 = 7 and 5 kHz, and 2 days later for 2f1-f2 = 3 kHz. Adult-like patterns of the acoustic responses were achieved by day 18 for 2f1-f2 = 3 kHz, by day 20 for 2f1-f2 = 5 kHz and by day 28 for 2f1-f2 = 7 kHz. These results are discussed in relation to the available anatomical and functional data on the cochlear development of the rat. The delayed appearance of the 3 kHz acoustic responses might be related to the basal-to-apical gradient of morphological cochlear maturation. The fact that the 2f1-f2 otoacoustic emissions reached adult characteristics from the low to high frequencies is consistent with the development of the tuning properties of the basilar membrane. The long development of the 2f1-f2 acoustic responses at 7 kHz suggests that the organ of Corti undergoes subtle changes well after the end of its apparent maturation.

Postnatal changes in the size of the avian cochlear duct.

The length of the cochlear duct was measured in chicks aged embryonic day 14 to post-hatch day 469. Chicks were anesthetized, decapitated and their cochlear ducts exposed under an operating microscope. Because of the very thin bone and cartilage surrounding the relatively straight tube of the papilla the entire cochlear duct could rapidly be exposed and measured without fixation or removal from the head. The length of the duct was measured using a computer based Zeiss Videoplan Image Analysis System. A total 41% increase in length was seen from embryonic day 14 to post-hatch day 469; 20% of this increase occurred after hatching. It is suggested that this increase in cochlear duct length could influence basilar membrane properties important to frequency coding mechanisms during development.

Morphological changes in the cochlea of the mouse after the onset of hearing.

The cochleae of 5, 10, 12 and 15 day old mice and of adults (Mus musculus, strain NMRI) were studied by light and electron microscopy. In each case the same part of the organ of Corti (2.8-3.3 mm from the helicotrema) was examined. The results were correlated with the development of auditory thresholds (in mice of the same strain) obtained by Ehret (Ehret, G. (1971) J. Am. Audiol. Soc. 1, 179-184). It was demonstrated that morphological development of the organ of Corti is by no means complete at the onset of behavioural responses to acoustic stimuli. After this event the following morphological changes occur: (1) The basilar membrane filaments stain more intensely and the tympanic cover layer is greatly reduced in thickness and almost completely disappears. (2) The filaments of the pillar cells stain more intensely and apparently increase in number, and the angle between the outer and inner pillar cells increases so that the cross-sectional area of the tunnel of Corti expands. (3) Hook-shaped connections (marginal pillars) between the reticular membrane and the tectorial membrane disappear. The consequences of these morphological changes for the cochlear mechanics are discussed, especially in respect to the increase of sensitivity of hearing.