Postnatal development of the rat organ of Corti

Anat Embryol (1992) 185: 571 581 Anatomy and Embryology �9 Springer-Verlag 1992

Postnatal development of the rat organ of Corti

II. Hair cell receptors and their supporting elements

Birgit Roth and Volkmar Bruns

Zoologisches Institut der J.W. Goethe Universit/it, Siesmayerstrasse 70, W-6000 Frankfurt am Main 11, Federal Republic of Germany

Accepted January 14, 1992

Summary. The development of cochlear receptor cells and their supporting elements was studied by means of semi-thin and ultra-thin sections during the first postnatal weeks in the rat. The temporal and spatial patterns of the receptor cell development were investigated between the 4th and 24th days after birth. At approx, ten equidistant positions along the entire cochlear duct length of inner and outer hair cells, width of outer hair cell triad and stereocilia-length of the outer hair cells were quantitatively analyzed. Striking maturational changes take place before the 12th day after birth, that is, when the onset of hearing occurs. These changes are the formation of the tunnel of Corti, of the Nuel spaces, the appearance of filaments within the supporting elements and the change in cell shape of the hair ceils. Between 4 days and 20 days after birth the maturation of outer hair cells is characterized by a decrease of organelles in the cytoplasm and establishment of the subsurface cistern. The quantitative analysis revealed a unique developmental pattern of the length of the outer hair cells, the width of the outer hair cell triad and the stereocilia length of the outer hair cells. Shortly after birth these structures have an almost constant size along the whole cochlear duct, but with increasing age the structures shorten at the cochlear base and enlarge at the apex. This pattern results in the establishment of a baso- apical gradient of the above mentioned structures. We assume that this baso-apical gradient is of central importance for the frequency representation.

Key words: Cochlea - Development Rat Hair cells Stereocilia

Introduction

The receptor cells of the organ of Corti, the inner and the outer hair cells, have been studied in numerous inves-

Offprint requests to: B. Roth

tigations (for review: Lim 1986, 1987). These studies reveal differences in the ultrastructure and the innervation pattern that indicate different functions in these two types of receptor cells. It is generally assumed that the inner hair cells are working as transducers, whereas the outer hair cells are thought to function as effectors (for review: Corwin and Warchol 1991). The frequency discrimination of the cochlea cannot be explained by passive effects only, but is a consequence of an active micromechanical process (Zenner 1986). Strong evidence for this active process is provided by the existence of oto-acoustic emissions, which are produced within the cochlea. They occur spontaneously but can also be evoked by acoustic stimuli. The evoked oto-acoustic emissions contain more energy, produced by the cochlea, than the originally applied stimulus. It is postulated that the active process amplifies the motion of the basilar membrane and influences the frequency selectivity (Zen- ner 1986). It is generally assumed that the active element is localized within the outer hair cells, so recent investigations focus on this type of receptor cell. The outer hair cells show specialized structures of lamellar endoplasmatic reticulum at the external cell membrane, the so-called subsurface cistern. The apical poles of the outer hair cells are covered by stereocilia bundles which are coupled to the tectorial membrane. One of the striking results of recent years was the discovery that the outer hair cells show motility, i.e. contractions and elonga- tions along their longitudinal axis (Brownell et al. 1985; Zenner et al. 1985). The motility is thought to enhance frequency selectivity in the cochlea, Brundin et al. (1989) demonstrated that the frequency of the outer hair cell motility depends on the cell length. Dannhof et al. (1991) reported a general relationship between the outer hair cell length and the frequency representation along the cochlear duct.

Most investigations of hair cell development focus on synaptogenesis and ciliogenesis (Lenoir et al. 1980, 1987; Lavigne-Rebillard and Pujol 1987, 1990). It is generally assumed that the development of the inner hair cells precedes that of the outer hair cells (Rubcl 1978).

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However , qua l i t a t ive and quan t i t a t i ve d a t a on the recep- to r cell d e v e l o p m e n t a long the ent i re coch lear duc t are still no t avai lable . We s tud ied the t e m p o r a l and spa t ia l pa t t e rn s o f the r ecep to r cell d e v e l o p m e n t in the ra t cochlea. Sys temat ic ana lys is us ing semi- th in sect ions o f approx . ten equ id i s t an t pos i t i ons a long the whole coch lea r duc t was pe r fo rmed . The analys is was c o m p l e t e d by ul- t r a - th in sect ions. Quan t i t a t i ve analys is a long the ent i re coch lea r duc t o f a re levan t n u m b e r o f s tages is o f cen t ra l i m p o r t a n c e for m o r p h o - f u n c t i o n a l i n t e r p r e t a t i o n o f the ha i r cell m a t u r a t i o n .

studied. From the second litter two animals each at the stages 4, 12, and 24 DAB were investigated. For ultrastructnral analysis, from the third litter two animals each at the stages 4 and 20 DAB were studied.

The animals were sacrificed with an overdose of Pentobarbital (Nembutal). After dissection the cochleae were fixed by immersion in phosphate-buffered glutaraldehyde (2.5%). The cochleae were postfixed with 2% osmium tetroxide, dehydrated and embedded in epoxy resin (Epon). During the dehydration process the cochleae for ultrastructural analysis were blockstained in 1% uranyl acetate. The cochleae were dissected according to the block surface technique (for detail: Roth and Bruns 1992). From the first litter radial

Material and methods

Cochleae from 17 rats of the Wistar strain ranging in the age between 4 and 24 days after birth (DAB) were investigated. The animals originated from three litters. From the first litter one animal at each of the stages 4, 8, 12, 16, 20, 24 days after birth was

4 DAB

8 DAB

12 DAB

16 DAB

20 DAB

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I I 100pro Fig. 1. Radial sections of the hair cell region in the rat at 5 mm from the base demonstrating the development from 4 days (above) to 24 days after birth (DAB," below)

0.8mm

2mm

4mrn i

6ram 1

8 r a m

1 0 m m

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lOOpm Fig. 2. Radial sections of the hair cell region in the rat demonstrating baso-apical differences at 24 days after birth. Base=0.8 mm; apex = i0 mm

573

Fig. 3. Radial sections of the outer hair cells at 4 days (left) and 20 days after birth (right) at 2 mm from the base. Note the kinocilia (arrow) at the 4th postnatal day. The subsurface cistern at 20 days

after bir th is marked by long arrows. DC, Deiters' cell; N, nucleus; OHC, outer hair cell; OSF, outer spiral fibres (afferent)

Fig. 4. Cross section through the outer hair cells at 20 days after birth, x 2750

semi-thin sections (1 gm) were taken at ten equidistant positions (from I mm up to 10 ram; I m m = b a s e , 10 m m = a p e x ) along the basilar membrane from cochleae of the stages 8, 12, 16, 20 and 24 DAB. Sections from the youngest stage (4 DAB) were cut semi- thin at seven equidistant positions from 1 mm up to 7 mm. Because of methodological problems, we did not succeed in taking sections at the apical two positions 8 mm and 9 mm (note that the basilar membrane is shorter than 10 mm at this stage). From the second litter two cochleae from two animals were sectioned at three to six distinct positions along the basilar membrane. Semi-thin sections were stained with methylene blue and covered with paraffin- oil. They were qualitatively and quantitatively analyzed. At all examined positions the length of inner and outer hair cells, the width of the outer hair cell triad (definition according to Burda 1985) and the stereocilia-length outer hair cells were measured. The length of stereocilia of inner hair cells was only measured in 24-day-old rats. In all three rows of outer hair cells the length of the hair cells themselves and of their stereocilia were measured.

Since development is identical in all three rows of outer hair cells, only the data of the second row were taken into consideration.

The standard deviation was calculated for three animals at stages 4, 12 and 24 DAB. The defined positions along the basilar membrane, where the semi-thin sections were taken, are not identical in all three animals of the same stage. Therefore for evaluation of the standard deviation a broader segment of 20% length of basilar membrane was considered. For observation of developmental changes along the cochlear duct the basilar membrane was divided at all stages into a basal (0 20%), a middle (40 60%) and an apical (70-90%) segment. In each segment the mean value and the standard deviation were calculated from measurements of all three animals.

Radial and horizontal (parallel to the basilar membrane) ultra- thin sections of the basal turn were stained with uranyl acetate and lead citrate, then studied in a ZEISS EM 902 electron micro- scope. The preparation technique used here is in conformity with the German law of animal protection.

574

Fig. 5. Cross section through one outer hair cell (second row) at 4 days (left) and 20 days after birth (right) at 2 mm from the base. The subsurface cistern is marked by arrows, x 10500

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Fig. 6. a Length of inner hair cells (IHC) and b length of outer hair cells (OHC) at 4 days, 16 days and 24 days after birth (DAB) along the basilar membrane (BM); base = 0%; apex= 100%. Note that observations of the extreme apical positions at 4 days after birth are missing, e Length of inner hair cells (IHC) and d length of outer hair cells (OHC) at a basal (0-20%), at a middle (40-60%) and at an apical (70-90%) cochlear position�9 At every position the left column represents four days, the middle column 12 days, and the right column 24 days after birth�9 The shaded area and the horizontal line in .it represent the standard deviation and the mean value

Results

General morphology o f the hair cell region

The r ecep to r cells and the suppo r t i ng cells f o rm a com- pac t epi the l ia l l ining on the bas i l a r m e m b r a n e 4 days

af ter b i r th ( D A B ) (Fig. 1). The ha i r cells and the sup- p o r t i n g cells can ha rd ly be dis t inguished�9 U p to 8 D A B few m o r p h o l o g i c a l changes occur , t h o u g h the ep i the l ium increases s l ight ly in height . A t 12 D A B the ex t race l lu la r f luid spaces, the tunne l o f Cor t i and the Nue l spaces a p p e a r for the first t ime. The tunnel o f C o r d is f o r m e d

between the inner and the outer pillar cells. The Nuel- spaces separate the outer pillar cells from the 1st row of outer hair cells (Nuel space 1), the three rows of outer hair cells (Nuel space 2 and 3) and the outer hair cells of the 3rd row from the border cells (Nuel space 4). Be- tween 8 and 12 DAB both the pillar cells and the Deiters' cells increased remarkably in length, so that finally the outer hair cell region marks the highest point in the organ of Corti. Simultaneously an incorporation of filaments in both cell types occurs. Between 16 and 24 DAB the lumen of the Nuel spaces increase, and during this period the Nuel space 4 becomes larger than the Nue l space 1. After the 16th DAB Deiters' cells and pillar cells increase in length and show a continuing incorporation of filaments. At 24 DAB the Deiters' cells and the pillar cells become gradually longer from base to apex (Fig. 2) while the content of filaments within these cells decreases towards the apex. The cross-sectional area of the tunnel of Corti and the Nuel spaces 1 3 are larger at the apex than at the base, while the Nuel space 4 shows the maximum area in the middle region of the cochlea.

Hair cell receptors

Already at 4 DAB the inner hair cells are arranged in one row and the outer hair cells in three rows (Fig. 3). The inner and the outer hair cells are separated from one another by the pillar cells. Both types of hair cells have large nuclei and the inner hair cells are somewhat larger than the outer hair cells. The cross-section of the outer hair cells revealed a more or less ovoid shape (Fig. 5). The outer hair cells within the same row are in close contact with each other, whereas the cells of different rows are separated from each other by the phalanges of the Deiters' cells. At 4 DAB the cytoplasm of the outer hair cells contains many organelles, while

575

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BM-length [~]

Fig. 7. Length of outer hair cells (OHC) at 4 days (above) and 24 days after birth (below)demonstrating in three animals individual measurements along the basilar membrane (BM). B a s e = 0 % ; apex = 100%

fragments of the developing subsurface cistern can be observed at their cell membranes. At 12 DAB both types of receptor cells have increased in length, the inner hair cells being bottle-shaped while the outer hair cells have a slender cylindrical shape. Because of the formation of the Nuel spaces, the outer hair cells within each row are apart. The phalanges of the Deiters' cells have reached their final slender shape. Now the outer hair cells are almost completely surrounded by extracellular fluid except at their basal and apical poles. After the 12th DAB few morphological changes occur. In cross- section the outer hair cells have a round shape (Fig. 4). In relation to the 4th DAB the diameter of the outer

4 DAB 20 DAB

IHC

I P C

OHC 1

OHC 2

O H C 3

Fig. 8. Top view to the reticular membrane at 2 mm from the base at 4 days (left) and 20 days after birth (DAB; right). IHC, inner hair cell; [PC, inner pillar cell; OHC, outer hair cell

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hair cells has diminished in the basal region of the cochlea (Fig. 5). During this period the number of organelles in the cytoplasm of the outer hair cells has been reduced, and a single layer of subsurface cistern is situated throughout the whole cell membrane except at the basal and apical poles.

At 4 DAB the length of the inner hair cell increases gradually from base (25 gin) to apex (33 gm). A baso- apical gradient appears in all stages. At 16 DAB the curve has shifted to higher values and corresponds to that at 24 DAB (Fig. 6a). At 24 DAB the length increases from 35 ~tm at the cochlear base to 55 gm at the apex. The mean values show an increase in length between 4 and 12 DAB at the basal (0-20% length of basilar membrane), the middle (40-60%) and the apical (70 90%) segment (Fig. 6c). The length of inner hair cells remains unchanged after 12 DAB.

At 4 DAB the length of the outer hair cells varies between 20 gm and 26 gm along the entire cochlear duct (Figs�9 6b and 7). At 16 DAB the length increases from 17 gm at the cochlear base to 35.5 gm at the apex. Up to 24 DAB the length of outer hair cells remains unchanged with the exception of the apical positions�9 The evaluation of mean values (Fig. 6a) revealed that in the basal region (0-20%) the length decreases between 4 and 12 DAB from 22.6 gm to 16.3 gm. In the middle segment of the cochlea (40-60%) the cell length (around 25 lam) remains unchanged during development. In the apical segment (70-90%) the length of outer hair cells increases continuously from 4DAB (22.7gm) to 24 DAB (36.7 gm). Comparing the course of the outer hair cell length at the 4th DAB and the 24th DAB, a different baso-apical pattern can be observed (Fig. 7). At 4 DAB all measurements parallel the x-axis, while at 24 DAB a baso-apical gradient of cell length is evident. The variation of the measurements is higher in the younger stages than in the older ones.

Reticular membrane

The top view of the reticular membrane at 4 DAB revealed a regular arrangement of the cuticular plates of the inner hair cells, the heads of the inner pillar cells and the cuticular plates of outer hair cells (Fig. 8). The cuticular plates of the receptor cells are somewhat square in shape and less electron dense than the cytoplasm (Fig. 3). At this stage the reticular membrane runs more or less parallel to the basilar membrane. Up to 20 DAB the distance between the inner hair cells and the first row of outer hair cells increases, while the distance between the inner hair cells and the third row of outer hair cells remains constant. The three rows of outer hair cells come closer (Fig. 8). The apical poles of the inner pillar cells increase so much that they cover the surface of the outer pillar cells: The cuticular plates have now a kidney-like shape and are more electron dense than the cytoplasm. At this stage the reticular membrane tilts towards the modiolus (Fig. 1).

According to Burda (1985) the width of the outer hair cell triad is defined as the radial width of the three

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Fig. 9. a Width of outer hair cells (OHC) triad at 4 days, 16 days and 24 days after birth (DAB) along the basilar membrane (BM); base=0%; apex=100%. Note that observations of the extreme apical positions at 4 days after birth are missing, b Width of outer hair cell (OHC) triad at a basal (0-20%), at a middle (40-60%) and at an apical (70-90%) cochlear position. At every position the left column represents four days, the middle column 12 days, and the right column 24 days after birth. The shaded area and the horizontal line in it represent the standard deviation and the mean value

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rows of cuticular plates in the outer hair cells. At 4 DAB the width of the outer hair cell triad decreases slightly from base (32 gm) to apex (24 gm; Fig. 9a). At 16 DAB the gradation of the width is reversed, as it increases from 20 gm at the base to 40 gm at the apex. The curve remains unchanged up to the 24th DAB. In the basal segment (0-20%) the width of the outer hair cell triad decreases between 4 DAB and 12 DAB (Fig. 9b). The development of the width at distinct positions along the

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basilar membrane revealed the most striking changes between a restricted period from 8 to 12 DAB (Fig. 10). At 4 and 8 DAB the width of the outer hair cell triad varies between 24 gm and 32 gm at all positions. Be- tween the 8th and the 12th DAB the width diminishes at the positions 1 mm and 3 mm, remains constant at 5 mm and increases at 7 mm and 9 mm. After the 12th DAB no further changes occur.

Stereocilia

At 4 DAB the surface of the receptor cells and the supporting cells is covered by microvilli. At this stage the receptor cells can be easily distinguished from the adja- cent supporting cells by their stereocilia and kinocilia. The stereocilia bundles are arranged in a W-like pattern typical for mammals (Fig. 11), and up to five rows of stereocilia are found. The kinocilium is situated lateral to the stereocilia bundle in the direction of the outer bony wall of the cochlea and at the periphery of the hair cells where the cuticular plate is absent. The stereocilia are anchored in the cuticular plate by means of rootlets. At this stage the interior of the stereocilia is less electron dense and both the stereocilia and the microvilli are embedded in filaments of the developing tectorial membrane. At 12 DAB the apical poles of the receptor cells are exclusively covered by stereocilia, the microvilli and the kinocilia having disappeared. The stereocilia bundles are standing free since the tectorial membrane is detached from the epithelium. In compari-

son to 4 DAB the stereocilia become electron dense at 20 DAB while at the cochlear base the diameter of the outer hair cell stereocilia becomes smaller (Fig. 11).

The stereocilia-length of all three rows of outer hair cells was measured. For simplification only the data of the second row were taken into consideration. In the basal segment (0-20%) the stereocilia-length decreases slightly between 4 DAB and 12 DAB (Fig. 12 a) then up to 24 DAB the length remains unchanged. In the middle segment (40-60%) the length is approx. 2.8 gm in the stages 4 DAB, 12 DAB and 24 DAB. In the apical segment (70-90%) the stereocilia increase in length between 4 DAB and 12 DAB and then remain constant. The baso-apical course of the stereocilia-length at 4 DAB differs considerably from that at 24 DAB (Fig. 12b). At 4 DAB the measurements vary between 2 gm and 4 gm along the entire length of the basilar membrane. The curve parallels more or less the x-axis. At 24 DAB the stereocilia-length of the outer hair cells increases from 1.9 ~tm at the cochlear base to 8.2 gm at the apex.

At 24 DAB the stereocilia-length of the inner hair cells and of all three rows of outer hair cells increases from base to apex (Fig. 13). With the exception of the extreme apical position (90% distance from the base) the stereocilia of the inner hair cells are always longer than those of the outer hair cells. The stereocilia of the inner hair cells increase from 3 gm to 8.6 gm. The stereocilia-length of all three rows of outer hair cells is 1.5- 2 gm at the cochlear base. At the apex the stereocilia achieve a length of 7.5 gm in the first row, 8.2 ~tm in the second row and 8.4 gm in the third row.

Fig. I1. Cross section of the stereocilia of outer hair cells at 2 mm distance from the base at 4 days (left) and 20 days after birth (right). Note the kinocilia (arrow) at 4 days after birth, x 50000

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Fig. 12. a Stereocilia-length of the outer hair cells (OHC) at a basal (0-20%), at a middle (4~60%) and at an apical (70-90%) cochlear position. At every position the left column represents 4 days, the middle column 12 days, and the right column 24 days after birth. The shaded area and the horizontal line in it represent the standard deviation and the mean value, b Stereocilia-length of the outer hair cells (OHC) at 4 days (above) and 24 days after birth (DAB, below) along the basilar membrane (BM). The individual measurements are separated in data from litter 1 (filled symbols) and from litter 2 (open symbols); base=0% ; apex= 100%

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Fig. 13. Stereocilia-length o f inner hair cells (IHC) and all three rows o f outer hair cells (OHC) along the basilar membrane (BM). n, inner hair cell; a , outer hair cell 1; e , outer hair ce l l2 ; m, outer hair cell 3; base = 0 % ; apex = 100 %

Discuss ion

General morphology of the hair cell region

Striking changes of the hair cell region occur until 12 DAB. These are the change in cell shape of the hair cells, the forming of Nuel spaces and the appearance of filaments within the supporting elements. The developmental stage of 12 DAB correlates with the onset of hearing, as the first action potentials in the cochlear nerve were obtained in the rat at about this stage (Uziel et al. 1981). The development of the Nuel spaces and of the filaments still continues up to 24 DAB. Using electron microscopy and immunohistochemistry Bannis- ter et al. (1989) showed that in the rat the microtubules are synthesized before the onset of hearing. The authors assumed that the filaments are the major determinants of cell shape changes. Within the hair cells and the supporting cells of the cochlea actin microfilaments, microtubules and tropomyosin have been identified (Anniko et al. 1987; Slepecky and Chamberlain 1987; Furness et al. 1990; Slepecky etal . 1990). The supporting elements form a mechanical framework around the outer hair cells and give rigidity to the organ of Corti. It is generally assumed that the function of rigidity is brought about by microtubules.

In accordance with the present result, the continuous increase in height of the epithelium in the rat after the onset of hearing was also observed by Wada (1923). He found the adult height of the organ of Corti (measured at the 3rd row of outer hair cells) at 20 DAB. Wada showed that the increase of the epithelium is chiefly de- pendent upon the development of the Deiters' cells, which grow up to the 20th DAB. Wada (1923) showed that there was a big increase in height of the lesser epithe- lial ridge (outer hair cell region) while the greater epithe- lial ridge (inner hair cell region) remained almost constant in height during development. This is in agreement with the present results.

Hair cell receptors

The development of the receptor cells is characterized by changes in shape, decrease of organelles in the cytoplasm, and the establishment of the subsurface cistern. The change in shape of the hair cells has also been reported by Thorn (1975) in the guinea pig, Pujol and Marty (1970) in the cat and Lenoir et al. (1980) in the rat, and so far seems to be a common feature during the maturat ion of the receptor cells. A reduction of organelles (i.e. mitochondria, endoplasmatic reticulum) has also been described in the guinea pig (Thorn 1975) and may indicate the diminishing general cell activity at the end of cytological differentiation.

The present study reveals that the maturat ion of the inner hair cells and the outer hair cells (except of the cochlear apex) ended with the onset of hearing. Wada (1923) observed that from 1 to 12 DAB the volume of the inner hair cells and outer hair cells increased rapidly and then remained unchanged. These results are in contradiction to the general hypothesis that the development of the receptor cells follows an inner hair cell to

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outer hair cell gradient (Sobin and Anniko 1984; Pujol 1985, 1986; Lavigne-Rebillard and Pujol 1987, 1990). In relation to synaptogenesis in the rat, the end of maturation differs as between the inner hair cells and the outer hair cells. The adult innervation pattern at the inner hair cell level is achieved about 12 DAB, and correlates with the onset of hearing, while the adult-type stage at the outer hair cell level is observed at 20 DAB (Lenoir et al. 1980; Pujol and Lenoir 1986). Similar findings have been reported in human fetuses. The adult innervation pattern and the adult shape of the inner hair cells is achieved long before those of the outer hair cells (La- vigne-Rebillard and Pujol 1990). The present results could not reveal a delayed maturation of the outer hair cells themselves. However, one should bear in mind that rat development is several times faster than human.

The most conspicuous change occurs during the development of outer hair cells and concerns the decrease in cell length and diameter at the cochlear base. A scru- tiny of the data of Wada (1923) revealed that our data are not entirely in contradiction to his. Wada studied the postnatal development of the rat organ of Corti at four positions along the basilar membrane. During the development he observed a constant volume of the outer hair cells in the first turn, while the volume at other positions had at least doubled. Since the first turn in- cludes approx. 50% length of the basilar membrane, Wada might have studied a more apical position than in the analysis presented here, so that he failed to observe the decreasing volume. The decrease of cell length at the base and the increase at the apex results in a baso- apical gradient of outer hair cell length. The outer hair cells are considered to enhance frequency selectivity of the cochlea by their active motor properties, which have been described recently by Brownell (1986) and Zenner (1988). Isolated outer hair cells respond to an electrical stimulus (Brownell et al. 1985) with contraction along their longitudinal axis (motility). Brundin et al. (1989) demonstrated that isolated outer hair cells of the guinea pig show a sharply tuned motile behaviour. They observed that "the motile responses of each cell varied with frequencies and demonstrated optimal sensitivity in a narrow frequency region". The best frequency of each cell is correlated to the cell length, and depends on the cochlear region from which the outer hair cell was obtained, Using a comparative approach Dannhof et al. (1991) reported a general relationship between the length of outer hair cells and frequency representation along the basilar membrane. On the basis of these results, the establishment of the outer hair cell gradient during development is of central importance.

Reticular membrane

During development considerable changes in the cellular pattern of the reticular membrane occur. The formation of the adult-type pattern is mainly connected with changes in the outer hair cell triad, whose width decreases at the cochlear base. The same development was observed in two different strains of rats (Sprague-Daw- ley and Lewis) by Burda (1985). His quantitative analysis

revealed that at birth the outer hair cell triad is wider at the cochlear base than at the apex. With increasing age the baso-apical course is reversed and results in a baso-apical increase in width. The decrease of outer hair cell triad at the cochlear base creates a greater distance between the inner hair cells and the outer hair cells of the first row. Consistent results have been reported in mice and cats using scanning electron microscopy (Lim and Anniko 1985, 1986). These studies showed a widen- ing of the headplates of the inner pillar cells, increasing the distance between the inner and the outer hair cells. Lim and Anniko suggest that this change is important for aligning the inner hair cells with the Hensen's stripe of the tectorial membrane and the outer hair cells with the Hardesty's membrane of the tectorial membrane. The exact arrangement of the hair cells and their stereocilia may be essential for establishment of the stereocilia- tectorial membrane-system. The tuning capacity of the stereocilia-tectorial membrane-system is much better than that of the single elements (Zwislocki and Kletzsky 1980).

Stereocilia

The development of the outer hair cell stereocilia bundle is characterized by the formation of three rows of stereocilia, the establishment of a baso-apical gradient in length, and the disappearance of kinocilia and microvilli. In the literature there are different accounts of the number and arrangement of stereocilia in immature animals. Lenoir et al. (1980) described, in contrast to the present results, a U-shaped organization of the stereociliary bundle in the rat at 2 DAB. They found nascent stereocilia restricted to the periphery of the stereociliary bundles and exclusively in the lower apical region of the cochlea. At 10 DAB the stereocilia are arranged in the characteristic W-pattern. In agreement with our results, Lim and Anniko (1985; 1986) found in mice a great number of mainly nascent stereocilia, which are slightly larger in diameter than the microvilli. They are already arranged in a W-formation at birth. The embry- onal stereocilia begin to disappear at 3 DAB and have completely receded at 10 DAB. The receding of the em- bryonal stereocilia coincides with the formation of the adult stereocilia, which are arranged in three rows of graded length. As in the present study, Lenoir et al. (1980) and Lira and Anniko (1985, 1986) observed a great number of microvilli on the surface of outer hair cells and supporting cells in immature animals.

The stereocilia of the outer hair cells reach their adult length at 12 DAB, that is, at the stage of the onset of hearing. The present results show a decrease in length at the cochlear base. The standard deviation is high when calculated for both litters (Fig. 12a), but within each litter the decrease in length is evident between 4 and 12 DAB. By separate consideration of the data from the two litters (Fig. 12b) it is shown that the values ascer- tained in the first litter are in general higher than those of the second litter. A similar decrease of length at the cochlear base has been described in the gerbil Pachyur- omys duprasi (Bruns et al. 1985).

580

In the present study the stereocilia of all four rows of hair cells show a baso-apical increase in the mature organ of Corti (24 DAB). Also in the chinchilla a baso- apical gradation of stereocilia-length was described (Lim 1980, 1986). Several studies show a tonotopic organization of stereocilia length in mammals (Lim 1980), in birds and in reptiles (for review see: Manley 1990). Mea- surements on ciliary stiffness revealed that the length of the stereocilia is inversely correlated with the stiffness (Strelioff and Flock 1982). Some investigations have demonstrated in the alligator lizard that the resonance frequency of the stereociliary bundle can be correlated with the stereociliary length (Frishkopf and DeRosier 1983; Holton and Hudspeth 1983). According to Zwis- locki (1980) the stereocilia together with the tectorial membrane may act as a resonating system which leads to an improvement of frequency analysis.

Developmental pattern

The maturation of the structures investigated here ends at the time when the onset of hearing occurs (12 DAB). The maturation of all structures connected with the outer hair cells show a unique pattern. The development of these structures depends on their baso-apical position. At birth the length of outer hair cells, the width of outer hair cell-triad and the stereocilia-length of outer hair cells have an almost uniform size along the entire cochlear duct (Figs. 14, 15). With increasing age, structures at the cochlear base decrease in size, while structures at the apex increase. Structures in the middle region of the cochlear remain more or less unchanged, and therefore have about the adult size already at birth. Fre- quency mapping of the adult rat cochlea shows that the region of maximum sensitivity is found in the middle of the cochlear duct (Mfiller 1991 a). This corresponds to the region where the structures mentioned above remained constant in size during development. This middle region of the cochlea responds first to acoustic stimuli (Mfiller 1991 b).

The developmental pattern described above results in an exact gradation of the structures along the basilar membrane. This gradation seems to play an important role in frequency representation. The development of

100-~ i

,~ 90 ~

c~ 80 - -T- o

70-

6 0 - E _~ 50-

40

24 DAB

4 DAB

6 2'o go do go 1 bo BM- length [%]

Fig. 14. Relative length of outer hair cells (OHC) along the basilar membrane (BM) at 4 days (dashed line) and 24 days after birth (DAB; solid line). Maximal value = 100 %

6 "1- o

ej

o

100-

90-

80-

70-

apical

60-

50- basal

40

6 ~ ~ r l's ~o 2'r DAB bi~h onset adul~

Fig. 15. Development of the length of outer hair cells (OHC) at a basal (10%) and at an apical (80%) position. Maximal value= 100%

a similar gradation was described by Cotanche (1987), who analyzed the maturation of stereocilia in the chick. His study reveals that the growth of stereocilia occurs in three phases, in which the cilia increase first in length, second in thickness and finally in length again. The ex- tent of each phase depends on the position of each hair cell along the basilar papilla. The development results in a position-specific gradient of length and thickness of the stereocilia. The precision achieved by these tempo- rally and spatially defined phases leads Cotanche to the conclusion that this process is controlled by one or more regulatory factors. These factors should influence the stereociliary development locally, and may also coordi- nate the stereocilia development throughout the whole basilar papilla. However, Cotanche (1987) thought that the gradation of structures during development is always achieved by an increase of these structures. Starting with a distinct immature size, the structures grow until the adult size is reached. In contrast to that, the present study shows several examples where the adult size is achieved by a unique developmental pattern. Starting with a uniform size along the entire basilar membrane, the structures at the cochlear base decrease until the adult size is reached. A decrease in size during development has, for the cochlea at least, not been reported up to now.

Conclusion of part I and part H

Three principles of cochlear development were postulated by Rubel (1978):

1. The developmental process begins at the base and proceeds apically.

2. The development of all elements at a given position in the cochlea occurs synchronously.

3. The development of the inner hair cells precedes that of the outer hair cells.

The development of the structures in the rat as revealed in this study clearly deviates from Rubel's principles.

1. A general baso-apical gradient of cochlear development could not be observed. The cochlear elements show the same maturational stage at all examined positions.

2. The development of the cochlear structures does not occur synchronously. We found three different tern-

581

poral-spat ia l pa t te rns of cochlear development . (i) There are parameters , such as the width of basi lar membrane , which are completely developed unt i l the onset of hearing (12 DAB). (ii) There are parameters , such as the length of basi lar membrane , which still develop after the onset of hear ing up to 20-24 DAB. (iii) In the present s tudy a third deve lopmenta l pa t te rn was observed. There are parameters , such as the length of outer hair cells, which show a posit ion-specific development . Structures in the middle region of the cochlea achieve adul t size already at b i r th while deve lopment at the base and the apex finished at 12 DAB. Structures at the base decrease in size and those at the apex increase.

3. A n inner hair cell to outer hair cell gradient could no t be found. Length, stereocil ia-length and cell shape of the inner and the outer hair cells reach their adul t characteristics s imul taneous ly at 12 DAB.

Acknowledgements. This investigation was supported by the Deutsche Forschungsgemeinschaft SFB 45/B21. The authors would like to thank Dr. B.J. Dannhof for comments on the manuscript.

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Postnatal development of the rat organ of Corti

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