Talk:Book - Contributions to Embryology Carnegie Institution No.31

By 0. Van der Stricht,

Professor of Histology and Embryology, Universily of Ghent, Lecturer in Anatomy, Johns Hopkins University.

With four plates.

CONTENTS.

PAGE.

Introduction Ill

Methods 112

Historical 112

Connections between the oviter supporting and hair-cells 116

First stage of development 117

Second stage of development 119

Third stage of devcIo])ment 124

Connections between the inner hair-cells and their sustentacular elements 125

Significance of some of the so-called cells of Hensen 128

Mitochondria and other structures in hair and supporting elements 129

Hair-cells 129

Supporting cells 132

Summary 135

Bibliography 139

Description of plates 141

General abbreviations 142

110

THE ARRANGEMENT AND STRUCTURE OF SUSTENTACULAR CELLS AND HAIR-CELLS IN THE DEVELOPING ORGAN OF CORTI.

By 0. Van der Stricht.

INTRODUCTION.

The arrangement of the outer sustentacular and hair-cells of the organ of Corti in adult mammals is rather well known. According to the investigations of Held (1902), N. Van der Stricht (1908), and Kolmer (1909), the body of the cell of Deiters is situated below that of its sensory element, so that the two are connected by a chalice-shaped, greatly modified segment, in the concavity of which hes the deep cytoplasmic portion of the supported hair-cell. In the embrj'onic stages, however, the relation between these two elements is entirely different, and it might be well worth while to trace accurately their connections through the whole devel- opmental stage. The same holds good for the inner and outer rods of Corti, the inner supporting cells, and even some of the so-called cells of Hensen.

Most investigators who have tried to clarify the arrangement of the sensory and sustentacular elements in embryonic material have made use of and describe vertical radial sections of the organ of Corti. Although very interesting, and in fact highly necessarj^, such preparations are hable to be deceptive and lead to misinter- pretation. Indeed, most authors incorrectly represent the cells of Deiters. Many authors, even Retzius (1884) and Held (1909), who describe the phalanx process of the sustentacular elements as running obliquely from the cell body towards the lamina reticularis, thus crossing two or three hair cells, generally picture it in illus- trations of vertical radial sections as an uninterrupted band connecting the nucle- ated portion of the cell with the free surface of the epitheUum. So, also, do most authors of text-books of histology, notwithstancUng the fact that in a radial vertical section this protoplasmic strand shows at least three interruptions.

By making use of sections tangential and alwaj^s somewhat obUque to the sur- face of the organ of Corti, N. Van der Stricht was able to accuratelj' locate the nucleated body of the supporting elements in successive stages of development and to determine the amount of gradual shifting. In this stud}' the same method of research was apphed in order to locate the more superficial portions of these cells between the sense-epitheUum elements, and to ascertain their exact relation to the hair-cells and the mechanical factors that cause the shifting of the sustentacular elements. Moreover, a series of preparations, exhibiting mitochondrial structures in the supporting cells and hair-cells, has rendered it possible to define the nature of coarser structures noted by previous observers.

HI

112 ARRANGEMENT AND STRUCTURE OF SUSTENTACULAR CELLS

METHODS.

Kittens, dogs, and rabbits, from birth to 12 days old, white rat fetuses, and young white rats about 2 days old, constituted the material used in these investiga- tions. The isolatetl cochlea was fixed by trichloracetic acid (5 per cent in water), by Bouin's or by Zenker's fluid, and subjected for many weeks to the mordant action of some drops of iodine in alcohol (70 per cent). Where necessary, after fixation by Bouin's or by Zenker's fluid, decalcification was completed by 2 per cent nitric acid in 70 per cent alcohol. Before embedding in paraffin the pieces were stained with borax carmine and the sections with iron hematoxylin, Congo red, and light green.

The mitochondria in the sustentacular and hair-cells were fixed in the following manner: Mixture of formalin and bichromate of Regaud (1910), according to the modifications indicated by Cowdry (1916), and subsequent staining with acid fuchsin and methjd-green ; treatment by a 1 per cent aqueous solution of osmic acid for about an hour, followed by immersion in trichloracetic acid, or Bouin's or Zenker's fluid; exposure of the cochlea, the bony wall of which had been previously provided with two or three small o[)enings, to vapors from a 2 per cent a([ueous solution of osmic acid for approximately 30 to 60 minutes, and subsequent treat- ment of the piece by one of the three above-mentioned agents; fixation for an hour in a 1 per cent aqueous solution of osmic acid, followed bj' immersion in a 1 per cent aqueous solution of silver nitrate for 3 hours. By these methods of fixation, and staining with iron hematoxylin and Congo red, the mitochondria can be brought into prominence within one or two turns of the cochlea, occasionally throughout its extent. Osmic vajjors have been recommended as a fixing agent for mitochondrial structures by M. R. Ijcwis and W. H. Lewis (1914). We are able to confirm tins statement, having for many years successfully used these vapors, and a subsequent treatment by another reagent, for the purpose of fixing the chondriomites in the ova of the dog. Henneguy (1895) was able to bring into view chondrioconts in the spermatocytes of Helix by the use of osmic vapors.

The description given herein is illustrated by figures representing three different series of sections:

(1) Radial, vertical sections of tho organ of Corti. These are cross-sections of the rows of hair and supporting colls, the knife cutting the latter along their length and from the axis toward the outer wall of the cochlea (figs. 14, 15, and 16).

(2) Spiral, vertical sections of the organ of Corti, these being longitudinal sections of the parallel spiral rows, the knife cutting the hair and supporting cells along their length, from the more apical to the more liasal part of the sjiiral organ (figs. 19, 20, 22, and 23).

(3) Sections tangential and always somewhat oblique to the surface of the organ of Corti, the knife cutting transversely the hair and sustentacular cells of the spiral parallel rows at all levels, from the surface of the epithehum towards the basilar membrane (figs. 2 tf) 13, 17 and 18), so that their arrangement and structure can be traced in cross-sections throughout their lengths (figs. 2 and 3).

HISTORICAL. The structure of the organ of Corti has been exhaustively studied. It is known to be made up of two kinds of cells, the hair-cells and the supporting elements, arranged in spiral, parallel rows: an inner row of inner hair cells on the medial side of the inner rods of Corti, and three rows of outer hair-cells on the lateral side of the outer pillars. According to Waldeyer (1872), Retzius (1884), Tafani (1884, 1885), who examined Cercopithecus viridus, and Kolmer (1910), four and occasionally five rows of outer hair-cells exist in man and monkeys; in oth(>r mammals a fourth row may appear in some parts of tlie cochlea. The hairs upon the free surface of all these elements were first described by Deiters (1860), and all authors agree that these sensory epithelial cells are cylindrical in shape and contain a single rounded nucleus in the deeper cytoplasmic portion. Deiters (1860), Hensen (1863, 1873), Middendorp (1867), Loewenberg (1868), Boettcher (1869, 1872), v. Winiwarter (1870), Krause (1876), and Nuel (1878), believed that the cell body of the sensory elements is connected with the basilar membrane by an intermediate deep process. That no such process exists has been conclusively proved by Rosenberg (1868), Retzius (1884), Denis (1901), Vernieuwe (1905), and by all of the more recent observers. All of the other elements of the organ of Corti are held to be supporting cells, and among these two types must be distinguished :

(1) The tivo rows of inner and outer pillar cells or rods of Corti: With the excep- tion of Loewenberg (1868), who finds more outer than inner pillars, most investi- gators—Claudius (1855), Boettcher (1856, 1859), Max Schultze (1858), Middendorp (1867), V. Winiwarter (1870), Krause (1876), Nuel (1878), and many others, com- pute about three inner pillars for two outer; while N. Van der Stricht (1908) proves that the number of inner pillar cells is !just double the number of the outer pillar cells, of the cells of Deiters of the first and second rows, and of the hair-cells of each row. Tafani (1884) had already noted that the number of outer pillars is exactly the same as the number of hair-cells of each outer row. He stated also that the phalanx apex of the outer pillars is located between two apices of neighboring hair-cells of the first outer row. Many misinterpretations have been published about the development of the pillars. Rosenberg (1868), Boettcher (1869, 1872), and Pritchard (1878) assert that one original cell divides into two, an inner and an outer pillar; whereas Loewenberg (1868), Gottstein (1870), Waldeyer (1872), and Hardesty (1915) regard each pillar as derived from two cells. The investigations of Hensen (1863, 1871), Middendorp (1867), Retzius (1884), Denis (1901), Vernieuwe (1905), and other more recent observers, have proved conclusively that each [lillar with its nucleus is originalh' developed from one cell (the pillar cell). The striated fibrillar structure of the pillars in the adult cochlea has been noted by M. Schultze (1858), Boettcher (1859, 1869), Deiters (1860), Loewenberg (1868), v. Winiwarter (1870), Gottstein (1870), Hensen (1871), Nuel (1872, 1878), Lavdowsky (1876), Retzius (1884) , and others; but their actual filamentous structure and the basal body of the outer pillars have been clearly brought out b3' Joseph (1900), Retzius (1900), V. 8pee (1901), Held (1902), N. Van der Stricht (1908), and Kolmer (1910).

(2) The outer supporting cells, or the cells of Deiters: According to the investiga- tions of all observers, these cells, hke the pillars, are stretched between the basilar membrane and the lamina reticularis. They are composed of a nucleated cell body

114 ARRANGEMENT AND STRUCTURE OF SUSTENTACULAR CELLS

and a more superficial portion, the phalanx process, the former running from the basilar membrane to the nucleated part of the hair-cell, the latter inclosed within the region of, and between the acoustic elements. Gottstein (1870), Waldeyer (1872), Nuel (1872), and Lavdowsky (1876) describe a true fusion of the apex of the nucleated part of the supporting element with the lower portion of the corre- sponding hair-cell, so as to produce a "twin or double cell." This view is to be regarded as a misinterpretation, and from the investigations of Tafani (1882, 1884), Retzius (1900), Held (1902), N. Van der Stricht (1908), and Kolmer (1910), it is plain that in adult animals a close connection exists between these two elements, the lower pole of the hair-cell occupying a cup-shaped depression in the upper pole of the supporting cell body. The wall of this pit is made up of a system of deeply staining fibrils which form a goblet- or chalice-shaped covering (Held) to sustain the sensory epitheUal cell. Tliis chalice extends down into a broad, fibrillar filament running obhquelj' toward the inner side of the nucleus, and coursing through the medial por- tion of the cj^toplasm to reach the base of the cell, where the filament enlarges into a conical, fibrillated foot, the base of which rests upon the basilar membrane. At the level of the nucleus the filament divides into two branches, one of which is connected with the chaUce, the other more slender one extending throughout the conical phalanx process of the cell of Deiters, to abut against the membrana reticularis. Parts of this filament have been noted by many previous observers. Its basal por- tion has been erroneouslj^ interpreted l)y Boettcher (1869, 1872), Lavdowsky (1876), and Nuel (1878) as a deep process given off from the neighboring hair-cell. The chalice and the branch of the filament beneath it were not observed by Retzius (1884, 1900), nor by v. Spee (1901), but these authors nevertheless describe a single fibrillar band extending throughout the cell body and its phalanx process. The chahce itself was first noted by Kishi (1902, p. 177), as "an dem unteren Ende der Haarzellen befindliches Gebilde von Kelchformiger Gestallt, " but misinterpreted as a nerve ending.

The real seat of the nucleated body of the cell of Deiters is illustrated and described by all authors in vertical radial sections, during the earliest stages in the development of the organ of Corti, as alternating with that of the more superficial hair-cells. Thus the supporting cell of the first row lies below an interstice sepa- rating the acoustic elements of the first row from those of the second; the sujiporting cell of the second row lies below an interstice separating the acoustic elements of the second row from those of the third; and the sui)porting cell of the third row hes outside the acoustic elements of that row. No alteration in the relation of the sup- porting elements to the hair-cells is mentioned. Even when representing each cell of Deiters beneath its respective acoustic element, Retzius (1884, plates xxii and xxiii), and many others, make no allusion to it in their descriptions. N. \'an der Stricht is the only \vriter who has given an accurate explanation of these mutual relations, which can be seen in sections tangential and somewhat oblique to the surface of the epithelium, the two kinds of cells being represented by long, nucleated columns, the real position of which can not be misinterpreted. According to his investigations three successive stages are distinguishable:

AND HAIR-CELLS IN THE DEVELOPING ORGAN OF CORTI. 115

(1) Outside the row of inner pillar cells there exists "une seconde rang^e nucleaire k noyaux plus irr^guliers, si^geant directement sous la rang^e des premieres cellules acous-

tiques externes; elle correspond aux noyaux des cellules a piliers externes [p. 580]

Primitivement, Ics cl(5ments de Deiters de la premiere rangee siegent nettement sous les cellules acoustiques externes de la seconde rangee, ceux de la seconde rangee sous les cellules acoustiques externes de la troisieme rangee, ceux de la troisieme rangee en dehors de la troisieme rang(^e des cellules acoustiques externes." (2) "Plus tard, k mesure que Torgane deCorti evolue, il s'opere un refoulement des elements de soutenement de dehors en dedans vers I'axe du limagon. La rangee nucleaire des piliers internes est refouMe sous celle des cellules acoustiques internes. La rangee nucleaire des piliers externes est refoulee en dedans de la premiere rangee sensorielle externe. La premiere rangee nucleaire de Deiters atteint I'interstice sejjarant les deux premieres rangdes sensorielles externes. La seconde atteint I'interstice separant les deux dernieres rangees sensorielles externes. La troisieme rangee nucleaire de Deiters est disposee a peu pres sous la derniere rangee sensorielle externe [p. 666] (3) Les cellules de Deiters, apres avoir subi un premier refoule- ment de dehors en dedans, amenant leur corps c>i:oplasmique en dedans de la rangee sensorielle, dans la quelle elles sont primitivement intercalees, en subissent un second dans le meme sens, amenant le corps cellulaire directement sous celui de la cellule sensorielle voisine, de sorte que I'element de Deiters, faisant partie prunitivement de la seconde rangee sensorielle externe, siege definitivement sous une cellule de Corti de la premiere rangee sensorielle. L'element de Deiters de la seconde rangee sensorielle arrive sous une cellule de Corti de la deuzieme rangee sensorielle et l'element de Deiters, siegeant primitivement en dehors de la troisieme rangee sensorielle, est refoule sous une cellule de Corti de cette derniere serie." [p. 670].

Held (1909), commenting upon tlie paper of N. Van der Stricht, says (p. 243):

"Siene reichen Einzelangaben tiber die Entwicklung der Sinneshaare, der Lamina reticularis, der Stiitzfasersysteme kann ich zum grossten Teil bestatigen. Womit ich nicht iibereinstiaime, will ich im folgenden kurz hervorheben."

Although no objections are found in his succeeding pages against the theory of shifting of the supporting cells, described by N. Van der Stricht, Held does not recognize at all the three stages just mentioned. Indeed, referring to the close con- nection between the acoustic and supporting cells he saj^s (p. 215) :

"Die allgemeine Stellung der Deitersschen Zellen zu den iiusseren Haarzellen wird wahrend dieser Prozesse in kiener prinzipiellen Weise geandert."

The superficial phalanx process of the cells of Deiters, as first pointed out by Hensen (1863), reaches the corresponding phalanx interpolated wdthin the lamina reticularis. In adult mammals it has been described by Nuel (1878), Retzius (1884, 1900), Tafani (1884), v. Spee (1901), Held (1902), and N. Van der Stricht (1908), as running obliquely and decussating with three, or even with four or five (v. Spee) hair-cells.

Many discordant theories have been advanced regarding the part plaj-ed by the two epithelial thickenings of the cochlea duct in the development of the organ of Corti. Boettcher (1869), Hensen, Baginsky (1866), and Hardesty (1908) regard the lesser ridge as the germ from w'hich all the supporting and hair-cells arise. Prentiss (1915) beheves that the inner supporting cells, and probably the inner hair- cells and inner pillars are derived from the greater ridge, and that the lesser epi-

116 ARRANGEMENT AND STRX'CTURE OF SUSTENTACULAR CELLS

thelial tliickening forms the external jiortion of the sj^iral organ. But at present no doubt exists as to the origin of these structures. According to Rosenberg (1868), Gottstein (1871), Boettcher (1872), Retzius (1884), Denis (1901), Vernieuwe (1905), N. Van der Strioht (1908), and Held (1909), the inner hair-cells originate from the greater ridge, all the pillars and outer hair and sujjjiorting elements from the lesser ridge; Rosenberg, Vernieuwe and Held locating the inner rods of Corti in an inter- stice outside the greater ridge between the two epithelial thickenings.

The superficial membrana reticularis of the organ of Corti has been referred to in a previous paper (1918).

It is evident from the above review that most of the structures of the s])iral organ are at i)resent well known. Nevertheless, many others retiuire furthci' inves- tigation, and of these, four types will be dealt with herein, /. e.:

(1) The connections between the outer supi)orting cells and the hair-cells. If the view taken by N. Van der Stricht be correct, and the nucleated body of the sustentacular cell undergoes gradual shifting, what happens to its more super- ficial segment? What is its exact location during the three successive stages of development? What mechanical factor causes this alteration in the position of the cell body? At what period of development, and in what manner, does the spiral shifting of the apical process of the cell of Deiters take place?

(2) The connections between the inner hair-cells and their sustentacular ele- ments.

(3) The significance of some of the so-called cells of Hensen.

(4) The nature and origin of peculiar, coarse structures in the cyptoplasm of the hair and supporting cells.

CONNECTIONS BETWEEN THE OUTER SUPPORTING AND HAIR-CELLS.

In the new-born dog the epithelium of that part of the spiral membranous duct which Ues close to the apex of the cochlea is still undifferentiated and is composed of elongated columnar cells, the apices of which reach the surface. A section tan- gential to these free ends shows a regularly formed mo.saic of small, undifferentiated polygonal fields, each of which contains a diplosome. The polygons are separated from one another by terminal bars (referred to in a previous paper, 1918). On tracing the pattern through a series of sections, more and more remote from the .summit of the cochlea, different structures are successively met with. First, within the greater ridge appear the inner hair-cells, recognizable by the enlargement of their cell bodies and nuclei, while in a section tangential to the surface their apices are seen in the form of rounded, sensorial fields, (juite different from {\w neighbor- ing supporting and non-differentiated polygons. Somewhat farther from the apex of the cochlea the outer hair-cells become differentiat(>d and constitute the lesser epithelial thickening; there is an increase in the cytojjlasm and the size of the nucleus, and the latter, hke that of the inner sensory cells, stands out jirominently in the vicinity of the epithelial surface, whereas the nuclei of the future su])porting elements persist near the basilar membrane.

AND HAIR-CELLS IN THE DEVELOPING ORGAN OF CORTI. 117

FIRST STAGE OF DEVELOPMENT.

In figure 2 is illustrated a section tangential to the surface of the apical or first turn of the cochlea in a dog 12 hours after birth. Between the inner and outer hair- cells is seen a column of distinct, nearly square fields — cross-sections of a series of inner pillar cells (ip). The lowermost third of this column is nucleated and belongs to sLx pillars cut at six different levels. From mutual compression, together with an enlargement of this basal i)ortion of the cell, the nucleus and its surrounding protoplasm are somewhat flattened out in a radial direction. The upper two-thirds of the column becomes gradually smaller and contains a darker, axial, granular strand. The clear superficial field of the column — the true apex of the cell — is the narrowest and incloses a central corpuscle. From this it is clear that the iimer rod of Corti is a columnar cell, a four-sided prism in shape, enlarged at its base and tapering to its free surface. In more advanced stages (figs. 3 and 4) the enlargement and flattening of the basal portion of the cell are more marked, the apical portion thinner, while the intermediate, true cytoplasmic part increases in size.

Close to this spiral row of inner pillar cells, and situated within the greater ridge, may be seen (figs. 2, 3, and 4) cross-sections of three different rows of cells; one row of inner hair-cells (ih), and two rows of inner supporting cells (is', is"). The former extend through the superficial two-tliirds of the epithehum and possess a dark, granular, and considerably increased cytoplasm with large nuclei in the lower portion of the cell. At the free surface of the cell is an eccentric, central corpuscle surrounded by a clear are? — the medullary zone of the attraction sphere of Ed. Van Beneden, and a deeply staining plate — the superficial cuticula or the cuticular plate (N. Van der Stricht), from which the hairs arise.

Just below the cell bodies of the inner hair-cells Ues the basal portion of the inner sustentacular cells of the first row (figs. 2, 3, 4 and 10, is'), composed of a smaller nucleus and a clearer protoplasm, which reaches the surface of the epitheUum by means of a long cytoplasmic process, running between and compressed by the enlarged hair-cells. Being a four-sided prism at the level of its nucleated basal part, this columnar cell becomes lamellar, flattened out in a radial direction at the level of the acoustic elements (figs. 2 and 10), and by compression between two neighboring cells is pushed toward the inner pillars, its form being triangular on section, at first near the nucleated portion (figs. 2 and 3), and later also near the superficial cytoplasmic part of the inner hair-cells (fig. 4) . Thus it is clearly seen that the inner supporting cells of the first row do not run laterally, either inside or outside of the inner hair-cells, but course through an interval between two acoustic elements, and by enlargement of the developing hair-cells are pressed towards the outer or lateral part of this interstice to take again their original position just beneath the superficial membrana reticularis.

The inner supporting cells of the second row (figs. 2 and 3, is") are four-sided prisms, composed each of a small basal nucleus and a clear c>i;oplasm. These cells undergo no compression from intermediate cells, but form a distinct spiral row, constituting the inner boundary of the organ of Corti, just as the cells of Deiters of the outer third row (figs. 2 and 3, d'") constitute the outer limit of the spiral organ.

118 ARRANGEMENT AND STRUCTURE OF SUSTENTACULAR CfiLLS

Outside of the inner pillars the organ of Corti, in the earliest stages of its devel- opment, when the outer superficial sensory fields (fig. 10, oh', oh", oh'") have attained about half the size of the inner (fig. 10, ih), is formed of four spiral rows (figs. 2, 3, and 10) : (1) The first row of outer hair-cells and outer pillar-cells (oh', op); (2) the second row of outer hair-cells and cells of Deiters of the first row (oh", d') ; (3) the third row of outer hair-cells and cells of Deiters of the second row (oh"', d") ; (4) the cells of Deiters of the third row (d'")- The outer hair-cells correspond with the inner in structure, except that their development and enlargement begin somewhat later, and that even in the adultcochleatheirsizeissmaller. The outer pillar-cells and the cells of Deiters of the first and second rows are similar to the inner supporting cells of the first row in number, arrangement, and structure. Their cytoplasm is clear, and their nuclei occupy the basal one-third of the cell.

In sections tangential to the free surface of the epithelium these nucleated portions of the supporting cells are juxtaposed into nuclear columns, and, hke the nuclear column of the inner sustentacular elements of the first row, lie directly beneath the acoustic cells of their respective columns. The superficial two-thirds of these outer supporting elements reach the membrana reticularis, running through the interval between two developing hair-cells by which they are compressed, so that the original four-sided prism (fig. 10, op) becomes flattened out in a radial direction (fig. 3, op, d', d"), and assume a lamellar, pentagonal (fig. 3) or triangular (fig. 2) shape on section. As the result of mechanical pressure from the neighboring hair- cells, wliich gradually increase in size chiefly in their nucleated portion, the super- ficial cytoplasmic processes of the outer pillars and the cells of Deiters of the first and second rows are compressed and pushed toward the medial side of their respec- tive spiral rows — that is, toward the inner pillars, the superficial portion of which is thereby flattened out. The subsequent shifting of this compressing process becomes conspicuous, first at the level of the enlarged nucleated parts of the hair- cells (fig. 3, op. d\ d"), afterwards in the more superficial region (fig. 2).

This gradual, mechanical sliifting of the upper two-thirds of the,se supporting cells ultimately results in their transposition into a system of new spiral rows or spaces as follows: (1) A spiral row of outer pillar cells situated between the spiral row of inner rods of Corti and the first row of outer hair-cells. This is illustrated in figure 4, showing the upper part of the outer pillars j^ushed inward from their original sjnral row. This apical portion is increased in size and i)entagonal in shape on section, and its outer angle still encroaches upon the original row, mainly close to the surface where it is connected with the phalanx or the apex of the outer pillar-cell, always interpolated within the primitive first outer spiral row. (2) A first outer interstice situated between the first and second rows of outer hair-cells (fig. 4, d'). (3) A second outer interstice situated between the second and third rows of outer hair-cells (fig. 4, d"). The first and second outer interstices contain respectively the phalanx i)rocesses of the cells of Deiters of the first and second rows. These processes for a time may encroach upon their original sensory row, but later are entirely incorporated within their ultimate iiiterstice except close to

AND HAIR-CELLS IN THE DE\T;L0PING ORGAN OF CORTI. 119

the surface, where they join their apex, the stationary phalanx, interpolated in the lamina reticularis.

The cells of Deiters of the third row (figs. 2 and 3, d"') are four-sided prismatic elements. In tangential section their nucleated basal portions form a nuclear col- umn situated obviously outside of the tliird outer sensory row, and their superficial cytoplasmic processes are seen as a column of clear, superposed fields often some- what flattened by the enlarged neighboring hair-cells. In figure 2 (d'") their number is apparently the same as that of the cells of Deiters of the second row, but in the bat, according to N. Van der Stricht, there are two cells of Deiters in the third row for each one in the second row. Views from the surface of the membrana reticularis, as illustrated in figs. 10, 13, and 18, enable one to comijute the apices of these cells (d"'), and prove that the cells of Deiters of the third row, if not double in number, are at least always more numerous than those of the second or first row.

The above-described seven sj^iral rows of the organ of Corti in the earliest stage of development can be distinctly seen in views of the surface of the membrana reticularis. As illustrated in figure 10, the arrangement of the apices of all the supporting and hair-cells is indisi)utably along seven spiral rows, each sharply demarcated. These rows are of two different types. The second row of inner sustentacular cells (is"), the row of inner pillars (ip), and the third row of Deiters cells (d'"), are jiurelj^ sustentacular in character; while the others — the inner (ih, is"), and the three outer rows (op, oh'; d', oh"; d, oh'") are mixed rows, supporting and sensory in character. At a shghtly more advanced stage of development, however, this primitive condition changes (figs. 3 and 4), the structures assuming a definite arrangement (fig. 18) excej^t for the apices of the pillar-cells, as seen in figure 13. The chief transformation, as compared with figure 10, consists in the appearance between the three mixed spiral outer rows of two interstices belonging entirely to supporting fields. The first is composed of parts of the apices of two varieties of cells, alternately the outer and the inner extremities of the phalanges of the respec- tive outer pillars (op) and the cells of Deiters of the first row (d') ; and the second interval is of a similar pattern, alternately the inner and outer extremities of the phalanges of the respective cells of Deiters of the first and second rows. The sen- sory fields are not altered. The sujjporting fields, or the phalanges, undergo an elongation and extend over the two interstices appearing in the depth of the epithe- hum. The apices of all the cells remain in situ, but the phalanges become elongated and cover parts of two developing supporting interstices.

SECOND STAGE OF DEVELOPMENT.

This stage is characterized chiefly by the shifting of the nucleated portions of the outer pillar and outer supporting cells inward and toward the axis of the cochlea, so that, as shown in figures 5, 6, and 7, the basal part of the outer pillars (op) is found to be entirely inside of the first row of outer acoustic elements; that of the first row of Deiters cells (d'), inside of the second row of acoustic elements; that of the second row of Deiters cells (d"), inside of the third row of acoustic elements; and that of the third row of Deiters cells (d"*) either partially or almost entirely beneath

120 ARRANGEMENT AND STRUCTURE OF SUSTENTACULAR CELLS

the third row of acoustic olomonts. The basal, nucleated portion of these support- ing cells follows the shifting of their more superficial part, and each of the three originally mixed outer spiral rows divides into two distinct rows: One, purely sensory, wliich does not reach the basilar membrane; the other, i)urely sustentacular, which is continuous throughout the thickness of the epithelium. Of all these elements the apices alone maintain their i)riniitive i)osition. At the same time f)ther imjjort- ant changes are occurring. The ba.sal, nucleated portion of the cells of Deiters undergoes a slight enlargement, while the increase in the superficial cytoplasmic part is inconspicuous; as a result of mutual compression between the neighboring hair-cells it is still flattened out in a spiral direction and thereby reduced in size.

In this respect the outer pillars differ from the outer supporting cells. IJke the inner pillars they enlarge rapidly, and three parts, without marked outUnes, become distinguishable; i. e., a nucleated basal portion, or foot, which is the largest; an intermediate, cyto])lasmic portion, of medium size, which tajjers off to a more sujierficial portion, the latter being the smallest (fig. 5). At its foot the outer pillar- cell assumes the form of a four-sided, and in its more superficial j^art a five-sided pyramid (figs. 4 and 5), the basal part of which is flattened out in a radial direction (fig. 5, op). A similar but more pronounced increase in size and a subdivision into three segments are noticeable in the inner rods of Corti (figs. 4 and 5, ip), the foot and nucleus being markedly flattened out in a radial direction. INIoreover, the foot extends considerably toward the foramina nervina, seemingly repelUng the bases of the neighboring inner cells. This process of extension has been pointed out by many observers — Hensen, Boettcher, Middendorp, Retzius and others. No true shifting of the base of any supporting cell, however, occurs. The expansion of the foot of the inner pillars inward and of the foot of the outer pillars outward is accompanied by a corresponding extension of the subjacent basilar membrane. Tliis has already been emphazised by Vernieuwe and others, and should be attrib- uted to abundant nutriment from the neighboring vas spirale. This elongation of the base of the inner pillar-cell is accompanied bj'^ an alteration in the inclination of the inner pillar itself, and causes Ukewise an alteration in the inchnation of the inner suj^porting and hair-cells. The degree of slant of these cells is difficult to determine accurately, although it is quite marked. Indeed, sections tangential to the surface of the organ of Corti, in the earliest stages of develojiment, rei)resent cross-sections through all of its elements (figs. 2, 3 and 4). When the inner pillars extend toward the foramina nervina such sections cut transverselj^ the components of the outer part of the spiral organ (figs. 6, 7, 8 and 9), while the inner pillars, the inner hair and supporting cells are frequently cut along their length. This can be explained only by their marked inclination outward and towards the surface, and the inclination of the outer hair and supporting elements in another direction, so that the axis of the inner supporting cells (fig. l(i, is') meets that of the outer at nearly right angles.

During the second stage of development of the organ of C'orti, and soon after the protoplasmic portions of the cells of Deiters reach their neighboring interstice, another remarkable shifting occurs — a shifting in the spiral direction of the organ,

AND HAIR-CELLS IN THE DE\TELOPI\G ORGAN OF CORTI. 121

as illustrated in figs. 19, 19', 20, 21, and 23. These are diagranas of vertical spiral sections through the outer spiral rows. In figures 19 and 19' there can be seen to the left a more basal portion of the second turn of the cochlea in a new-born kitten, and to the right a more apical portion, consisting of the following elements: A series of acoustic cells of the first outer row (f)h') rocognizal)le by the presence, between the apices of the hair-cells of a bundle of fibrils (oj)) emanating from the heads of the outer pillars; a series of acoustic elements of the second outer row (oh"), and a series of hair-cells of the third row (oh'")- The clearer intervals filled with longitudinal tangential sections through the lateral surfaces of acoustic elements, and situated between the first and second, between the second and tliird row, and at the end of the third row (compare with figs. 20 and 21) are respectively the first, second, and third outer sustentacular interstices.

Below these sensorj^ elements are seen three series of the nucleated portion of the cells of Deiters of the first (d'), second (d") and third (d'") rows; these are con- nected with the membrana reticularis bj' a thin, superficial phalanx process, running obliquely through their respective interstices (fig. 20, d', d"). In the three turns of the cochlea in the kitten, young dog, and young rabbit these apical processes always course reguhirlj' below uj^ward, and from a more basal to a more apical por- tion of the turn, crossing the medial side of three hair-cells (figs. 19, 21, oh", d'; oh'", d"), or the lateral side of the hair-cells of the third row (fig. 19', oh'", d'").

From the above description it is clear that during the second stage of devel- opment the cells of Deiters undergo a shifting in two directions — one axial, from without inward ; the other spiral, from the base of the cochlea upward and toward the more apical portion of it. What mechanical factors are involved in this process? The cliief framework around which the constituents of the organ of Corti are built up is undoubtedly the original single spiral row of inner pillar-cells (fig. 2, ip). Very early this framework becomes stronger, owing to the ajipearance of an additional row of outer pillars (figs. 4 and 5, ip, op); and although the inchnation of these two kinds of rods of Corti is hable to change, the axis of the framework (figs. 1, 3, 4, 5, and 14), even when the tunnel space is present (figs. 15, 16, and 17, t), is more or less perpendicular to the basement membrane and passes through a part of the lumen of the vas spirale (vs). As development progresses, this spiral framework becomes more and more wedge-shaped or triangular in sections, the summit of the triangle being formed bj' a verj^ narrow field — the apex of the inner pillar (figs. 2, 5, and 15, ip), so long as the tunnel space is not developed, and the broad base of the triangle consisting of the adjoining feet of the inner and outer rods of Corti. As the result of this rapid and considerable extension of the base, the deepest part of the cells of Deiters, although immobile on the basilar membrane, is removed farther from the axis of the framework. On the other hand, by comparing the illustrations of the membrana reticularis in figures 4, 10, and 18, the lateral extension of this is rather i nconspicuous and the apices of the outer supporting cells do not follow the lateral shifting of their bases. The result is that the direction of the nucleated supporting columns seen in figures 2, 3, and 4, (op, d', d, d"'), lying just beneath their respective colunms of supported sensory elements (oh', oh", oh'") wUl change, their bases

122 ARRANGEMENT AND STRUCTURE OF SUSTENTACULAR CELLS

resting upon the membrana basilaris, and farther removed from the axis of the framework than their more superficial portion in connection with the lower pole of the hair-cells. Due to this pressure from the axia" framework, and especially from the feet of the outer pillars, the nucleated portion of the cells of Deiters tends to follow an obh(iue direction upward and inward. In other words, the superficial part of the nucleated cell body will tend to join the contiguous spiral interstice into wliich its phalanx process has already been projected during the first stage of devel- opment (fig. 5, oj), d', d") and the nucleated part of the cells of Deiters of the tliird row will be pushed beneath the lower pole of the hair-cells of the tliird row (fig. 5, d'"). Another mechanical factor plays an important part in this shifting of the outer supi)orting cells. On enlarging and elongating, the outer hair-cells not only expel from their spiral row the supporting elements and efface the intercellular spaces left by them, but they also extend laterally so that each sensorj^ row becomes thicker, i)articularly at the level of the nuclei, wliile the diameters of the apices of the hair-cells do not change during the second stage of development. This broad- ening of the three outer sensory rows results in a more obvious enlargement of this part of the lesser ridge, which in turn causes a bulging towards the outer wall of the cochlea (figs. 5 and 15), since the presence of the tough axial framework renders expansion towards the axis impossible. Hence the outer hair-cells run obh(iuely (fig. 14), downward and outward. While the upper pole of the nucleated portion of the cells of Deiters tends to turn inward, the lower pole of the sensory elements tends to turn outward, so that the axis of the interstices between the sensory rows gradually blends with that of the basal portion of the supporting elements (fig. 6). This result is obtained by the concomitant action of a third mechanical factor, the elongation of the hair-cells, and probably by the elongation of the cells of Deiters. In figure 5 the nucleated colums (d', d") do not encroach upon the more superficial interstices, while in a more advanced stage (fig. 7) they obviously invade the lower region of these spaces. This invasion is caused primarilj- by the elongation of the hair-cells between the subjacent cell bodies of the supporting elements. No true intercellular spaces exist at the level of the latter; nevertheless between them are found spiral nerve bundles (N"', N, N") connected with the lower pole of the hair- cells by ascending branches. Such a disiiosition is, of course, apt to facilitate the invasion of these intercellular nerve spaces bj' the elongating sensory elements. The explanation of the shifting of the phalanx processes of the cells of Deiters in a spiral direction and toward the apex of the cochlea appears to be more difficult. Before attempting any interpretation it must be borne in mind that the extremities of these elements, the basal resting u])on the membrana basilaris, and the superficial interpolated in the membrana recticularis, always remain fixed. Of course these membranes may increase in size, regularly or irregularly, so that a point .4 upon the former, just beneath a point A' uijon the latter, may, bj' a process of unequal devel- opment, become situated just below a point jS' or D', the point A' being removed farther from basal part of the cochlea; but point A' will never take the place of l)()int B', such as has alreadj^ lieen proved is the case with portions of the cell bodies

AND HAIR-CELLS IN THE DE\T;L0PING ORGAN OF CORTI. 123

that are pushed out from a mixed spiral row, or from beneath the hair-cell, into a new space. If this view is correct it must be recognized that the membrana reticu- laris undergoes a process of extension somewhat different from that of the membrana basilaris. It is a well-known fact that the development of the spiral organ of Corti proceeds from the base of the cochlea towards its apex and the sense epithelium may be well develope din the third turn, while in the second it is still in process of differ- entiation, and as yet undifferentiated in the first. Moreover, it has already been mentioned that during the first stage of development the growth and enlargement of the hair-cells, hence of the superficial two-thirds of the epithelium, are more marked than that of the cell bodies of the sustentacular elements constituting the basal third of the epitheUum. From this it is evident that the farmer, and its superficial membrana reticularis, extend more rapidly than the latter, not only towards the lateral side of the organ of Corti, but a:o in a spiial direction from the third turn toward the second, and from the second toward the first; that is to saj', from points where the pressure is high to j^laccs where it is lower. According to this principle it is not surprising that during the first stage of growth of the organ the apices of the Deiters cells precede their bases in extending toward the apex of the cochlea, so that the deeper part of the cell remains behind three hair-cells, crossed by the phalanx process.

The second stage of growth, characterized by the pecuUar arrangement of the cells of Deiters, lasts a long time, and many other important transformations occur before the final, third stage is reached. The tunnel space between the rods of Corti maj^ appear, but it is noteworthy that the definite arrangement of the outer supporting cells, which is pecuhar to the third stage, may be attained before an}^ formation of a tunnel space (fig. 9) .

The most important role in the production of the third stage of development is played by the cells of Deiters, chief!}- by their extension in length. During the first and the beginning of the second stage the sensory elements undergo a rapid increase in size and attain their full volume. Afterwards the nucleated body of the Deiters cell becomes gradually longer, as illustrated in figures 7, 20, 19, 19', 14, 15, and 16 (d', d", d'") . In earUer stages (figs. 1, 2, 3 and 4) their nuclei, Uke those of the rods of Corti (op, ip) are situated close to the basilar membrane, and the nucleated cell body is very short. Later (fig. 19) it gradually extends and soon attains about the length of the acoustic elements (fig. 21, d', d", d'"), its nucleus becoming farther removed from its base (figs. 19 and 19', d', d", d"'), reaching its top rather abruptly (figs. 20, 21, d', d", d'")- Finally, when the nucleated body of the Deiters cell attains two- thirds (figs. 16, 15, d', d", d"') or more (fig. 23, d') of the thickness of the epitheUum, its superficial cytoplasmic end appears to lengthen out more rapidly than its deeper parts and to give rise to a very important segment of the cell, which, for the sake of its phj'siological function, may be termed the supporting segment, within which a pecuhar framework or sustentacular apparatus makes its appearance, the nucleus occupj-ing about the middle (fig. 16) or lower part (fig. 23) of the superficial one- third of the cell body.

124 ARRANGEMENT AND STRUCTURE OF SUSTENTACULAR CELLS

THIRD STAGE OF DEVELOPMENT.

This is characterized by the peculiar arrangement of the cells of Deiters. Instead of being situated between the interstices of the rows of outer acoustic elements, the body of each sustentacular cell lies just beneath that of its respective supported hair-cell, so that each acoustic element of the first, second, and third rows is supi)orted respectively by the body of a cell of Deiters of the first, second, and third row, as shown in figures 8, 9, 11, and 17 (d', d", d'", and oh', oh", oh'")- In other words, the axes of the nucleated columns of hair-cells, hence of each individual hair- cell, blends more or less (figs. 8 and 17), or even exactly (figs. 9 and 11), with that of the nucleated columns of supporting elements — that is, of the cells of Deiters.

In order to obtain a true picture of the mechanical factors which take part in the final arrangement of the sustentacular elements it is only necessary to examine and compare figures 14, 15, and 16, especially the latter, representing the period of rapid elongation of the body of the Deiters cell. In figure 14 the supporting segments (d", d") embrace one half of the lower pole of each neighboring hair-cell ; in figure 16 they show varying connections with these poles which can onh' be ascribed to their varjdng capacity for extension. Owing to the peculiar disposition of the elements of Deiters and the obliquity of their axes, the elongation of the cell body and its segment of supjjort is possible in one direction only — i. e., towards the pole of the hair-cell which will eventually be supported by it. Indeed, on lengthen- ing out the segment, d', imjiinges upon oh", and from this pressure the inclination of the latter may be more pronounced ; but the extension of d' proceeds towards and around the outer part of the lower pole of oh', so that ultimately the axes of the acoustic elements of the first row will blend with those of the cell bodies of the sus- centacular elements of the first row (figs. 8 and 17, oh ', d'), and the axes of the hair tells of the second and third rows will blend respectively with those of the cell bodies of the sustentacular elements of the second and third rows (figs. 8 and 9, oh", d"; oh'", d'"). It may be mentioned that during this process of shifting, the spiral nerve bundles, originally situated outside their respective rows of outer hair-cells (fig. 3, N'", N'", N") are pushed inward, along with the neighboring supporting elements, and now occupj^ the interstice inside of these sensory rows (fig. 9, N"', N"', N").

The upper portion of the body of the Deiters cell — that is, the apex of the segment of support, extending around the lower pole of the hair-cell — becomes converted into a cui)-likc depression which surrounds and enwrai)s the deeper cyto- plasmic part of the acoustic element, particularly at its lower and outer jiortion, its inner portion for a time remaining free. Whereas in figure 9 this depression is still inconspicuous, in figure 11 (owing to a considerable enlargement of the segment of support) it is seen to be deeper and its margins separate the inclosed pole of the hair- cells from the neighboring elements except those situated inside of the ]w\c. In a more advanced stage this denuded part of the hair-cell will in turn be surrounded by the expanding segment of support.

During these transformations there appears within the cell of Deiters a part of its apparatus of support, originally consisting of a bundle of fibrils, or a fibrillated

AND HAIR-CELLS IN THE DEVELOPING ORGAN OF CORTI. 125

filament, extending uninterruptedly from the basilar membrane to the membrana reticularis. This filament rests upon the basilar membrane with a fibrillated trian- gular foot, and runs through the nucleated cell body in a direction parallel with the axis of the cytoplasma, but occui)ying the medial part of the latter. The filament is not axial, but paraxial (fig. 16, d', d", d'"), and is in direct continuity with another, which occupies the axis of the cytoplasmic phalanx process (fig. 21, d', d", d""), and may be termed the apical filament. Whereas the paraxial filament originally (fig. 16) courses through the inner part of the body of the supporting element, curves about its apex, and continues along with the apical filament (fig. 21, d", d"), it later traverses the cell body obhquely, since above the nucleus (fig. 18, d', fig. 8, d', d", d"')) and in still more advanced stages at the level of the nucleus (fig. 9, d', d"), it blends with the axis, reaching the lateral part of the cytoplasm and extending into the apical process. This singular modification in the course of the paraxial filament at the level of the segment of support is undoubtedly due to the latter's peculiar process of development and furnishes striking evidence that it extends unequally (fig. 16, d'), as above mentioned; that is, more toward the hair cell supported by it in the third stage of development (oh') than towards the one supported by it in the first stage (oh"), hence more inwardly than outwardly.

The apparatus of support is completed by the appearance of a third fibrillated filament, the axial filament of the sustentacular segment of the cell of Deiters, expanding into a fibrillated, chalice-like enlargement which develops in the wall of the cup-shaped depression. The first trace of this axial filament and its apical chaUce is illustrated in figure 1 1 (d') . On tracing the paraxia' filament of the cell of Deiters of the first row (d'), from the region of the nucleus towards the hair-cell (oh'), it is seen to divide into two branches just at the point where it reaches the axis of the cell. One branch (the outermost) runs outside the acoustic element and represents the apical filament; another ver}^ short branch (the innermost, visible in three cross-sections) is the axial filament which blends with and is replaced by a deeply staining semicircle, partially surrounding the cytoplasmic pole of the acoustic element (oh') and imperfect on the inner side of the latter. This incomplete ring is the cross-section of an elongated, still imperfect goblet — the chalice in process of development. The cells of Deiters of the second and third rows also exhibit the apical filament, the axial filament, and the chalice (fig. 11, d", d'"). The appa- ratus of support is thus composed of a paraxial filament or stem, which divides into two branches, an apical filament and an axial filament with its chalice. Figure 23 shows parts of these structures (d') at a more advanced stage when the susten- tacular segment of the cell of Deiters has reached nearly its fuU extent and contains a much longer axial filament in continuity with the chahce.

CONNECTIONS BETWEEN THE INNER HAIR-CELLS AND THEIR SUSTENTACULAR

ELEMENTS.

All authors agree that the greater epithelial thickening of the cochlea is made up of elongated, columnar cells, which undergo many changes and become con- verted, at the level of the sulcus spirahs, into a simple row of rather low, columnar

126 ARRANGEMENT AND STRUCTURE OF SUSTENTACULAR CELLS

or cubical olomonts (fig. 15, esp), while close to the inner hair-cells a few retain their original, elongated form (figs. 15 and 17, nd), the latter being erroneously termed by some investigators, iimer supporting cells. Indeed, they represent undifferentiated epithelial elements, and in the cochlea of most adult mammals their numl)er is reduced to that of the true inner supporting cells. The character of these elements and the relation they bear to the neighboring sensory elements should be more accurately investigated. Most authors agree also that, during this stage of develop- ment, when the sulcus spiralis is being built up, many of the columnar cells undergo degeneration and disintegration, although very few observers are able to jiicture or even describe such a process. In the cochlea of the dog, cat, rabbit, and ox two distinct regions should be. distinguished. One of these is medial, the true sulcus spiralis (fig. 15, esp), where not the shghtest evidence of cell disintegration is ever seen, the high, columnar cells becoming gradually shorter, larger, and flattened out to cover the sulcus, which at first is narrow, but which subsequently, as the lumen of the duct enlarges, acquires considerable size. The other is lateral, just inside of the inner hair-cells (figs. 15 and 17, ih) or the foramen nervinum (N), where the columnar cells (nd) persist for a long time, and where occasional elements may undergo chromatolysis (fig. 17, ch), while most, if not all, of the others become con- verted into larger and shorter Uning-epithelium cells.

External to, or even partly encroaching upon the foramen nervinum (figs. 15 and 17, N), are found the two rows of inner supporting cells already referred to as belonging to the first inner mixed and second inner sui;)i)orting spiral rows. In the course of development the inner supporting cells undergo no marked shifting or cytoplasmic differentiation. In this respect they differ from the cells of Deiters, although resembling the latter and possessing many of the same essential charac- teristics; for example, (1) original connections with the hair-cells, (2) number, and (3) the shape of their free apices.

(1) The first inner row, like-the three outer spiral rows, or'ginally is a mixed row of sensory and sustentacular elements, and remains so, the supporting elements being entirely inclosed within the spiral row and running through the intervals between the hair-cells. Moreover, the free apex of the sustentacular elements, interpolated within the membrana reticularis, is more or less phalanx-shaped. The inner supporting cells of the second row, like the cells of Deiters of the third row, belong to a spiral row ]nirely sustentacular in character; but whereas the latter, after a process of shifting, become transformetl into a true sustaining framework for the neighboring hair-cells, the former preserve their original jw.'^ition. In other words, the two rows, the second inner and fourth outer, are originally boundary rows of the organ of Corti. The former does not change in its nature, but the latter develops into a true sustentacular row, at least as regards the cell bodies of its components.

(2) As to the inner sui)porting cells of the first row, no doubt can be entertained, the sjiiral row being composed of alternating sensory and sustentacular elements, and their number exactly the same as that of the su])porting cells of each of the three

AND HAIR-CELLS IN THE DEA^LOPING ORGAN OF CORTI. 127

outer spiral rows. The number of cells of the two boundary rows is more difficult to determine, although in surface views of the membrana reticularis their free apices can be readily enumerated. In this respect the number of inner supporting cells of the second row (figs. 10, 12 and 13, is") corresponds to that of the neighboring acoustic elements (ih), while in the cochlea of the bat, according to N. Van der Stricht, and also as illustrated in figure 10, the original number of apices of the cells of Deiters of the third row (d"') is just double that of the neighboring hair-cells (oh'"), wliile in figure 5 (d'") it is the same as the latter, and in figure 13 there are four sensory apices (olV") in contact with five sustentacular fields (d'"). On the other hand, in the adult bat there are just as many sensory apices as sustentacular fields; hence it is plain that in the course of development the number of cells of the outer spiral boundary row is reduced. Further investigation is required to clear up the significance of this reduction. It seems possible, even probable, that this row represents a fourth mixed spiral row, which in man and in some animals may become differentiated into hair and supporting cells.

(3) The apices of the inner sustentacular elements of the first row resemble those of the cells of Deiters of the first and second rows; they are also even more compressed between the apices of the inner acoustic elements, so that from being originally phalanx-shaped (figs. 12 and 18, is') they become much thinner (fig. 13 is'), and later are veiled bj' a deeply staining covering derived, according to previous investigations (O. Van der Stricht, 1918), from the surrounding terminal bars.

The apices of the sustentacular elements of the second inner row resemble those of the cells of Deiters of the third outer row. Indeed, in the first stage of develop- ment both are represented by polygonal fields (fig. 3, is", d'") of about the same size, but the former (is") may be a Uttle larger, particularly more elongated in a spiral direction (figs. 10 and 12, is") owing to higher pressure from the inner hair cells (ih). In more advanced stages (fig. 13, is") they are represented by a very narrow, lanceolate field containing a central corpuscle and circumscribing the inner border of the acoustic elements. In the adult cochlea of the bat (Vespertilio fuscns, Pipistrellus subflavus), of the dog, and of the rat, this streak or line is covered also by a deeply staining veil produced by the terminal bars (the extension of the terminal bars over these fields is noticeable in figure 13, is"), and the spiral row of apices of cells is then seen in the form of a dark homogeneous streak deeply stained by iron hematoxyhn, which constitutes a very sharp demarcation between the membrana reticularis and the large polygonal fields (fig. 13, nd) belonging to undifferentiated columnar cells.

The apex of the third row of cells of Deiters maintains more or less its original, polygonal shape (fig. 13, d"') ; it covers a very dark triangular, fibrillated band or plate, the summit of the apical filament of the sustentacular element, as described by Held (1902) and N. Van de Stricht (1908).

Baginsky (1886) recognizes below the deep extremity of the inner hair-ceUstwo other elements, one situated internal and the other external to the inner acoustic cell ; both are connected with the surface, the former by a process running along the inner

128 ARRANGEMENT AND STRUCTURE OF SUSTENTACULAR CELLS

side of the hair-coll, the latter alonp; the outer side. This clement, close to the inner pillar, should be refjarrled as homologous to the cell of Deiters.

The "inner i)halanx cells," or supi)ortinfj; cells of the first inner row, with their superficial phalanx-shajied apices, are accurately descrih(>(l in the adult or};an of Corti by Held (1902), who mentions in connection with the phalanx the existence of a plate or process extending from the head of the inner pillar between two neigh- boring apices of inner hair-cells. This plate ("Innenschnabel der Innenpfeiler- zellen") has been observed also by Waldeyer (1872), Nuel (1878), Retzius (1885), and Kolmer (1909). In reality it does not exist, and what has been mistaken for it is probably only the dark, more superficial veil derived from the terminal bars.

According to N. Van der Stricht (1908), the nucleated bodies of the inner sup- porting cells, located in the greater ridge in the embryo bat, are found beneath the inner acoustic elements, and (p. 611) "de ce segment basal, renfermant le noyau, part un proplongement superficiel effile, qui s'engage entre deux cellules acoustiques internes. i)our atteindre la surface du neuroepithelium." This author did not recog- nize the shifting of the intermediate portion of their cytoplasm towards the inner pillars.

Referring to the development of the membrane of Corti, Held (1909, p. 219) mentions, inside of the row of inner phalanx cells, a row of " Grenzzellen, deren freie Zellenfliichen die Kopfe der inneren Haarzellen an ihrem axialen Umfang umgrei- fen," but he does not give any further description of these elements. In his figure 18 Kolmer (1909, p. 309) pictures " doppelzeilige Stellung der inneren Haarzellen, Kopfe der Innenphalangen und Grenzzellen, mehrreihige Anordnung der Haare," but he, likewise, gives no further explanation.

SIGNIFICANCE OF SOME OF THE SO-CALLED CELLS OF HENSEN.

All observers are in agreement concerning the arrangement of the c(>lls of Hen.sen, representing them as elongated, columnar elements, extending from the membrana basilaris to the free surface of the epithelium; but further investigation is necessar.y to prove the correctness of this statement. In the earhest stage of differentiation of the organ of Corti, at which time the apices of the outer hair-cells attain only half the size of those of the inner (fig. 2), there can be seen in sections tan- gential to the surface and outside the third row of Deiters cells, a column of elements exhibiting nuclei at two different levels; two near the membrana liasilaris (ad'"), and two others nearer the free surface of the epithelium (aoh"). The arrangement of tlie.se two sets of nuclei or nuclear groups is very remarkable; one (ad") runs parallel to the nuclear column of Deiters cells (d"'), the other parallel to the nuclear column of hair-cells (oh"'). This pecuhar disposition would suggest that the deeper nuclei belong to the sustentacular elements and the more superficial nuclei to the acoustic elements, although there is no evidence of any difTerentiation into sensory cells at the level of their apices.

Similar structures are seen in later stages of develo])nicnt . and arc all \hr more remarkable because here (fig. 5) the cytoplasma surrounding 1 he nuclei has under-

AND HAIR-CELLS IN THE DEVELOPING OKGAN OF CORTI. 129

gone a jjeculiar differentiation. Around the nuclei of the deeper groui) (ad") it appears clear or vacuolated hke that of the cells of Deiters (d'") ; around the nuclei of the superficial group (aoh*') it is dark and granular, hke that of the hair-cells (oh"') . From this it is plainly evident that the protoplasm of these two cells (ad'" and aoh'") is beginning to differentiate respectively into supporting and sensory elements; but their apices, as can be seen on close examination of serial sections, are represented by a mosaic of undifferentiated jjolygons similar in character to those of the true neighboring cells of Hensen. In reality, therefore, these cells should be regarded respectively as aborted supporting (ad) and sensory (aoh'") elements of a fourth spiral row, which in man, especially, and also in some animals, become completely developed. In most animals, however, they retain their primitive character of simple, lining epithelial, or atrophied acoustic elements. Quite excep- tionally a fifth spiral row of aborted hair-cells inaj' be observed (fig. 7, aoh') external to the fourth (aoh).

The atrophied acoustic elements are characterized by their arrangement into a spiral row situated external to the third row of cells of Deiters, and by the fact that their lower ends never reach the membrana basilaris (fig. 11, aoh'^'). The apex of the cell is represented b}^ a large polygonal field containing a diplosome (fig. 13, aoh""), not unlike the apex of the true cell of Hensen. The atrophied hair-cells of the fourth outer row jjersist during the course of development and are illustrated in sections tangential to the surface in figures 8, 9, 17, and especially in figure 11 (aoh'"), and also in vertical, radial sections in figures 15 and 16 (aoh"). The atrophy continues, apparently, for in adult animals these cells are much shorter than in embryonic stages, although they are never absent.

As regards the atrophied cells of Deiters (figs. 2 and 5, ad'^) these belong to the spiral row of atrophied hair-cells and, contrary to the normally developed susten- tacular elements, do not reach the surface of the epithelium. As seen in vertical, spiral sections (fig. 21, ad'"), they alternate with the more superficial elements (aoh'") without passing between them. The phalanx process is absent, so that at this point the epithelium is formed of two strata of atrophied cells. Further investi- gations will have to be made before the arrangement of the atrophied sustentacular cells in more advanced stages of development can be determined. The atrophy of these elements seems to be accompanied and caused by the lack of corresponding nerve fibers.

MITOCHONDRIA AND OTHER STRUCTURES IN HAIR AND SUPPORTING ELEMENTS.

HAIR-CELLS.

In the earliest stages of development of the organ of Corti (fig. 1) the cytoplasm of the acoustic elements is denseh' packed with mitochondria, which are Uned up into granular chondriomites, or, as is usually the case, fused together into uniform filaments or chondrioconts. These are seen to run parallel with the axis of the cell and produce a longitudinal striation; in more advanced stages of development they still constitute the bulk of the protoplasm (figs. 19 and 20, oh', oh", oh'"; fig. 9, ih).

130 ARRANGEMENT AND STRUCTURE OF SUSTENTACULAR CELLS

In transverse sections of the cells the cytoplasm apears granular, the filaments being cut across (fig. 9, oh', oh", oh'")- Due to their presence, the protoplasm, even in specimens where the mitochondria are not stained, is always compact and dark. In the cochlea of the white rat, before birth and also two days after birth, these chondriosomes stained red by fuchsin and the nuclei green by methyl- green, after fixation by a mixture of formalin and bichromate.

Densely accumulated and packed chondriomites give rise to coarser structures, the so-called bodj^ of Retzius, the bodj' of Hensen, and the superficial cuticula and hairs. The presence of the bodj^ described by Retzius (1884, p. 3()()) as "eine grossere, kornig erscheinende Proto])lasmaansammlung," has been confirmed by Held (1902), N. Van der Stricht (1908), and Kolmer (1909), in the outer hair-cells. It exists within these elements in white rats before birth and in other new-born mammals (fig. 1, oh) in the form of a deeply staining granular mass of chondrio- mites. which incloses in its concavity the lower i)ole of the nucleus. In later stages of development (fig. 21, oh', oh") the subnuclear, cytoplasmic portion of the hair- cells becomes much longer and larger, and near its lower extremity contains a rounded, not sharply marked-off mitochondrial body, formed of an outer zone of chondriosomes and a clear central fluid, in the center of which a coarse granule may be noticed. The body of Retzius is described by N. Van der Stricht in the cochlea of the adult guinea pig and bat (p. 653) as formed "d'une couche compacte renfer- mant generalement un ou plus rarement deux cori)UScles." The significance of the central granule is uncertain, but the ])resence of such an accumulation of chondrio- somes near the nucleus, and particularly at the lower pole of the cell, where the nutritive supply affects and penetrates into the cell body, seems natural and logi- cal. Moreover, the inconstant occurrence of this body may be ascribed to a trans- ference of its chondriomites toward the more superficial portions of the cytoplasm. In other words, it seems to be a source for developing mitochondria, whence they migrate into other parts of the cell.

The " vSpiralkorper " of Hensen is described by Retzius (1884, 1900) as granu- lar; it has also been observed by v. Spee (1901), Held (1902), N. Van der Stricht (1908), and Kolmer (1909). V. Spee regards this body as a transformed "central corpuscle" surrounded by pigment granules. N. Van der Stricht considers it as a "centrosome" derived from an accessory central cori)U8cle of the attraction sphere, this corpuscle becoming shifted from the surface into the dejith of the cell. The body of Hensen is not apparent in the earliest stages of development (fig. 1), but makes its appearance later in some of the outer acoustic elements beneath the free apex of the cell (fig. 20, oh', oh", oh'"; fig. 19, oh"). Like the body of Retzius, it is not a constant, permanent structure. It is essentially mitochondrial in nature, being formed of densely accvunulated and packed chondriomites (fig. 20), and some- times contains a kind of centrosome, a clear central area surrounding a si)ecial granule. Tliis mitochondrial body .should not be confounded with the long axial cone composed of closely arranged chondriomites or chondrioconts, wliich is often visible between the apex and nucleus of the cell. The base of this cone contains the body of Hensen and is applied against the free surface of the cell.

AND HAIR-CELLS IN THE DEVELOPING ORGAN OF CORTI. 131

The superficial mitochondrial body is closely connected with another allied structure— the superficial " cuticula "— f rom which the hairs arise, and which appears to be a center of mitochondria, developing probably around an accessory centro- some and capable of supplying the cuticula with chondriomites. The superficial plate of the acoustic elements from which the hairs arise is mentioned by Retzius (1884) as "ein ausserst diinnen Hautscliicht," the inner surface of which is beset by "beim Kaninchen dicht, bei der Katze und dem Menschen mehr zerstreut, sehr feine, gleich grosse Korner" (p. 366). It is termed " Kopfeinlage " by v. Spee, "Haarplatte" by Held (1909), and "cuticula" by N. Van der Stricht. At the surface of the cuticula the latter author describes an "implantation plate or implan- tation crescent," which gradually gives rise to a cone of fibrils or hairs. Although this crescent is closely connected with the diplosome, it is not formed by the central corpuscle, but is derived from the cuticula. Hence the sensory cell is to be regarded as a ciliated but not a vibratile element. It is worthy of note that Fiirst (1900), many years before, had seen at the surface of the hair-cells of the crista and macula acustica in an embryo salmon aged 90 days, a disc deeply stained by iron hematoxylin, which seemed to be composed of granules ("Basalkorperchen"), centrosomic in character, from which arose the ciUa or haii's. Hence Fiirst regards the hairs as specialized, differentiated vibratile elements.

In most preparations the superficial cuticula of the acoustic elements is seen in the form of a homogeneous plate which intensely stains dark blue, Uke the hairs, by iron hematoxylin (fig. 4, oh', oh", oh'", ih). On tracing the mitochondrial struc- tures from the level of the nucleus toward the cuticula (figs. 4 and 9), the chondrio- mites are found to be more and more condensed and numerous, running close to the surface and giving rise to a superficial circular plate. This plate covers the largest part of the apex of the cell, leaving space within a notch on its lateral border for the diplosome (fig. 3). Tliis mitochondrial cuticula usually appears homogeneous and uniform, although in some specimens obviously granular in structure (fig. 9, oh', oh", oh""), and in a few selected preparations the arrangement of the mitochondria into granular filaments (true chondriomites) is conspicuous. This is best illus- trated by figure 12, showing a series of inner hair-cells (ih) cut transversely at suc- cessive levels near their apices, so that from right to left one sees three superficial granular crescents, or the ciUa or implantation plates; three dark, circular, granular cuticulse, with the neighboring central corpuscle, and wdthin the second cuticula three radiating chondriomites ; three masses composed of still deeply stained, coarse granules, situated directly beneath the cuticula; and three finer, granular fields of finer mitochondria of a somewhat deeper portion of the superficial cj'toplasm of the acoustic elements. Such figures justified the statement in a previous paper (1918, p. 63) that "this proves that cuticular formations belonging to the first series men- tioned above (p. 57) may be of mitochondrial origin, but in addition it is a striking proof of the mitochondrial nature of the acoustic hairs formed by this plate."

In other specimens where the mitochondria are also brought prominently into view by suitable fixing agents and stains, mainly by the mixture of formalin and

132 ARRANGEMENT AND STRUCTURE OF SUSTENTACULAR CELLS

bichromate, and staimng l)y fuchsin and mothyl-greon, tho sujicrficial outipula appears homogeneous and, Hke the hairs, remains unstained. After fixation with osmic vapors or osmic acid, and staining with iron hematoxj^lin, pale, clear cuticuhe may l)e observed. From these observations it would ajjpear that the chemical constitution of the hairs and cuticuhe is somewhat different from that of the original chondriosomes. Similar chemical changes have been noted in the devel- opment and evolution of mitochondrial structures. Without emphasizing those observed in glantl-cells, it may be recalled that during the period of growth and genesis of the yolk of the ovum the pseudochromosomes (O. Van der Stricht, 1904), and the vitellogenic bands (1909), both mitochondrial in nature, undergo analogous alterations.

SUPPORTING CELLS.

From the earliest stages of their differentiation (fig. 1) the jMllar-cells fip, op), the inner (is') and outer (d) sustcntacular cells contain numerous chondriomites, and like the acoustic elements are longitudinally striated, owing to the longitudinal disposition of the chondriosomes. The young cells of Deiters (d) possess a mitochon- drial body in close contact with the lower pole of the nucleus. In more advanced stages, before the aj^pearance of coarser permanent structures, the arrangement of the chondriosomes differs according to the nature of the sujiporting element. In the inner sustentacular elements, within which no jieculiar structures are forminl. the chondriomites remain scattered more or less regularly throughout the length of the cytoplasm (figs. 9 and 15, is').

As illustrated in figure 2, within the upper part of the pillar cells (ip, op) and the cells of Deiters of the first (d') and second (d") rows, appears an axial, darker granular strand which is mitochondrial in nature (fig. 4), formed of closely arranged chondriomites. The band is surrounded by a clearer (>xoplasm within which the chondriosomes are less numerous. In figure 4 this mitochondrial strand reaches the ba.se of all these sustentacular elements except that of the cells of Deiters of the third row (d'"), and the clearer cytoplasm becomes obviously reticulated or vacuo- lated, being pervaded bj'^ a network along which fewer mitochondria are scattered. In the meshes of the network are seen vacuoles formed of a pale, more fluid material. The mitochondrial bundle, longitudinally striated (figs. 15 and 9, ip) in vertical sec- tions, occupies the portion of the protoplasm within which the sui)porting fibers will appear in a more advanced stage (fig. 8, ip), these filaments being produced by a fu.sion of the juxtaposed ends of the chondrioconts.

In the inner jMllar-cells the chondriomites become densely groupinl anuuid tlie axial strand at the level of the ajncal jiortion of the cytoi^lasm, .so that on cro.ss- .section they form a mitochondrial ring or circle (figs. 3, 4, and 12, ij)) inclosing the central part of the band, within which they are thinly scatt(>red. This ring corre- s|)onds to the transverse section of a tube, the "Fibrillenrohre" of Held (1909), deveh)ped according to that observer (p. 209) "von der freieii Seite her und von dem ganzen Rand und I'mfaiig ihrer Schlussleisten." Il would be more correct to say that this mitochondiiMJ tiiix' extends from the superlicial ceiitrosome towards

AND HAIR-CELLS IN THE DEVELOPING OUGAN OF CORTI. 133

the deeper portions of the coll, where, at the level of the intermediate and basal parts it is converted into a solid latcro-medial lamellar strand (figs. 3 and 4, ip) reaching the basilar membrane. In more advanced stages the tube itself becomes transformed into a solid, cyhndrical (fig. 21, ip), and later into a flattened, lamellar mitochondrial bundle (fig. 18, ip) which also soon occupies the medial half of the superficial segment of the pillar (fig. 20, ip). This longitudinal, lamellar mito- chondrial bundle, extending throughout the latero-medial portion of the jiroto- plasm, is illustrated in vertical sections (figs. 15 and 9, ip), and by coalescence of its chondrioconts gives rise to the fibrillar supporting apparatus of this inner pillar (figs. 14, 17, and 8, ip).

Besides this bundle, the bulk of the inner pillar during the second and third stages of development is formed, at the level of its basal and intermediate segment, of a clear, vacuolated cytoplasma (figs. 4, 5, 14, and 16, ip), occupying its axial and lateral jjortions. Its apical segment is composed of a compact, almost homogeneous protoplasma (fig. 5, ip) external to (fig. 20, ip) or even surrounding (fig. 18, ip) the supporting bundle.

The axial mitochondrial strand of the outer piUar-cells (fig. 2, op) also becomes converted into a paraxial or lateral, solid bundle, flattened or lamellar in shape, which originally runs more or less vertically throughout the three segments of the cytoplasm (figs. 4 and 5, op). In more advanced stages, while the apical segment undergoes an enlargement and develops the head of the pillar, the mitochondrial strand becomes obviously interrupted at the level of the junction of the two upper segments. The superposed chondrioconts (fig. 20, op) fuse together and give rise to two distinct fibrillar bundles; a lower bundle, belonging to the basal and interme- (hate parts of the pillar (figs. 9, 14, 15, and 16, op), and a superficial one, belonging to the apical segment — that is, the fibrillar supporting band connecting the phalanx with the head of the pillar (fig. 13, op). Tliis filamentous band, originally vertical, later runs obUquely towards its phalanx between the superficial cytoplasmic por- tions of two neighboring outer hair-cells (fig. 14, op), its course becoming more hori- zontal as development progresses (figs. 16 and 17, op). Thus, during the second and third stage in the development of the organ of Corti, the outer pillar, at the level of its basal and intermediate segments, is composed of a lateral mitochondrial or fibrillar, lamellar bundle, and an axial and inner, large, vacuolated portion (figs. 4, 5, 14, 16, 7, and 9, op), representing at a certain time (fig. 9, op) the bulk of the cytoplasm. At the level of its apical segment the pillar is composed of its oblique or nearly horizontal fibrillar bundle and a compact cytoplasm — the bulk of the head.

It is worthy of note that prolongations from the superficial terminal bars separating the apices of the inner and surrounding those of the outer pillars, and constituting part of the membrana reticularis (figs. 18 and 13, tb), extend between the future heads of the inner and outer rods of Corti and sever not only their adjoining surfaces (figs. 14, 15 and 16, tb), but also the contiguous surfaces of the heads of the two rows (fig. 9, tb). Under the influence of this modified cellular cement (the terminal bars) there appear to develop firm, compact bodies, originally

134 ARRANGEMENT AND STRUCTURE OF SUSTENTACULAR CELLS

in direct contact with the bars, and described by Joseph (1900) as " Einschliissen " of the heads of the inner and outer rods of Corti.

The supporting apparatus of the cells of Deiters, its stem or paraxial, fibrillar bundle, and its two branches — the apical filament and the axial strand with the chalice — is laid down by the original mitochondrial band extending throughout the length of the outer sustentacular elements (fig. 4, d', d", d"'). The chondriomites of this strand are clearly illustrated in figures 9 and 19 (d', d", d'"). By a process of coalescence of the chondrioconts (fig. 10, d', d", d) the fibrils of the apical (fig. 21) and of the paraxial, medial (figs. 25 and 16, d', d", d'") filaments are produced. The axial filament, with its chahce, appears in more advanced stages, when the segment of support of the cell of Deiters becomes enlarged and elongated below the hair-cell. At tliis time a long, axial, compact, and mitochondrial band is formed within the upper part of the nucleated cell body (fig. 23, d*), and by accumulation and coales- cence of the chondrioconts the short axial filament, along with its superficial chahce, is built up (fig. 11, d\ d", d'")- The chondriosomes of this axial part of the segment of support of the sustentacular elements have been described by Retzius (1900, p. 80), as "eine schwarz gefarbte Korneransammlung," which do not represent centro- somes and bear no relation to filaments. After examining these preparations M. Heidenhain stated that the granules may be either multiple, central corpuscles, or structures analogous to the " Basalfilamente " in other cells.

Besides their supporting apparatus, the cells of Deiters are composed of a much more abundant, clear, and often distinctly vacuolated (figs. 5, 7, 9, and 11) cyto- plasm, which is less conspicuous within their phalanx process, although recognizable in some preparations (figs. 5 and 14). The paraxial filament alwaj^s occupies the medial half of this vacuolated protoplasm (fig. 16, d', d", d'"), and the latter be- comes reduced around the axial band and its chaUce (fig. 23, d'), when these struc- tures make their appearance.

From the above description it is clear that the fibrillar framework of the rods of Corti and of the cells of Deiters appears in the earhest stages of development and is mitochondrial in nature. Similar filamentous structures have been described in fining epithelium cells by Firket (1910), Favre and Regaud (1910), and Sakae Saguclii (1913). The fibrillated ajjjjaratus and vacuolated cytoplasm of the sus- tentacular cells seem to be related to two different functions of these elements, the former to an absolutely conspicuous supporting function, the latter to a nutritive function. In order to get a true picture of the capacity of nutrition of the rods of Corti and the outer supporting cells, we should bear in mind that during the earhest stages of development of the organ the epithehal constituents undergo a rapid enlargement. Peculiar and complex structures— the superficial cuticula with its hairs, the membrana reticularis, and a part of the membrana tectoria— si)ring from the surface of the neuro-ei)ith('lium, wliich is destituti^ of blood-cai)illaries. There exist no intercellular channels to convey the nutritive material from the subjacent vascular tissue of the membrana basilaris to the superficial portions of the epi- thelium. Minute intercellular spaces urtabubtedly exist, and their cement gives

AND HAIR-CELLS IN THE DEVELOPING ORGAN OF CORTI. 135

rise to the thick, firm terminal bars which close the spaces and separate them from the endoljonph of the cochlea duct. By close examination of figures 3, 4, 5, 7, 9, and 11, it is obvious that the vacuohzation of the sustentacular elements becomes more pronounced as the basilar membrane is approached, and that the chief sup- porting cells— the inner and outer pillars — at first undergo considerable enlargement and acquire a markedly vacuolated structure quite close to the subjacent va.s spirale (vs). which rapidly increases in size along with the transformations just described. Later, when the definite structures of the organ of Corti are nearly built up, the vas spirale becomes reduced in size (figs. 4, 16, and 15, vs). These facts furnish abundant evidence to support the theory that the fluid contained within the meshes of the network, or the contents of the vacuoles within the cyto- plasm of the supporting elements, is derived from the blood of the vas spirale by means of a process of exudation through its endothehal waU and the adjoining base- ment membrane. This food supjjly, stored in the cji;oplasm of the sustentacular cells, may undergo further glandular or chemical alteration before being utiUzed in the building up of the apparatus of support of the rods of Corti and the cells of Deiters, and transferred to the neighboring hair-cells.

SUMMARY.

1 . The first stage in the development of the organ of Corti is characterized by the existence of seven sharply marked, spiral rows of cells. Of these the inner pillar cells and two boundary rows — i. e., the second inner row of supporting elements and the third row of Deiters cells — are purely sustentacular in character. The remain- ing four are mixed rows, being composed of alternating sensory and sustentacular elements, and constituting the row of so-called inner huir-cells and the three rows of so-called outer hair-cells. The hair-cells extend through the superficial two thirds of the epithelial layer, and are separated from each other by the cj'toplasmic portions of their respective sustentacular elements, the basal, larger, nucleated part of which fills the space between the hair-ceUs and the basilar membrane, as illus- trated in figure 22 (is', ih). In sections tangential to the surface each of the four mixed rows is seen in the form of two nucleated columns; one, the column of exclu- sively supporting elements, near the basilar membrane; the other, the column of alternating sensory and sustentacular elements, near the surface of the epitheUum.

2. The original four mixed spiral rows are formed as follows: (a) The row of inner hair-cells is composed of inner acoustic elements and innej- supporting ele- ments; (b) the first row of outer hair-cells, of outer acoustic elements and outer pillar-cells; (c) the second row of outer hair-cells, of outer acoustic elements and the first row of Deiters cells; (d) the third row of outer hair-cells, of outer acoustic ele- ments and the second row of Deiters cells.

3. The superficial cytoplasmic two- .urds of the supporting cells of the niLxed spiral rows undergoes compression from the rapidly enlarging neighboring acoustic elements. This pressure results in a flattening and subsequent shifting of these portions of the inner supporting cells towards and into the lateral part of the spiral

136 ARRANGEMENT AND STRUCTURE OF SUSTENTACULAR CELLS

row, and of those of the outer sustentacuhir elements towards the axis of the cochlea and into interstices between the primitive rows; so that three new sjnral rows or spaces make their appearance. Indeed, each original mixed spiral row becomes divided into two — a medial or inner supporting column, and a lateral, purel}'^ sen- sory column. Hence the first mixed spiral row of outer hair-cells is divided into a column compo.sed of parts of outer pillars and a column of acoustic elements; the second mixed spiral row of outer hair-cells is divided into a column composed of parts of the cells of Deiters of the first row and a column of acoustic elements; the third mixed spiral row of outer hair-cells is divided into a column composed of jxarts of the second row of Deiters cells and a column of acoustic elements.

4. The original membrana reticularis is formed of seven very distinct spiral rows of fields, the apices of the subjacent cells, which are separated from one another by terminal bars. When each of the three outer mixed spiral rows becomes double near the surface of the epithelium, two spiral intervals appear within the lamina reticularis, one between the apices of the hair-cells of the first and second rows, and the other between the apices of the acoustic elements of the second and third rows, these intervals being covered by portions of the neighboring elongated phalanges.

5. A fundamental principle governs the arrangement of the supporting elements during the earUest stages of development of the organ of Corti. By compression from neighboring constituents the cell body may become shifted, but the base, which is attached to the membrana basilaris, and the apex interpolated within the membrana reticularis, remain fixed. These may enlarge with the extension of the membranse, but there is never any sliifting.

6. The second stage of development is characterized by the shifting of the nucleated cell bodies of the outer supporting elements from their original spiral row ; that is, from beneath the hair-cells of that row, inward and to a point just below the superficial, supporting interstices appearing in the first stage of development; so that the latter now constitute complete spiral rows extending throughout the tliick- ness of the epithehum, whereas the three original mixed outer rows are converted into a purely sensory one, occupying the superficial two-thirds of the epithelium. External to the inner pillars, from the axis of the cochlea towards the outer part of the lesser ridge, there exist the following sjiiral rows: One of outer pillars, the first row of outer liair-cells, the first row of cells of Deiters, the second row of outer hair- cells, the second row of cells of Deiters, the third row of out(>r liair-cells, and the third row of cells of Deiters, the nucleated cell bodies of which show a tendency to shift below the neighboring acoustic elements.

7. The shifting of the nucleated portions of the outer sui)porting cells is the result of several mechanical factors: (a) The appearance of an axial framework within the organ of Corti represented by two kinds of rods of Corti. liy the basal extension f)f this framewcjrk the bases of the cells of Deiters are jjressed outward much more than is the apical jwrtion of their nucleated cell bodies. (6) The broadening and lengthening of the outer hair-cells, which is jx^ssibie only towartls the outer side of the lesser ridge. The summit of the nucleated i)ortioii of the

AND HAIR-CELLS IN THE DEVELOPING OUGAN OF CORTI. 137

supporting elements is, therefore, repelled inward and ultimately incorporated into and comi)leting the superficial sustentacular interstices, which already contain the phalanx processes of the supporting elements, (c) This shifting is facilitated by the previous transference of the phalanx processes and by the peculiar connections between the lower pole of the hair-cells and the subjacent spiral nerve bundles.

8. Pressed out from their original mixed row, and having reached their respec- tive interstices, the phalanx processes of the first and second rows of Deiters cells, together with those of the third row, undergo a shifting in a spiral direction, their apices moving from a basal to a more apical portion of the cochlea, so that they cross three hair-cells in their course towards the membrana reticularis. Whereas the acoustic elements follow a nearly vertical course, the cell bodies, and particu- larly the phalanx processes of the cells of Deiters, run more obli(iuely (figs. 19, 19'). This spiral shifting is due to an unequal development or extension towards the apex of the cochlea of the two membransE to which are attached the two extremities of the cells of Deiters, the membrana reticularis extending more rapidly than the mem- brana basilaris.

9. The third stage of development is preceded by a considerable elongation of the nucleated cell bodies of the elements of Deiters and the appearance of a jiecuHar portion — the segment of supjjort or sustentacular segment. On lengthening out obliciuely from the nucleated part of the cj^toplasm towards the lower pole of the hair-cell which will eventually be supj^orted by it, the apex of the cell body enlarges and extends into a long segment surmounted by a cup-like depression surrounding the lower pole of the acoustic element; so that the axes of the hair-ceUs of the first, second, and third spiral rows ultimatel\- blend respectively with those of the cells of Deiters of the first, second, and third rows.

10. Lateral to the spiral row of inner pillars the final structure of the organ of Corti is composed of the following supporting and mixed spiral rows: The row of outer pillars; the first mixed row of superficial acoustic elements supported by the subjacent bodies of the cells of Deiters; the superficial interstice containing the phalanx processes of the Deiters cells of the first row; the second mixed row of superficial acoustic elements supported by subjacent bodies of the cells of Deiters; the superficial interstice containing the phalanx processes of the cells of Deiters of second row; the third mixed row of superficial acoustic elements supported by the subjacent bodies of the cells of Deiters; and the superficial interstice containing the apical processes of the cells of Deiters of the third row.

11. At the third and final stage of development the spiral nerve bundles be- tween the ceils of Deiters, which originally were situated lateral to the primitive outer mixed rows (fig. 3, N"', N, N') occupy a place in.side of the mixed spiral rows (fig. 9, N"', N'^ NO.

12. The supporting apparatus of the cells of Deiters is originally formed of a stem or paraxial fibrillated filament, running straight throughout the inner portion of the prismatic cell body, at the apex of which it curves around and continues as an obUque apical fibrillar filament in the phalanx process of the cell. In later stages

138 ARRANGEMENT AND STRUCTURE OF SUSTENTACULAR CELLS

of development the summit of the cell body becomes lengthened out into its segment of support; the paraxial filament, at the level of the nucleus, courses obhquely through the lateral part of this segment and gives off another branch — the axial filament — thus expanding into a fibrillar chahce located within the wall of the cup- shaped depression.

13. Contrary to the opinion of many authors, the columnar epitheUum of the greater epithehal thickening of the cochlea duct does not undergo disintegration during the development of the sulcus spiralis. At the level of the latter no cells are lost, but all become flattened out to cover the greatly extending furrow. Near the organ of Corti the columnar cells persist longer, and occasionally exhibit evidences of chromatolysis.

14. Medial to the inner mixed row of hair-cells there exists a second row of inner sustentacular elements with differentiated apices. These resemble in many respects the cells of Deiters of the third outer row, although in the course of devel- opment the cell bodies of the latter are shifted beneath and suiijwrt the neighboring acoustic elements. Like the sustentacular cells of the first inner row, those of the second remain in their original row; the apices of both take part in the formation of the membrana reticularis, that is, the apparatus of support of the apices of the hair- cells. Hence they represent true sustentacular elements. Like the cells of Deiters of the tliird row, the sustentacular elements of the second row also represent boundarj' elements.

15. Among the so-called cells of Hensen there exists, lateral to the third row of cells of Deiters, a fourth outer, mixed row, formed of alternating atrophied susten- tacular cells and more superficial hair-cells. The atrophied hair-elements become shorter in the course of development, but persist, even in the adult cochlea. As a rule, the absence of nerve fibers seems to result in atrophy of the constituents of this fourth row, but in man these become normally developed.

16. From the earUest stage of development the cytoiilasm of the hair and sup- porting cells contains innumerable chondriomites and chondrioconts which, by their pecaUar arrangement, give rise to a longitudinal striation. By their aggregation the chondriosomes form the body of Retzius and the body of Hensen, each one rep- resenting a center of develoi)ing chondriomites, whence the chondriomites migrate into other portions of the cell. The superficial body of Hensen is closely connected with the cuticula of the hair-cell. This superficial plate is derived from coarse, coalescing chondriomites wliich appear to undergo a chemical alteration. Hence the hairs arising from the cuticula should be regarded as of mitochondrial origin.

17. The outer and inner rods of Corti and the cells of Deiters contain a fibril- lated framework which, from the earliest stages of development, ai)pears in the form f)f mitoclK)ndrial bands or strands. Each fibril is the result of fusion of the super- jKjsed chondrioconts. In ad(Htion to the sustentacular function of the framework, these elements po.ssess also a function of nutrition. Indeed, the bulk of their cytoplasm is clear, and contains, besides a few chondriosomes, a serous fluid which exudes from the subjacent vas spirale and permeates the protoplasm of the cells in

AND HAIR-CELLS IN THE DEVELOPING ORGAN OF CORTI.

139

the form of clear vacuoles. The number, ^ize, and distinctness of these vacuoles decrease progressively from the base of the supporting element towards the surface of the epitheUum, where important phenomena of metabolism and elaboration of pecuUar structures take place, not only within the sustentacular elements, but also within the hair-cells and at the surface of the sensory epithelium.

The present investigation was started in the anatomical laboratory of the Med- ical School of the Western Reserve University, Cleveland, Ohio. It is my agreeable duty to express my sincere and profound gratitude to Professor T. Wingate Todd and for the gracious hospitality afforded me, and for all the material, reagents, and instruments which he placed at my disposal.

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+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

DESCRIPTION OF PLATES.

All figures were outlined with a Zeiss camera lucida at the level of the stage of the microscope, with the aid of ocular No. 3 and 2 mm., homog. immersion. Apcrt. 1.30.

Plate 1. Fig. 1. Radial vertical section of organ of Corti through the apical jjart of the cochlea. Dog 18 hours after

birth. Fixation: osraic acid, trichloracetic acid. Stain: iron hematoxylin and Congo red. Fig. 2. Section tangential to surface of organ of Corti through first turn and near apex of cochlea. Dog 12 hours

after birth. Bouin's fluid, iron hematoxylin, and Congo red. Fig. 3. Section tangential to surface of organ of Corti through first turn of cochlea. New-born dog. Trichloracetic

acid, iron hematoxyhn, Congo red. Fig. 4. Section tangential to surface of organ of Corti through first turn of cochlea. New-bom kitten. Osmic acid

and Zenker's fluid; iron hematoxylin and Congo red. Fig. 5. Section tangential to surface of organ of Corti throiigh third turn of cochlea. Rabbit 10 hours after birth.

Osmic vapors and Bouin's fluid; iron hematoxylin and Congo red. Fig. 6. Section tangential to siu-face of organ of Corti through basal part of third turn of cochlea. Dog 3 days and

10 hours after birth. Bouin's fluiil, iron hematoxylin, and light green.

Plate 2. Fig. 7. Section tangential to surface of organ of Corti through middle of third turn of cochlea. Dog 12 hours after

birth. Bouin's fluid, iron hematoxj'lin, and light green. Fig. 8. Section tangential to surface of organ of Corti through third turn of cochlea. Kitten 12 hours afterbirth.

Trichlorac<?tic acid, iron hematoxylin, and light green. Fig. 9. Section tangent ia' to surface of organ of Corti through second turn of cochlea. Kitten 3 days and 12 hours

after birth. Osmic vapors and trichloracetic acid; iron hematoxylin and Congo red. Fig. 10. Section tangential to surface of organ of Corti through first turn of cochlea. New-born dog. Trichloracetic

acid, iron hematoxylin, and Congo red. Fig. 11. Section tangential to surface of organ of Corti through third turn of cochlea. Ivitten 12 days after birth. Osmic acid and trichloracetic acid ; iron hematoxylin and Congo red.

Pl.\te 3. Fig. 12. Section tangential to surface of the organ of Corti through first turn of cochlea. New-born kitten. Osmic

acid and Zenker's fluid. Iron hematoxylin and Congo red. Fig. 13. Section tangential to surface of organ of Corti through third turn of cochlea. Kitten 12 days after birth.

Osmic acid and trichloracetic acid; iron hematoxylin and light green. Fig. 14. Radial vertical section of organ of Corti through first turn of cochlea (before appearance of tunnel space).

Dog 3 days and 18 hours after birth. Trichloracetic acid, iron hematoxylin, and Congo red. Fig. 15. Radial vertical section of organ of Corti through third turn of cochlea (basal part). Kitten 3 days and 12

hours after birth. Osmic vapors and trichloracetic acid; iron hematoxylin and Congo red. Fig. 16. Radial vertical section of organ of Corti through second turn of cochlea. Dog 3 days and 18 hours after

birth. Trichloracetic acid, iron hematoxylin and Congo red. Fig. 17. Section tangential to surface of organ of Corti through second turn of cochlea. Kitten 12 days after birth.

Trichloracetic acid, iron hematox-ylin, and Congo red.

Pl-\te 4. Fig. 18. Section tangential to surface of organ of Corti through apical part of second turn of cochlea. Kitten 16 hours

after birth. Osmic acid and Bouin's fluid; iron hematoxylin and light green. Figs. 19 and 19'. Vertical spiral section of organ of Corti through second turn of cochlea. New-born kitten: Osmic

acid and Zenker's fluid ; iron hematoxylin and Congo red. Fig. 20. Vertical spiral section of organ of Corti through third turn of cochlea. Rabbit 2 days after birth. Osmic

acid and silver nitrate; iron hematoxylin and Congo red. Fig. 21. Vertical spiral section of organ of Corti through second turn of cochlea. Dog 3 days and IS hours after birth.

Zenker's fluid, iron hematoxylin, and light green. Fig. 22. Vertical spiral section of organ of Corti through second turn of cochlea. Dog 3 days and 10 hours after birth.

Bouin's fluid, iron hematoxylin, and light green. FiQ. 23. Vertical spiral section of organ of Corti through second turn of cochlea. Kitten 12 daj's after birth. Osmic

acid and trichloracetic acid; iron hematoxylin and light green.

141

GENERAL ABBREVIATIONS,

ad'» Atrophied cells of Dciters of fourth

fipiral outer row.

aob^ Atrophied hair cells of fourth spiral

outer row.

bm Membrana basilaris.

C Cells ol Claudius.

ch Cliromatolysis of cells of greater epithe- lial ridRC.

d Cells of Deiters.

d', d>', d"' Cells of Deiters respectively of first, sec- ond, and third row.

esp Epithelium of sulcus spiralis.

h Hairs.

H Cells of Ilensen.

ih Inner hair-cells.

ip Inner pillar-cells.

isi, is" Supporting cells respectively of first and

second inner row.

N Nerve bundles passing through fora- mina nervinum.

N' Spiral nerve bundles running between

inner sustentacular cells and inner . pillars.

N'i Spiral nerve bundles running between

inner and outer pillars.

N'i' Spiral nerve bundles running between

outer pillars and cells of Deiters of first row.

N'v Spiral nerve bundles running between

cells of Deiters of first and second rows.

Nv Spiral nerve bundles running between

cells of Deiters of second and third rows.

nd Non-diflerentiated cells of greater epithe- lial ridge.

oh Outer hair-cells.

oh', oh, oh'" Outer hair-cells respectively of first, sec- ond and third row.

op Outer pillar-cells.

SN Space of Nuel.

T Tunnel space.

tb Terminal bars.

Vs Vas spiiale.