SELECTIVE and HISTOCHEMICAL STAINING of the OTOLITHIC MEMBRANES, CUPULAE and TECTORIAL MEMBRANE of the INNER EAR K by GEORGE B

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SELECTIVE AND HISTOCHEMICAL STAINING OF THE OTOLITHIC MEMBRANES, CUPULAE AND TECTORIAL MEMBRANE OF THE INNER EAR k By GEORGE B. WISLOCKI AND AARON J. LADMAN* Department of Anatomy, Harvard Medical School, Boston, Massachusetts t The present investigation reveals that the tectorial membrane, the gelatinous substance of the otolithic membranes and the cupulae of the internal ear are stained intensely and selectively by the alum-haematoxylin element of Gomori's chrome alum-haematoxylin and phloxine stain and by his aldehyde fuchsin stain. They react strongly also with the periodic acid-Schiff technique following exposure to saliva. These reactions are similar to those shown by several other structures of the central nervous system, namely, the hypophysial Herring substance (Bargmann, 1949; Bargmann & Scharrer, 1951), the subcommissural organ and Reissner's fibre (Wislocki & Leduc, 1952 a, b; Bargmann & Schiebler, 1952) and the ocular ciliary zonula (Wislocki, 1952). Brief mention of the selective staining of the tectorial membrane (Wislocki, 1952) and of the otolithic membranes and cupulae has ap- peared elsewhere (Wislocki & Ladman, 1954). The present paper gives a more com- plete account of the staining of these otic structures by a number of histochemical methods and selective stains and discusses the possible significance of the results. MATERIAL AND METHODS The material consisted of two foetal mouse heads of 17 days of gestation, six heads of newborn mice and the temporal bones of two 34-month-old mice. It was supple- mented by two temporal bones obtained from a fresh human foetus of 17 cm. crown- rump length. The material was fixed according to the prescriptions of the different staining pro- cedures. The temporal bones of the 34-month-old mice were fixed in a saturated solution of mercuric chloride in 10 % formalin and decalcified in 5 % trichloracetic acid before sectioning them. The less densely calcified heads of the foetal specimens and newborn mice proved soft enough to section them without resort to a special decalcifying agent. The heads were embedded in paraffin and sectioned at 5p, either in the horizontal or the sagittal plane. The former proved more favourable for encompassing all the structures in question within a single section. Gomori's chrome alum-haematoxylin and phloxine method (1941), and his aldehyde fuchsin stain counterstained with orange G and light green as modified by Halmi (1952), were applied to deparaffinized sections of material fixed in mercuric chloride and formalin, Rossman's fluid (a saturated solution of picric acid in absolute alcohol: 90 ml.; formaldehyde: 10 ml.), Bouin's fluid, Orth's fixative and Zenker's acetic acid fixative. * Research Fellow of the American Cancer Society, Inc., upon recommendation of the Com- mittee on Growth of the National Research Council, 1952-4. 1-2 4 George B. Wislocki and Aaron J. Ladman The periodic acid-Schiff method of McManus (1946) and Hotchkiss (1948) was applied to deparaffinized sections of the same blocks. Prior to staining them, the sections were placed in saliva for 2 or 3 hr. to remove glycogen. Other saliva-treated sections were stained with Schiff's reagent without previous oxidation with periodic acid. Pap's ammoniacal silver nitrate method for the demonstration offibrous reticulum, as modified by Mitchell & Wislocki (1944), and Weigert's resorcin-fuchsin stain for elastic tissue were applied to sections from the same blocks. Other deparaffinized sections of internal ears fixed in 4 % basic lead acetate or Zenker's acetic acid fixative were stained in either a 1 % aqueous solution of methylene blue or toluidin blue for the demonstration respectively of possible cytoplasmic basophilia and metachromasia. A series of sections of one of the labyrinths of the human foetus listed above was stained in solutions of methylene blue of graded pH as described by Dempsey, Bunting, Singer & Wislocki (1947). Sections of two heads of newborn mice fixed in a solution of 1 % trichloracetic acid in 80 % ethanol and in Zenker's acetic acid fluid were stained by the methods of Barrnett & Seligman (1952, 1954) for protein-bound sulph-hydryl and disulphide groups.

OBSERVATIONS Staining with chrome alum-haematoxylin and aldehyde fuchsin. Similar staining results were obtained by both methods at the three stages of development of the mouse and in the human tectorial membrane. Consequently, it is unnecessary to represent each structure at every stage by both methods. PI. 1, fig. 1, illustrates a typical horizontal section through the internal ear of a newborn mouse, revealing a crista ampullaris, the utricle and saccule with their maculae, and a portion of the cochlea with the developing organ of Corti. The section was stained by the chrome alum-haematoxylin and phloxine method. The three regions of the section contained in rectangles coincide approximately with the areas of figs. 2 and 19, 3 and 14, and 6 and 18 in Pls. 1-3. Reference to PI. 3, figs. 14, 18 and 19, illustrates that the otolithic membrane of the macula of the utricle, the developing tectorial membrane and the cupula of a crista ampullaris are stained selectively blue by the alum-haematoxylin component of Gomori's stain. PI. 1, fig. 4, and PI. 3, fig. 15, of the macula sacculi of a 38-month-old mouse reveal a similar selective staining of the otolithic membrane by the aldehyde fuchsin stain. These representative illustrations will serve to show that the tectorial membrane, cupulae and otolithic membranes are selectively stained. Precisely what elements of these membranes are stained is not so easy to judge. The tectorial membrane is generally accepted as being composed of a fibrillar matrix and a jelly-like ground substance. The otolithic membrane is believed to consist of a layer of a gelatinous substance in the outer part of which there are numerous small bodies, the otoliths or otoconia, which are composed of a mixture of calcium car- bonate and a protein. The cupulae are described as consisting of a jelly-like mass of substance containing pores or canals into which the hairs of the sensory cells of the cristae project. The cupulae are also described under some conditions as possessing a finely striated texture, an appearance which, according to Kolmer (1927). may be Selective and histochemical staining 5 a fixation artifact of the colloids of which they are composed. From examination of PI. 3, figs. 14, 15, 18 and 19, it is apparent that no critical distinctions between the several components of these membranes can be made upon the basis of their staining by means of chrome alum-haematoxylin or aldehyde fuchsin. In the case of the otolithic membranes (PI. 3, figs. 14, 15) it could be the protein of the otoliths that is stained, but it is equally possible that the gelatinous matrix precipitated upon the otoliths could account for the staining. Fibrillar shreds of selectively stained material appear to extend from the otolithic membrane on to the flagellae and the outer surface of the cells of the macula. The same relation of stained material to cells is faintly seen in the case of the cupula and crista (PI. 3, fig. 19). In the developing organ of Corti, quite heavily stained, blue fibrils appear to connect the under surface of the tectorial membrane with the adjacent surface of Corti's organ, and thence bluish strands seem to penetrate between the columnar cells (PI. 3, fig. 18). On the other hand, the bluish fibrils visible quite generally in the subepithelial connective tissue of the labyrinth (PI. 3, figs. 14, 18 and 19) are apparently collagenous fibrils which are known to stain variably strongly with chrome alum-haematoxylin. The periodic acid-Schiffreaction. By this procedure the tectorial membrane (P1. 2, fig. 7), the otolithic membranes (PI. 3, fig. 16) and the cupulae (PI. 1, fig. 5; PI. 3, fig. 17) are intensely stained. Since this staining occurs in sections which were ex- posed to saliva, it cannot be attributed to the presence of glycogen. Here, as with the previous stains, a decision cannot be reached in regard to which components of the membranes-whether the gelatinous substance or the fibrillar component, or both-are specifically stained. It is also possible that the protein of the otoliths may be stained. A basement membrane upon which the epithelium of the maculae and cristae rests also stains intensely (PI. 3, figs. 16, 17). Similar staining is observed in the foetal and newborn mouse heads, as well as in the human tectorial membrane. Sections stained with Schiff's reagent without prior oxidation with periodic acid reveal mild staining of the tectorial membrane, but none of the otolithic membranes. Metachromasia and basophitia. For the investigation of these manifestations of staining, sections were used from ears fixed in 4 % basic lead acetate, which is re- commended for the preservation of acid mucopolysaccharides (Holmgren & Wil- ander, 1937; Wislocki, Bunting & Dempsey, 1947), or fixed in Zenker's acetic acid fluid. In sections of ears of newborn mice so fixed and then stained with toluidin blue, no metachromasia was observed in the tectorial and otolithic membranes or in the cupulae. In the ear of a 3+-month-old mouse which was decalcified in 5 % trichloracetic acid there was similarly no metachromasia of the structures in question, except in one section in which the staining was allowed to run overnight, when a somewhat reddish blue coloration developed. In all of the sections referred to, car- tilage matrix and the granules of mast cells showed intense metachromasia, attri- butable to their acid mucopolysaccharide content, thus serving as test-objects for comparison with the membranes under investigation. Basophilic staining, carried out on a series of sections stained in solutions of methylene blue of ascending pH (Dempsey et al. 1947), provided a further means of characterizing acid substances and of investigating the properties of these mem- 6 George B. Wislocki and Aaron J. Ladman branes. Only a foetal human organ of Corti was stained in this manner. It shows moderate nuclear and some cytoplasmic basophilia at pH 483, but exhibits no basophilia of the tectorial membrane (PI. 2, fig. 12). This indicates that the substance of the tectorial membrane is considerably less acid than the nucleoproteins which are moderately stained at this pH. Furthermore, the tectorial membrane does not appear to consist of a sulphated mucopolysaccharide, because these compounds stain intensely with basic dyes at pH 4 (Dempsey et al. 1947). Protein-boundsulph-hydryl and disulphidegroups. As afurther means of attempting to characterize the substance of the membranes in question, sections were stained by the methods of Barrnett and Seligman for sulph-hydryl and disulphide groups. Upon staining for sulph-hydryls the membranes react only slightly. By the procedure for the demonstration of disulphide groups the response is greater. In the maculae and cristae the reaction tinges the gelatinous material of the otolithic membranes and cupulae, and it also colours the respective epithelia, particularly the distal margins of the cells and the hair bundles. PI. 2, fig. 13, shows this for the macula and otolithic membrane. The tectorial membrane stains more deeply for disulphide groups (P1. 2, fig. 8) than the substance of either the otolithic membranes or cupulae. Of particular interest is the relatively intense staining of the hair cells of Corti's organ in the newborn mouse (PI. 2, figs. 8 (arrow), 10 and 11). The supranuclear cytoplasm and the developing hairs of these cells react intensely for disulphide groups (PI. 2, figs. 10, 11). The strength of the reaction for disulphide groups varies somewhat depending upon the fixative used; the deepest staining was obtained in internal ears fixed in Zenker's acetic acid fluid. Weigert's resorcin-fuchsin stain for elastic tissue. By this method the otolithic membrane and cupulae arenegative, but the tectorial membraneismoderatelystained. However, in the photograph illustrating this staining (PI. 2, fig. 9), the tectorial membrane appears darker than in the actual histological section. Moreover, under the microscope it is perceived that the membrane stains reddish rather than the usual bluish black colour characteristic of typical elastic tissue. Argyrophil reticular fibres. Stained with ammoniacal silver nitrate for argyrophil fibrous reticulum, all of these otic membranes assume pale brownish or tan colours depending upon the fixatives used. In contrast to this, true collagenous reticular fibres, present in the stroma surrounding the labyrinth, are typically argyrophilic, assuming, variously, a deep brown or black appearance.

DISCUSSION From the preceding observations it is apparent that the tectorial and otolithic membranes and the cupulae of the internal ear have very similar histological and histochemical properties. They stain similarly and selectively with the alum-haematoxylin component of Gomori's chrome alum-haematoxylin and phloxine stain and with Gomori's aldehyde fuchsin stain. In addition, they react intensely with the periodic acid-Schiff procedure. They also react mildly to moderately for protein- bound disulphide groups, but do not exhibit metachromasia with toluidin blue or stain at low pH with methylene blue. They stain poorly with the ammoniacal silver nitrate method for collagenous reticulum and negatively with Weigert's resorcin- Selective and histochemical staining 7 fuchsin stain for elastic tissue, excepting the tectorial membrane which stains moderately and atypically by the latter method. The chemical nature of the selective staining with chrome alum-haematoxylin and aldehyde fuchsin is poorly understood. The intense staining with the periodic acid- Schiff technique, combined with saliva controls, precludes glycogen and indicates the presence in these structures ofa mucopolysaccharide orglycoprotein (cf. Leblond, 1950). The mild reaction of the tectorial membrane with the Schiff reagent alone, without oxidation with periodic acid, indicates the presence in this carbohydrate of some free carbonyl groups. That the carbohydrate present in these structures is not an acidic or sulphated mucopolysaccharide seems probable from the observations that the membranes neither stain metachromatically with toluidin blue nor exhibit a strong degree of basophilia with methylene blue. In this respect our observations differ from those of B6langer (1953) who states that when stained with toluidin blue these membranes exhibit metachromasia, whereas the 'Chevremont and Fredericq reaction for the sulph-hydryl groups of keratin was negative'. These findings, B6langer concludes, point to the presence of 'sulphomucopolysaccharides' in these membranes. Our own observations are contrary to his, in that we have not observed metachromasia but have obtained a moderate reaction for protein-bound disulphide groups, the latter especially in the tectorial membrane. Parenthetically it should be noted that in agreement with B6langer we also failed to find any reaction by Chevrement and Fred6ricq's method in the tectorial membrane of a rat's internal ear which we had occasion to examine. In commenting on these differences, it should be pointed out that the Barrnett and Seligman reaction for sulph-hydryl and disulphide groups is a specific histochemical test, whereas the nature of Chevremont and Fr6dericq's Prussian blue reaction is unknown. Moreover, metachromasia may under some conditions of fixation be produced by substances other than acid mucopolysaccharides (Wislocki, Bunting & Dempsey, 1947), a circumstance possibly accounting for B6langer's positive finding, since he used both formaldehyde and alcohol. Although disulphide groups are especially common in keratins, it would be hazardous, without further study of the protein present in these otic structures, to conclude that it was keratinous in nature. It should, however, be noted in passing that Hardesty (1908), in his study of the tectorial membrane of the pig, states that it possesses 'a hyaline matrix, probably keratin'. He gives no substantiating evi- dence for his conjecture, and in the comprehensive chapters of Kolmer (1927) and Shambaugh (1932) on the structure of the internal ear we find no mention of keratin. In conclusion, although we agree with Belanger that there is sulphur in these membranous structures, we believe that it is in the form of disulphides of cystine rather than as sulphates of an acid mucopolysaccharide. From our observations we would conclude that these membranes contain a substance of protein nature which is characterized by its selective staining with alum-haematoxylin and aldehyde fuchsin, by a strong periodic acid-Schiff reaction indicative of a mucopolysaccharide or glycoprotein, and by the possession of disulphide groups associated with cystine. Moreover, it does not stain characteristically with the ammoniacal silver nitrate method for collagenous reticulum or with Weigert's resorcin-fuchsin stain for elastic tissue. 8 George B. Wislocki and Aaron J. Ladman Of further interest is the observation that in the eye and brain there are structures which stain almost identically. In the eye the ciliary zonula reacts similarly (Wis- locki, 1952), and in the brain Reissner's fibre which arises from the subcommissural organ (Wislocki & Leduc, 1952a, b, 1954; Bargmann & Schiebler, 1952), and the Herring substance of the hypothalamus and neurohypophysis (Bargmann, 1949; Bargmann & Scharrer, 1951; Barrnett & Seligman, 1953) exhibit almost identical staining properties. Thus a substance possessing a distinctive constitution appears to characterize these several neural structures. The Herring material of the hypothalamus is believed to be involved in neurosecretory processes, whereas the ocular zonula and the respective membranous structures of the labryinth are regarded as subserving purely mechanical functions. The functional role of the subcommissural organ and Reissner's fibre has been the subject of much inconclusive speculation. It is noteworthy, however, that Kolmer (1921) called attention to some anatomical similarities between Reissner's fibre, the tectorial membrane and the cupulae of the labyrinth. He drew attention to what he regarded as their similar modes of forma- tion as well as to other histological resemblances, pronouncing them to be 'cuticular structures'. Bargmann & Schiebler (1952), in discussing the selective staining of Reissner's fibre with alum-haematoxylin, qualify its staining by saying that after all there are various other cells and elements in the body which are similarly stained, for example elastic tissue and the beta granules of the pancreatic islets. They overlook, however, that the two tissue elements which they have selected react quite differently in some other respects from the particular neural substance in question. Thus, elastic tissue stains deeply with Weigert's resorcin-fuchsin stain and by Unna's orcein technique which are reported as being negative in the case of Reissner's fibre (Nicholls, 1912; Wislocki & Leduc, 1952b), while the beta granules of the islets are periodic acid- Schiff negative (Halmi & Davies, 1953). In this connexion it should be noted that none of the neural structures under con- sideration reacts with orcein or resorcin-fuchsin, if one ignores faint and atypical staining of some Herring bodies and of the tectorial membrane. Although the substance of these structures does not stain with resorcin-fuchsin or orcein and hence is not identical with the substance of elastic tissue, the fact that both substances do stain with alum-haematoxylin and aldehyde fuchsin bespeaks a certain similarity, the chemical basis of which is completely obscure. The neural structures in question apparently contain disulphide groups ranging from a faint reaction in the case ofthe otolithic membranes and cupulae to moderately strong staining of the Herring substance, the secretion of the subcommissural organ (Wislocki & Leduc, 1954), and the tectorial membrane. The ciliary zonula has not been examined. Using aldehyde fuchsin, Halmi & Davies (1953) list all of the substances in the body, known to them, which it stains. These they subdivide into groups on the basis of whether or not they also exhibit metachromasia and give a periodic acid-Schiff reaction. The category which interests us is the one which lists tissues which stain with aldehyde fuchsin, react orthochromatically with toluidin blue and give a positive periodic acid-Schiff reaction. Amongst the substances so characterized, they list epithelial mucoid of some cells of the gastrointestinal tract, gastric chief cell Selective and histochemical staining 9 cytoplasm, lipofuscin pigment in various cells, thyroid colloid, beta granules of the anterior pituitary, some adenohypophysial colloid and neurohypophysial neurosecretory substance (Herring material). Although the substances which they have listed react similarly with the three stains which they used, the group is disparate on other grounds, with most of its members differing from the neural substances under discussion. In contrast to the substance of the neural structures, lipofuscin pigment would at once be ruled out, for besides its being coloured and giving sundry lipid reactions, it reacts negatively for disulphides. The beta granules of the anterior pituitary cells are negative for disulphides (Ladman & Barrnett, 1954). The thyroid colloid, in its intense staining with both acid and basic dyes and its exceptionally strong reaction for disulphide groups (Barrnett & Seligman, 1954), would also seem to differ somewhat from the neural substance. Similarly, the colloid of the hypophysial residual lumen reacts more strongly for disulphide groups than the neural protein in question. We have no comment to offer about the intestinal mucoid cells and the gastric chief cells. From these considerations it seems likely that the substance of the group of structures in brain, eye and inner ear differs in some respects from the majority of the similar, non-neural materials listed by Halmi & Davies. Despite the close similarities of the histological and histochemical reactions of the neural structures considered in this study, it should be pointed out in conclusion that they are not absolutely identical. For example, the reaction for disulphide groups is much stronger in the Herring substance, the tectorial membrane and the secretion of the subcommissural organ than in the substance of the otolithic membranes and cupulae. Furthermore, the Schiff reaction without previous oxidation is stronger in the Herring substance and tectorial membrane than in Reissner's fibre (Bargmann & Schiebler, 1952), and is absent in the otolithic membranes and cupulae.

SUMMARY The tectorial and otolithic membranes and the cupulae of the internal ear have similar histological and histochemical properties. These structures contain a substance of protein nature which is characterized by its selective staining with alum- haematoxylin and aldehyde fuchsin (Gomori's methods), by a strong periodic acid-Schiff reaction, and by the presence of disulphide groups (Barrnett & Seligman's method) associated with cystine. The positive periodic acid-Schiff reaction indicates the presence of a mucopolysaccharide or glycoprotein, but the absence of metachromasia with toluidin blue and of basophilic staining with methylene blue at low pH militate against its being a sulphated mucopolysaccharide. The selective reactions of these otic structures by the methods cited relate them to several neural structures which exhibit similar staining properties. These are the ciliary zonula, the secretion of the subcommissural organ of the epithalamus, Reissner's fibre, and the Herring substance of the neurohypophysis. These structures are more closely allied to one another with respect to their staining properties than to other substances of the body (e.g. elastic tissue, mucus), from which they can be shown to differ in one or more important ways. With regard to function, there is no apparent similarity between the structures: the several otic membranes in question and the ocular zonula are believed to exercise mechanical functions and the 10 George B. Wislocki and Aaron J. Ladman Herring material to bear a relationship to neurosecretory processes, while the functions of the subcommissural organ and of Reissner's fibre are unknown. This study was aided by a grant from the Eugene Higgins Trust of Harvard University. The authors wish to express their appreciation to Mr Arthur Mitchell for pre- paring the material and to Miss Etta Piotti for the coloured illustrations.

REFERENCES BARGMANN, W. (1949). Ober die neurosekretorische Verknupfung von Hypothalamus und Hypo- physe. Klin. Wschr. 27, 617-622. BARGMANN, W. & SCHARRER, E. (1951). The site of origin of the hormones ofthe posterior pituitary. Amer. Scientist, 39, 255-259. BARGMANN, W. & SCHIEBLER, T. H. (1952). Histologische und cytochemische Untersuchungen am Subkommissuralorgan von Saugern. Z. Zellforsch. 37, 582-596. BARRNETT, R. J. & SELIGMAN, A. M. (1952). Histochemical demonstration of protein-bound sulfhydryl groups. Science, 116, 823-327. BARRNETT, R. J. & SELIGMAN, A. M. (1953). Investigations of the histochemical localization of disulfides. J. Histochem. Cytochem. 1, 392-893 (Abstract). BARRNETT, R. J. & SELIGMAN, A. M. (1954). Histochemical demonstration of sulfhydryl and disulfide groups of protein. J. nat. Cancer Inst. 14, 769-803. BELANGER, L. F. (1953). Autoradiographic detection of S35 in the membranes of the inner ear of the rat. Science, 118, 520-521. DEMPSEY, E. W., BUNTING, H., SINGER, M. & WISLOCKI, G. B. (1947). The dye-binding capacity and other chemohistological properties of mammalian mucopolysaccharides. Anat. Rec. 98, 417-430. GoMORI, G. (1941). Observations with differential stains on human islets of Langerhans. Amer. J. Path. 17, 395-406. HALMI, N. S. (1952). Differentiation of two types of basophils in the adenohypophysis of the rat and the mouse. Stain Tech. 27, 61-64. HALMI, N. S. & DAVIES, J. (1953). Comparison of aldehyde fuchsin staining, metachromasia and periodic acid-Schiff reactivity of various tissues. J. Histochem. Cytochem. 1, 447-459. HARDESTY, I. (1908). On the nature ofthe tectorial membrane and its probable role in the anatomy of hearing. Amer. J. Anat. 8, 109-179. HOLMGREN, H. & WILANDER, 0. (1937). Beitrag zur Kenntnis der Chemie und Funktion der Ehr- lichschen Mastzellen. Z. mikr.-anat. Forsch. 42, 242-278. HOTCHKISS, R. D. (1948). A microchemical reaction resulting in the staining of polysaccharide structures in fixed tissue preparations. Arch. Biochem. 16, 131-141. KOLMER, W. (1921). Das Sagittalorgan der Wirbeltiere. Z. ges. Anat. 1. Z. Anat. EntwGesch. 60, 652-717. KOLMER, W. (1927). Geh6rorgan. In von M61lendorff's Handbuch d. mikr. Anat. d. Menschen, III/I, 250-478. Berlin: $Springer. LADMAN, A. J. & BARRNETT, R. J. (1954). Histochemical demonstration of protein-bound sulfhydryl and disulfide groups in cells of the anterior pituitary. Endocrinology, 54, 355-360. LEBLOND, C. P. (1950). Distribution of periodic acid-reactive carbohydrates in the adult rat. Amer. J. Anat. 86, 1-49. McMANus, J. F. A. (1946). Histological demonstration of mucin after periodic acid. Nature, Lond., 158, 202. MITCHELL, A. J. & WISLOCKI, G. B. (1944). Selective staining of glycogen by ammoniacal silver nitrate: a new method. Anat. Rec. 90, 261-266. NIcHoLLs, G. E. (1912). The structure and development of Reissner's fibre and the subcommissural organ. Part I. Quart. J. micr. Sci. 58, 1-116. SHAMBAUGH, G. E. (1932). Cytology of the internal ear. In Special Cytology, 3, 1334-1367. Edited by E. V. Cowdry. WISLOCRI, G. B. (1952). The anterior segment of the eye of the rhesus monkey investigated by histochemical means. Amer. J. Anat. 91, 233-262. Selective and histochemical staining 11 WISLOCBI, G. B. & LADMAN, A. J. (1954). Selective staining of the otolithic membranes, cupulae and tectorial membrane of the inner ear. Anat. Rec. (Abstract), 118, 416. WISLOCKI, G. B. & LEDUC, E. H. (1952a). Vital staining of the hematoencephalic barrier by silver nitrate and trypan blue, and cytological comparisons of the neurohypophysis, pineal body, area postrema, intercolumnar tubercle and supraoptic crest. J. comp. Neurol. 96, 371-413. WISLOCKI, G. B. & LEDUC, E. H. (1952b). The cytology and histochemistry of the subcommissural organ and Reissner's fiber in rodents. J. comp. Neurol. 97, 515-544. WISLOCKI, G. B. & LEDUC, E. H. (1954). The cytology of the subcommissural organ, Reissner's fiber, periventricular glial cells and posterior collicular recess of the rat's brain. J. comp. Neurol. (In the Press). WISLOCKI, G. B., BUNTING, H. & DEMPSEY, E. W. (1947). Metachromasia in mammalian tissues and its relationship to mucopolysaccharides. Amer. J. Anat. 81, 1-38.

EXPLANATION OF PLATES PLATE 1 Fig. 1. Low-power photomicrograph of the internal ear of a newborn mouse stained by Gomori's chrome alum-haematoxylin and phloxine method. The three areas delineated by rectangles contain respectively a crista ampullaris, the macula of the saccule and a portion of the developing organ of Corti. In P1. 3, figs. 14, 18 and 19, these areas are shown at higher magnifications. x 40. Fig. 2. A crista ampullaris surmounted by its cupula. Newborn mouse. Chrome alum-haematoxylin and phloxine stain. Compare with P1. 3, fig. 19, of a similar section which shows the selective blue staining of the cupula by the haematoxylin component of the stain. x 200. Fig. 3. The macula of the saccule of a newborn mouse with the otolithic membrane. Chrome alum-haematoxylin and phloxine stain. Observe the darkly stained otolithic membrane and compare it with P1. 3, fig. 14, which illustrates in colour its selective staining. x 200. Fig. 4. The macula of the utricle of a 3i-month-old mouse. Gomori's aldehyde fuchsin stain. Observe the darkly stained otolithic membrane and compare it for detail with a drawing of the same (P1. 3, fig. 15). x 200. Fig. 5. A crista of a 3i-month-old mouse surmounted by its cupula. Periodic acid-Schiff reaction. Observe the intense coloration of the cupula and compare it with a drawing of the same (P1. 3, fig. 17). x 200. Fig. 6. A portion of the cochlea of a newborn mouse showing the developing organ of Corti. Chrome alum-haematoxylin and phloxine stain. Compare with P1. 3, fig. 18, which shows the selective staining of the developing tectorial membrane in relationship to the underlying cells. x 150. PLATE 2 Fig. 7. The organ of Corti of a 3j-month-old mouse with the tectorial membrane. Periodic acid- Schiff reaction. Observe the intense staining of the tectorial membrane. x 200. Fig. 8. The developing organ of Corti of a newborn mouse with the tectorial membrane. Barrnett & Seligman's method for disulphide groups. Observe the moderately deep staining of the tectorial membrane, especially of its outer part. The arrow points to the developing hair cells. x 300. Fig. 9. The developing organ of Corti of a newborn mouse with the tectorial membrane. Weigert's elastic tissue stain. Although appearing relatively dark in the photograph, the staining of the tectorial membrane is atypical, as explained in the text. x 250. Fig. 10. The developing hair cells of the organ of Corti of a newborn mouse. Method for disulphide groups. Note the moderately strong reaction for disulphide groups in the supranuclear cytoplasm of the hair cells. The arrow points to the tectorial membrane which is also moderately stained. x 650. Fig. 11. A drawing of the previous field at a higher magnification, showing stronglydisulphide- positive hairs developing within the cytoplasm of the hair cells. x 900. Fig. 12. The developing organ of Corti and the tectorial membrane of a human foetus (17 cm. crown-rump length). Section stained in a methylene-blue solution at pH 4-3. The nuclei are deeply stained at this pH because of the presence of acidic desoxyribonucleoprotein, whereas, the tectorial membrane is unstained because of its relative lack of acidity. x 300. / 12 George B. Wislocki and Aaron J. Ladman Fig. 13. A portion of a macula of the utricle of a newborn mouse. Method for disulphide groups. Compare the staining of the distal cytoplasm, hairs and flagella of the sensory epithelium and of the otolithic membrane with the stronger reaction of the tectorial membrane (fig. 8). The black appearance of some parts of the basement membrane beneath the epithelium is due to the presence of melanin. x 300. PLATE 3 (Figures 14-19 are drawings prepared with a camera lucida) Fig. 14. A portion of the macula of the saccule of a newborn mouse, illustrating, in colour, the selective staining ofthe otolithic membrane by the alum-haematoxylin component of Gomori's stain. x 450. Fig. 15. A portion of the macula of the utricle of a 3i-month-old mouse showing the selective staining of the otolithic membrane by Gomori's aldehyde fuchsin stain. x 800. Fig. 16. A portion of the macula of the utricle of a 31-month-old mouse, stained by the periodic acid-Schiff method. x 800. Fig. 17. A cupula with part of a crista ampullaris from the previous specimen, stained by the periodic acid-Schiff method. x 450. Fig. 18. The organ of Corti of a newborn mouse illustrating the selective staining of the substance of the tectorial membrane by the alum-haematoxylin component of Gomori's stain. x 300. Fig. 19. A crista ampullaris of a newborn mouse surmounted by its cupula; the latter is stained selectively by the alum-haematoxylin component of Gomori's stain. x 200. Journal of Anatomy, Vol. 89, Part 1 Plate 1

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