Cesar D. Fermin

Developmental Gradients in the Embryonic Chick's Basilar Papilla

The basilar papilla, the avian counterpart to Cortis organ, was examined cytologically in the embryonic chick (Gallus domesticus) from stages 31 through 46 (hatching). Developmental reconstructions were prepared by using light microscopy to analyse serial sections (1-5 microM) along the entire lagenar length and electron microscopy to examine representative thin sections from the proximal, medial and distal regions. We identified two gradients, the lateral and longitudinal. In the longitudinal gradient, the mid-basal basilar papilla, which lies directly above the statoacoustic ganglion, develops before the proximal and distal regions as evidenced by the maturation of hair cells and afferent synapses. Similarly, in the lateral gradient, tall hair cells and afferent synapses develop before short hair cells and their respective afferent terminals. Efferent synaptogenesis follows the same two gradients several days later than the afferents. Afferent synaptogenesis corresponds with the opening of the perilymphatic spaces and completion of the tectorial membrane.

Development of the embryonic chick's tectorial membrane

The nascent tectorial membrane (TM) is identifiable as early as stage 33 (7th day) as thin, wispy material. By stage 37 (11th day), the dense mesh of the immature TM and fibrous webs (subtectorial threads) that attach the TM to the basilar papilla are distinct but scanty. The TM condenses slightly in its upper face. The growth of the columnar cells and basilar papilla during the following days pulls the TM, lifting it upward, and resembling the cables on a suspension bridge in cross-section. As a result, a large hollow wedge forms. During stages 40-44 (14th-18th days), the columnar cells secrete large amounts of fibrous material, which fills the hollow wedge and condenses into the dense meshes. The honeycombed patterns appear at this time. The supporting cells secrete the fibrous webs. Their secretory activity closely corresponds to that of the columnar cells. The secretory material from both cell types remains attached to the apical ends of their respective cells after secretory activity ends. By hatching (stage 46-21 days), the columnar cells have filled with fibrous material and their cytoplasmic organelles are restricted to the apices. The cytoplasm of supporting cells is relatively clear, with few cytoplasmic remnants of their intense secretory activity earlier.

Development of the embryonic chick's statoacoustic ganglion.

During stage 25 (4 1/2 days of incubation), the embryonic chicks statoacoustic ganglion appeared as a homogeneous cell mass. By stage 29 (6 days)- perikarya could be distinguished from Schwann cells because the latter contained more endoplasmic reticulum and ribosome. Granular Schwann cells processes have formed loose boundaries around groups of neurites. During the next 7 days, the Schwann cells clutched the neurites more tightly and divided them into successively smaller bundles. By the 13th day (stage 39) the first compact myelin (2-6 layers) surrounded the larger dendrites, and by the 14th day (stage 40) compact myelin appeared regularly. Until the 17th day (stage 43), the perikaryal were covered by a few layers of loose myelin. Perikaryal compact myelin appeared a day later (stage 44); the larger perikaryl diameter (15-30x) probably accounts for the lag. At hatching (20-21 days), 12-18 myelin layers surrounded many dendrites, though the extent of myelination varied markedly among dendrites in adjacent bundles. By the 2nd week after hatching, large myelinated perikarya outnumbered the smaller and loosely myelinated ones. Perikaryonal myelination required the cooperation of several Schwann cells. The myelin regularly alternated from compact to loose around each perikaryon.

Ultracytochemical Study of Ouabain-sensitive, Potassium-dependent p-Nitrophenylphosphatase Activity in the Inner Ear of the Squirrel Monkey

The localization of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase (K(+)-NPPase) activity of the Na(+),K(+)-ATPase complex was studied ultracytochemically in the squirrel monkey inner ear. In the stria vascularis the reaction products showing K(+)-NPPase activity were limited to the cytoplasmic side of the plasmalemmal infoldings of the marginal cells. In the spiral prominence, a weak reaction was also found on the cytoplasmic process of the stromal cell, while no or little reaction was detected on the spiral prominence epithelium. In the dark cells of vestibular labyrinth the reaction products were observed on the basolateral interdigitation of the plasmalemma. In contrast, no reaction was observed on the apical cell surface. K(+)-NPPase activity was most intense in the strial marginal cell, followed by the dark cell of the ampulla and the utricle. The present results revealed that the dark cells in the vestibular labyrinth are involved in endolymph homeostasis.

Development of otoconia in the embryonic chick (Gallus domesticus).

In the chick (Gallus domesticus) embryo, otoconium formation started first over the macula sacculi around the 4th day of incubation, and a day later over the macula utriculi. It was determined that each otoconium formed as a result of the segmentation of the immature otolithic membrane, and that the calcium responsible for otoconium calcification was incorporated into the organic matrix of each otoconium in the form of small electron-dense granules (20-150 nm in diameter). The presence of calcium in these granules was confirmed by histochemical staining with osmic-potassium pyroantimonate, by EDTA chelation, and by X-ray microanalysis under the electron microscope.

Vestibular Ganglion of the Squirrel Monkey

The vestibular ganglia of adult squirrel monkeys (Saimiri sciureus) were studied with the aid of light and electron microscopy. The vestibular ganglia are formed by small and large neurons (perikarya). The perikaryas outermost surface is surrounded by 2–20 lamellae of compact myelin. The compact myelin sheath varies its thickness around one perikaryon. The sheath generally thins out in the vicinity of the axon hillock. Occasionally, the compact myelin transforms completely into loose myelin for a length of 2–5 μ. Two to eight lamellae of loose myelin usually face the cytoplasm of the neurons. This myelin arrangement is constant in the majority of the cells examined regardless of shape and/or size. Large myelinated perikarya form the majority of the ganglion cells. There are, however, a few neurons that lack a true compact myelin sheath. Others are surrounded by very few lamellae of loose myelin. The general morphology of the neurons was compared to the available literature on other species. Possible variation on the myelination pattern due to fixation differences, and variation on perikaryal size due to the angle of cut and irregular shape of the cells are also discussed.

Ultrastructural changes of statoconia after segmentation of the otolithic membrane

The chick vestibule transformed from a homogeneous epithelial layer at day 2 (stage 15) into a pseudo-stratified epithelial layer at day 4 (stage 24). The apical columnal appearance of sensory cells was evident by day 6 (stage 29). In the supporting cells of the saccule and utricle large rough endoplasmic reticulum cisterns filled with material similar to the primitive organic matrix. Fibrillar material of the otolithic membrane remained attached to the supporting cells and accumulated over the saccule and utricle. The primitive otolithic membrane acquired stress-like lines and statoconial units emerged from the upper surface without a central core. Statoconia thickened at the periphery and a central core formed. Calcium was deposited between the fibrils of older statoconia which were located on top of the segmenting membrane. DIAMOX inhibited statoconia formation and/or prevented calcium and the matrix from associating. Large statoconia (100-200 microns diameter) were formed in embryos injected with this carbonic anhydrase inhibitor. Gel electrophoresis of immature statoconial complexes yielded at least 5 major protein bands between 25 and 210 kDa. Ouabain-sensitive potassium-dependent p-nitrophenylphosphatase activity was demonstrated in the endolymphatic sac of newly hatched chicks.

Aminoglycoside ototoxicity in the chick (Gallus domesticus) inner ear: I. The effects of kanamycin and netilmicin on the basilar papilla†

A single dose (100 mg/kg of egg weight) of kanamycin or netilmicin was injected into the yolk sacs of 7-day-old chick (Gallus domesticus) embryos. Embryos were collected every 24 hours and processed for light and transmission electron microscopy. Morphologic study of the medial basilar papilla disclosed that both kanamycin and netilmicin are toxic to the hair cells in this region. Intoxication was manifested by an increased number of dense osmiophilic bodies, swollen mitochondria, agglomerated chromatin, and occasional disorganization of the kinocilium basal bodies. The cytologic changes observed in the hair cells of embryos injected with netilmicin and kanamycin were similar. However, mitochondrial damage was more severe in the chicks after kanamycin than after netilmicin injection. Some of the cytologic alterations described here are comparable to those already reported for aminoglycoside-intoxicated hair cells in several mammalian species. This study and previous work indicate that the chick embryo provides a satisfactory developmental model for testing ototoxicity of drugs in vivo.

Morphometry and ultrastructure of the squirrel monkey (Saimiri sciureus) vestibular nerve

Vestibular nerves of squirrel monkeys (Saimiri sciureus) embedded in plastics and epoxies were examined with light microscopy (LM) and transmission electron microscopy (TEM), and computerized measures were obtained and analyzed statistically. An average of 12,412 perikarya and 12,005 myelinated nerve fibers was obtained. Approximately 0.7% of the perikarya appeared unmyelinated under LM. About 500 unmyelinated fibers were counted. The cross-sectional area of 1,864 perikarya was 200-650 micron 2. The cross-sectional area of 1,346 nerve fibers was 3-11 micron 2 for the axoplasm and 11-12 micron 2 for the myelin sheath of the same fiber. Myelin thickness was directly proportional to the axoplasm cross-sectional area of the nerve fibers. The cross-sectional area of central axons and peripheral dendrites differed significantly (p less than 0.001). The initial segments of peripheral dendrites were usually smaller, but longer than the initial segments of the central axons. Both initial segments increased in diameter after the first node of Ranvier. Schmidt-Lantermann incisures were more abundant in thick and heavily myelinated fibers than in thin and lightly myelinated fibers. Larger perikarya usually had larger fibers and vice versa, within the first 100-200 micron from the first node of Ranvier. No major ultrastructural differences were found between myelinated and unmyelinated perikarya, except at the hillock region. The Nissl substance was preferentially located in the peripheral cytoplasm.

Dendritic Growth Following Labyrinthectomy in the Squirrel Monkey:Light and Electron Microscopic Studies

In the squirrel monkey (Saimiri sciureus) peripheral vestibular dendrites underwent degeneration after destruction of the vestibular end organs (labyrinthectomy). The dendrites subsequently grew into the remodelled area where the end organs, surrounding tissues and inner ear fluid spaces were previously located, and were progressively occupied by connective tissue, blood vessels and also new bone. This finding indicates that peripheral vestibular dendrites of adult squirrel monkey can grow, that Schwann cells migrate along with these dendrites, and that myelin can be formed in vivo in locations not previously destined for myelination. The importance of these findings in relation to the plastic properties of the squirrel monkey vestibular nerve is discussed.