Acetylcholinesterase activity in the embryonic chick's inner ear
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Hearing Research, 28 (1987) 57-63
Acetylcholinesterase activity in the embryonic chicks inner ear
Glenn M. Cohen Dept. of Biological Sciences, Florida Institute of Technology, Melbourne, U.S.A.
Putative cholinergic efferent endings, as demonstrated by the presence of a localized acetylcholinesterase reaction product and visualized by phase-contrast microscopy, appeared in the lagenar macula and cristae ampullares on the 1 Ith embryonic day (ED) and in the basilar papilla by the 13th ED. During the course of development, the reaction product became denser and more sharply defined, reflecting the maturation of efferent endings.
Acetylcholinesterase; Basilar papilla; Crista ampullaris; Efferent terminal; Lagenar macula; Developing inner ear
Acetylcholinesterase (AChE) histochemistry, electron microscopy, and various tracing methods have identified some cochlear and vestibular effer- ent nerves in mammals and birds as being of putative cholinergic nature (Gacek et al., 1965; Takasaka and Smith, 1971; Smith, 1973; Tanaka and Smith, 1978; Firbas and Miiller, 1983).
In the present study, the developmental ap- pearance of AChE, as evidenced by AChE
histochemistry (Karnovsky and Roots, 1964) and visualized under a phase-contrast microscope, was
used to demonstrate cholinergic efferent endings in the embryonic chicks basilar papilla (cochlea) and vestibule.
Materials and Methods
Fertilized White Leghorn eggs were obtained from Bronson Farms, Sorento, FL. They were incubated at 37-38.5 o C and sacrificed from stages 39-46 (embryonic days [ED] 11-21) using Ham- burger and Hamiltons (1951) staging scheme.
Correspondence to: G.M. Cohen, Department of Biological Sciences, Florida Institute of Technology, 150 W. University Blvd., Melbourne, FL 32901-6988, U.S.A.
Three to five embryos were used for each stage;
two were used for stage 37. The inner ears were preserved using a technique
described elsewhere (Otto et al., 1984). The fixa- tive contained 3.5% glutaraldehyde, 0.1 M
cacodylate buffer, 5 mM Ca2+, at pH 7.0. The inner ears were exposed to the fixative for 4 h. The
basilar papillae and vestibules (lagenar maculae and semicircular canal endorgans) were then dis- sected out. To facilitate penetration of the reac- tion medium, the lagenae were opened along the length of the tegmentum vasculosum; the ampullary roofs were opened to expose the end-
organs. The specimens were rinsed for 4-16 h in 0.1 M cacodylate buffer, and then rinsed in 0.1 M maleic acid buffer, pH 6.0, immediately before
incubation. Kamovsky and Roots (1964) procedure was
used at room temperature (23-25 C) for 2 h. In each experimental run, specimens of different stages were incubated from aliquots of the same medium. The specimens were kept in separate capped vials, covered with aluminum foil, and
rotated to ensure sufficient agitation. Control ears were soaked in buffer solution containing eserine for 30 min prior to placement in the reaction medium containing eserine (3.5 x 10m3 M).
After the incubation, the specimens were rinsed in 0.1 M maleic acid buffer, transferred to the
0378-5955/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)
Figs. 1-9. Reaction product (deposits) indicates AChE activity. Arrowheads point to putative efferent endings touching hair cells. The unstained sections (1-2 pm) were photographed using phase microscopy.
Fig. 1. Crista ampu11aris. A small number of putative efferent synapses touch the hair cell bases. Dense deposits, possibly representing a tangle of efferent fibers and/or terminal fields, extend across the middle of the sensory epithelium. These deposits end
abruptly at the boundary of the sensory epithelium. 11th ED. x 850.
Fig. 2. (A) Lagenar macula. Deposits form a line that extends across the sensory epithelium. 11th ED. x 340. (B) Lagenar macula, higher magnification. Putative efferent synapses touch hair ceils. The densest deposits he midway within the epithelium and extend
across it. X 850.
Fig. 3. Lagenar macula. Putative efferent endings, though still infrequent, have increased in number. Dense deposits are clustered midway within the sensory epithelium. 13th ED. x 340.
Fig. 4. Basilar papilla. A few putative efferent endings have formed. Dense deposits are concentrated midway and extend across the width of the basilar papilla. 13th ED. x 340.
Fig. 5. Crista ampullaris. Efferent endings on hair cells, as judged by AChE reaction product, are more frequent. Deposits are denser than at earlier stages. 14-15th ED. x 340.
Fig. 6. Basilar papilla. Efferent endings touch all hair cells. It was not determined whether some hair cells were multiply innervated by efferents or whether a single synapse showed multiple sites of activity. Intermediate hair cells are located on the right and short hair cells on the left. (Reaction medium damaged some specimens, particularly the expanded afferent terminals.) 16-17th ED. X 850.
Fig. 7. Lagenar macula. There are numerous contacts with hair cells and probably some on afferent terminals. Deposits are dense. 16-17th ED. A, x 340; B, x 850.
Fig. 8. Basilar papilla. Efferent endings touch all hair cells. Deposits are dense. Short hair cells are located on right. 17th ED. X 850.
Fig. 9. Basilar papilla. The efferent endings resemble their mature form. In short hair cells (right), the larger efferent endings are now more obvious. 18-19th ED. X 850.
cacodylate buffer, and then postfixed in 1% osmium tetroxide, buffered in 0.1 M cacodylate (pH 7.0). The specimens were embedded in Aral- dite 502. Some sernithin sections (1-2 pm) were stained with either paraphenylenediamine (Esta- ble-Puig et al., 1965) or toluidine blue. To visual- ize the AChE reaction product, unstained sections were viewed and photographed under a phase- contrast microscope.
Results at each stage represent observations from several specimens. The controls showed no reaction.
Stage 37 (I1 ED)
Basilar papilla No efferent endings were identi- fied. A light reaction product zig-zagged above the basilar membrane at the bases of the supporting cells. In another specimen, the reaction product, which was localized midway within the epi- thelium, extended across the width of the basilar papilla.
Cristae ampullares The identity of a few putative efferent endings was inferred by the presence of a light reaction product that touched the hair cell bases. These were rare (Fig. 1).
Lugenar macula Though rare, a few putative efferent endings were identified in some hair cell areas, but not in others (Fig. 2B). A light reaction product, possibly representing a tangle of fibers and/or terminal fields, was local&d midway within the sensory epithelium (Fig. 2A). It ended abruptly at the boundary of the sensory epi- thelium.
Stage 39 (13 ED)
Basilar papilla Reaction product, possibly repre- senting efferent endings, was identified on the hair cell bases, but was infrequent (Fig. 4). Dense deposits of reaction product formed midway within the epithelium of the basilar papilla. The hyaline cells, which abut the inferior papillary edge next to the last short hair cells, showed a strong reac-
tion. The latter was a constant feature through hatching. Heavy, distinct deposits formed on the perikaryal surfaces of the ganglionic neurons, but it was not determined whether the reaction prod- uct originated in the perikarya or satellite cells.
Cristae ampullares Efferent endings were identi- fied more frequently. Putative efferent endings also appeared beneath and around presumptive afferent terminals. Although there were no overt developmental differences among the canal end- organs, the deposition was uneven within a single endorgan, with some sensory epithelial areas showing more efferent endings than others.
Lugenar macula Some efferent endings touched hair cell bases and others the presumptive afferent endings. However, some hair cell areas showed no deposits. Dense deposits appeared midway within the sensory epithelium (Fig. 3).
Stages 40-41 (14-15 ED)
Basilar papilla The number of efferent endings increased. Deposits appeared midway within the epithelium of the basilar papilla. In the statoacoustic ganglion, heavy deposits were in- vested between the perikarya and satellite cells, but the neuronal cytoplasm was clear.
Cristae ampullares More efferent endings were identified than at earlier stages (Fig. 5). Some deposits surrounded afferent terminals on Type I hair cells.
hzgenar macula The number of hair cells show- ing deposits at their basal ends increased. Distinct, well-defined deposits were insinuated beneath and to the sides of many afferent fibers and terminals.
Stages 42-43 (16-17 ED)
Basilar papilla Distinctive efferent endings were visible throughout the basilar papilla (Figs. 6, 8). Deposits covered the statoacoustic ganglion.
Cristae ampullares All hair cells showed deposits beneath them, but those on Type I hair cells were apparently transient.
Lugenar macula Almost all hair cells showed de- posits at their bases (Fig. 7A, B), though a few scattered hair cells had none. Some fibers in the macular nerve bundle also showed discrete de- posits.
Stages 44-4.5 (18-19 ED)
Basilar papilla Efferent endings touched the bases of all hair cells (Fig. 9). Ganglionic deposits were reduced and many neurons showed no reac- tion.
Stages 45-46 (19-20 ED)
Basilar papilla Efferent endings formed on the sides and bases of hair cells. The efferent endings enlarged from earlier stages, especi