Hearing Research, 28 (1987) 57-63
Elsevier
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HRR 00929
Acetylcholinesterase activity in the embryonic chick’s 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
Introduction
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 chick’s 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 Hamilton’s (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)
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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.
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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
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.
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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, especially those on the bases of short hair cells.
Cristae ampullares Dense deposits touched the hair cell bases in all sensory areas and were evenly distributed. Type II hair cells, which are directly innervated by the efferent endings, showed de- posits on their bases; Type I hair cells, which have large chaliced afferent terminals covering their bases, showed deposits on the sides.
lkgenar macula Along with increased sizes, the efferent endings were denser than in earlier stages, but their distribution remained the same.
Discussion
Basilar papilla The afferent nerves form synapses several days
before the efferents, as demonstrated by both electron microscopy (Cohen and Fermin, 1978; Fermin and Cohen, 1984a) and Golgi silver stains (Whitehead and Morest, 1985a,b).
On the 11th ED, AChE histochemistry showed that light deposits extended transversely across the basal region of the basilar papilla. However, no AChE reaction product was visualized at the hair cell bases. On the 13th ED, the first efferents appeared, identified by deposits touching the hair cell bases (Fig. 4). Using electron microscopy, Rebillard and Pujol (1983) identified efferent en- dings by the 14th ED ‘, though they were scant in
number. Moreover, both tall and short hair cells displayed efferent endings with all the accompany- ing structural features: presynaptic vesicles, sub- synaptic cisterns, and membrane thickenings. By the 16-17th ED, efferent endings on hair cells were readily visible by both AChE histochemistry (Fig. 6) and by electron microscopy (Fermin and Cohen, 1984a; Rebillard and Pujol, 1983). By the 1819th ED, the large chaliced efferents were conspicuous (Rebillard and Pujol, 1983; Cohen and Fermin, 1978; Fermin and Cohen, 1984a). AChE hi&chemistry reflected the growth of efferent endings, particularly among short hair cells (Figs. 8, 9). Although these semithin (1 pm) sections gave a delicate, localized staining, they did not reveal the differing sizes and shapes of efferent endings on the three hair cell types as effectively as thick (14 pm) sections (Firbas and Mtiller, 1983).
The statoacoustic ganglion (SAG) showed an intense staining among many of its neurons (peri- karyal surfaces), but the staining subsided as hatching neared. Since the perikarya are covered by several layers of myelin by these later stages (Fermin and Cohen, 1984b), the reduced staining might indicate that either the reaction substrates did not penetrate through the myelin sheaths (Iurato et al., 1971, p. 152) or the AChE per- formed different enzymatic functions during the earlier stages (Chubb et al., 1980; Chubb and Millar, 1984; Greenfield, 1984). Light AChE stain- ing also occurred in the SAG of the adult pigeon (Takasaka and Smith, 1971) and budgerigar (Firbas and Mtiller, 1983).
Vestibule The vestibule develops earlier than the auditory
system. Vestibular hair cells and their nerve sup- plies are identifiable before comparable structures in the auditory system (Arey, 1965).
In the chick’s cristae ampullares, afferent syn- apses form as early as 5i ED and fully formed
* It is not known whether strain differences among chickens influence the timing of developmental events. For example, Rebillard and Pujol (1983) used Hubbard x Hubbard; Meza et al. (1984) used Rhode Island Red; Cohen (this paper) used White Leghorn.
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synaptic ribbons begin to appear in hair ceils during 6f ED (Ginzberg and Gilula, 1979) but those in the basilar papilla do not form until the lo-11th ED (Rebillard and Pujol, 1983; Cohen and Fermin, 1978). Not all afferents in the cristae form at the same time. For example, in the cat fully developed afferent chalices are often flanked by nascent ones (Favre and Sans, 1979). A similar pattern of staggered development occurs in the embryonic chick’s efferent endings. Meza and Hinojosa (1987) showed by electron microscopy that the first efferent endings form in the cristae ampullares as early as the 10th ED, but in very small numbers. Their numbers increased rapidly during development. AChE histochemistry closely corresponded with the timing and pattern of their findings: AChE was evident on the 11th ED (Fig. l), the earliest stage described in the present study, and steadily increased during development (Fig. 5). Choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine, was detectable with biochemical techniques by the 18th ED (Meza and Hinojosa, 1987).
The lagenar macula, located at the distal end of the lagena, shares many structural features of the otolithic organs, including hair cell types and pat- terns of afferent and efferent innervation (Jorgen- sen, 1970). It might also serve an auditory func- tion (Boord and Rasmussen, 1963). On the 11th ED, when this study began, the macula already displayed a small number of efferent endings touching the hair cell bases (Fig. 2). By the 13th ED, the number of hair cells showing efferent endings noticeably increased (Fig. 3), and con- tinued at an accelerating pace throughout develop- ment (Fig. 7).
The early appearance of the AChE reaction product in both the cristae and macula suggests that cristal and lagenar macular efferents form earlier than those in the basilar papilla. The effer- ent endings showed an uneven distribution in the cristae and lagenar macula from the 11-14th EDs, which probably indicated regional differences in maturity, but might also represent artifacts of the technique.
A dense AChE reaction product appeared ap- proximately midway within the sensory epithelia of both the vestibule and basilar papilla prior to the formation of efferent endings. The reaction
product dissipated and eventually disappeared as
the efferents formed in large numbers, suggesting that it represented tangles of efferent fibers and/or terminal fields.
Onset of function Nascent afferent terminals and hair cells in the
cochlea (Rubel, 1984) and vestibule (Romand and Dupres, 1987) gradually acquire function. Audi- tory and vestibular thresholds, which are initially high, decline during development, reflecting a combination of maturational events (Rebillard and Rubel, 1981; Rubel, 1984; Romand and Dupres, 1987). The cholinergic efferents of both the audi- tory and vestibular systems presumably acquire function in a similarly gradual manner. However, unlike the afferents, the cholinergic efferents con- tain known enzyme markers for demonstrating biochemical correlates of function. For example, functional efferent cholinergic endings in the inner ear must contain ChAT to synthesize the neuro- transmitter, as occurs in the ACh-containing syn- apses in the central nervous system (Hebb and Whittaker, 1958; Hebb, 1963). These cholinergic endings must also contain AChE, so that ACh can be degraded (inactivated) upon its release. During development, levels of both AChE and ChAT correspond with the growth and differentiation of efferent fibers in the chick’s vestibule (Meza and Hinojosa, 1987; and Figs. 1-3, 5, 7) and along the turns of the mouse’s cochlea (Emmerling and Sobkowicz, 1986; Sobkowicz et al., 1986). Thus, the coexistence of ChAT and AChE in efferent endings provides evidence that functional activity is close at hand.
Acknowledgements
I would like to thank Mr. Joseph S. Grass0 for his technical help. This research was supported by institutional funding.
References
Arey, L.B. (1965) Developmental Anatomy, 7th edn, pp. 541-546. W.B. Saunders Co., Philadelphia.
Boord, R.L. and Rasmussen, G.L. (1963) FYojection of the co&ear and lagenar nerves on the cochlear nuclei of the pigeon. J. Comp. Neurol. 120, 463-471.
63
Chubb, I.W. and Millar, T.J. (1984) Is intracellular
acetylcholinesterase involved in the processing of peptide
neurotransmitters? Clin. Exp. Hypertens. 6, 79-89.
Chubb, I.W., Hodgson, A.J. and White, G.H. (1980)
Acetylcholinesterase hydrolyzes substance P. Neuroscience
5, 20652072.
Cohen, G.M. and Fermin, C.D. (1978) Development of hair
cells in the embryonic chick’s basilar papilla. Acta Oto-
Laryngol. 86, 342-358.
Emmerling, M.R. and Sobkowicz, H.M. (1986) Differential
distribution of choline& enzymes in the developing mouse
cochlea. Sot. Neurosci. Abstr. 12, 1048.
Estable-Puig, J.F., Bauer, W.C. and Blumberg, J.M. (1965)
Paraphenylenediamine staining of osmium-fixed, plastic
embedded tissue for light and phase microscopy. J. Neuro-
pathol. Exp. Neurol. 24, 531-535.
Favre, D. and Sam, A. (1979) Embryonic and postnatal devel-
opment of afferent innervation in cat vestibular receptors.
Acta Oto-Laryngol. 87, 97-107.
Fermin, CD. and Cohen, G.M. (1984a) Developmental gradi-
ents in the embryonic chick’s basilar papilla. Acta Oto-
Laryngol. 97, 39-51.
Fermin, CD. and Cohen, G.M. (198413) Development of the
embryonic chick’s statoacoustic ganglion. Acta Oto-
Laryngol. 98,42-52.
Firbas, W. and Miiller, G. (1983) The efferent innervation of
the avian cochlea. Hear. Res. 10, 109-116.
Gacek, R.R., Nomura, Y. and Balogh, K. (1965)
Acetylcholinesterase activity in efferent fibers of the stato-
acoustic nerve. Acta Oto-Laryngol. 59, 541-553.
Ginzberg, R.D. and Gilula, N.B. (1979) Modulation of cell
junctions during differentiation of the chicken otocyst
sensory epithelium. Dev. Biol. 68, 110-129.
Greenfield, S. (1984) Acetylcholinesterase may have novel
functions in the brain. Trends Neurosci. 7, 364-368.
Hamburger, V. and Hamilton, H.L. (1951) A series of normal
stages in the development of the chick embryo. J. Morphol.
88, 49-92.
Hebb, C.O. (1963) Formation, storage and liberation of
acetylcholine. Handb. Exp. Pharmacol. 15, 55-88.
Hebb, C.O. and Whittaker, V.P. (1958) Intracellular distribu-
tion of acetylcholine and choline acetylase. J. Physiol.
(London) 192, 187-196.
Iurato, S.. Luciano, L., Panuese, E. and Reale, E. (1971)
Acetylcholinesterase activity in the vestibular sensory areas. Acta Oto-Laryngol. 71, 147-152.
Jorgensen, J.M. (1970) On the structure of the macula lagenae
in birds with some notes on the avian maculae utriculi and
sacculi. Vidensk. Meddr Dan. Naturhis. Foren. Khoben-
havn 133, 121-147.
Kamovsky, M.J. and Roots, L. (1964) A ‘direct-colouring’
thiocholine method for cholinesterases. J. Histochem. Cyto-
them. 12, 219.
Meza, G. and Hinojosa, R. (1987) Ontogenetic approach to the
cellular localization of neurotransmitters in the chick
vestibule. Hear. Res. 28, 73-85.
Meza, G., Lopez, I. and Ruiz, M. (1984) Possible cholinergic
neurotransmission in the cristae ampullares in the chick
inner ear. Neurosci. Lett. 49, 93-98.
Otto, J.V., Park, J.C., Fermin, C.D. and Cohen, G.M. (1984) A
new method for improved fixation of the chick’s inner ear.
Fla. Sci. 47, 251-256.
Rebillard, M. and Pujol, R. (1983) Innervation of the chicken
basilar papilla during its development. Acta Oto-Laryngol.
96, 379-388.
Rebillard, G. and Rubel, E.W. (1981) Electrophysiological
study of the maturation of auditory brain responses from
the inner ear of the chick. Brain Res. 229, 15-23.
Romand, R., Dupres, G. and Giry, N. (1987) Factors affecting
the onset of inner ear function. Hear. Res. 28, l-7.
Rubel, E.W. (1984) Ontogeny of auditory system function.
Annu. Rev. Physiol. 46, 213-229.
Smith, C.A. (1973) The efferent neural supply to the vertebrate
ear. Adv. Oto-Rhino-Laryngol. 20, 296-310. Sobkowicz, H.M., Emmerling, M.R. and Scott, G.L. (1986)
Biochemical and morphological differentiation of efferent
fibers in the mouse cochlea. Sot. Neurosci. Abstr. 12, 1048.
Strutz, J. and Schmidt, C.L. (1982) Acoustic and vestibular
efferent neurons in the chicken (Gal/us domesricur). A
horseradish peroxidase study. Acta Oto-Laryngol. 94.
45-51.
Takasaka, T. and Smith, C.A. (1971) The structure and
innervation of the pigeon’s basilar papilla. J. Ultrastruct.
Res. 35, 20-65.
Tanaka, K. and Smith, C.A. (1978) Structure of the chicken’s
inner ear: SEM and TEM study. Am. J. Anat. 153.251-272.
Whitehead, M.C. and Morest, D.K. (1985a) The development
of innervation patterns in the avian cochlea. Neuroscience 14, 255-276.
Whitehead, M.C. and Morest, D.K. (1985b) The growth of
cochlear fibers and the formation of their synaptic endings
in the avian inner ear: A study with the electron micro-
scope. Neuroscience 14, 277-300.
