Hearing Research 75 (1994) 61-M

AChE-staining of type II ganglion cells, processes and terminals in the cochlea of the mustached bat

D.H. Xie, M.M. Henson *, O.W. Henson, Jr

(Received 24 June 1993; Revision received 18 October 190.7: Accepted 20 December 199.3)

Abstract

Thcrc have been a number of reports showing that ganglion cells of sensory neurons may be stained by traditional acetylcholincsterase (AChE) histochemical techniques commonly used to demonstrate efferent nerve fibers and terminals. AChE-staining has been described for cell bodies in the vestibular and spiral ganglia; staining of peripheral and central processes. howcvcr, is rare and the presence of reaction product in afferent terminals has not been reported. The outer hair cells of mustached bats, Pteronotzcs purnellii, differ from those of most mammals in that they typically have a single, large efferent terminal surrounded by S-7 small, afferent terminals. In this animal an AChE-positive reaction was found not only in effcrcnt fibers and terminals but also in type II ganglion cells, their peripheral and central processes and in outer hair cell terminals. The stained cell bodies were smaller than the unstained type I ganglion cells and they were much fewer in number. The proccsscs of the stained cells could be followed from the soma. The central processes were dispersed throughout the VIIIth nerve trunk. Stained peripheral processes were evident in the osseous spiral lamina, floor of the tunnel of Corti and first space of Nuel and in the outer spiral plexus along the sides of the outer phalangcal (Deiters’) cells. AChE-stained affcrent terminals were easy to identify after transection of the crossed olivocochlear bundle (COCB) and subsequent degeneration of large cffcrent terminals. These results are of interest in that assessments of effcrcnt nerve histochemistry after COCB transection riced to recognize the potential contribution of AChE reaction product in afferent terminals. The functional significance of AChE-positive sensory nerves is not known: the positive reaction of the type II neurons does not mean that they are cholinergic.

Key w~ords: Mustached bat; Type II ganglion cells; Acetylcholinesterase; AChE

1. Introduction

There have been many reports showing that gan- glion cells of sensory neurons are sometimes stained by traditional AChE histochemical techniques (see Cauna and Naik, 1963; Palouzier et al., 1987; Malatov6 et al., 1985); this has also been noted for spiral ganglion cells in a number of mammals, including man, cat, rat, bat, guinea pig (Cauna and Naik, 1963; Firbas, 1972, Ishii et al., 1967; lurato et al., 1974, 1975). In a few cases fibers along the floor of the tunnel of Corti, outer spiral fibers or fibers in the core of the cochlear nerve have been stained (Stopp and Comis, 1979). The usual interpretation is that these fibers are cholinergic effer- ents or they have been classified as fibers of unknown origin.

* Corresponding author. Fax: (9 19) 9h6- 1 X56.

O.i7X-5YS5/Y4/$07.0~ CT) 1994 Elsevier Science B.V. All rights reserved

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In our laboratory we routinely stain surface prepara- tions of the cochlea to study efferent fibers and termi- nals and to assess the contribution of crossed vs. un- crossed components after transection of the crossed olivocochlear bundle (COCB). In these preparations we noticed an unusual distribution pattern of AChE- staining in the organ of Corti. The purpose of this report is to illustrate that AChE-staining in the cochlea

may not only be characteristic of the OHC efferent terminals but also may bc evident in type II ganglion cells, their central and peripheral processes and even the terminals associated with outer hair cells.

2. Methods

The animals used in this study were mustached bats, Pteronotus parnellii parnellii, from Jamaica, W.I. Most of the animals had been used in neurophysiological

and behavioral studies on the efferent auditory system. For tissue fixation, animals were deeply anesthetized with methoxyflurane (Metofane, Pitman-Moore, Inc.) and killed by decapitation; the heads were cut in the midsagittal plane, the cochleae rapidly removed and washed in 0.9%) NaCl. The stapes and membrane cov- ering the round window were removed and the tissue was placed in cold (4°C) fixative for 8-12 hours. The latter consisted of 4% paraformaldehyde, 0.5% glu- taraldehyde, and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4. After fixation, cochleae were decalci- fied in 0.1 M EDTA in 0.1 M phosphate buffer, pH 7.4 for four days in the cold. The EDTA solution was changed each day. The cochleae were then cut into four segments (hook, basal turn, second turn and apex). stained for AChE and viewed as surface preparations.

All steps in the staining procedure were carried out at room temperature and followed the method of Tago

Fig. 2. Drawing of surface preparation of cochlear nerve trunk and

spiral ganglion (near beginning of second turn). The AChE-stained

(black), type II ganglion cells are within the osseous spiral lamina;

their central processes (0 spiral within the core of the VIIIth nerve

as they ascend toward the cochlear nucleus. The peripheral pro-

cesses (PI accompany unstained type I processes toward the organ of

Corti within radially oriented bundles (arrowheads). Outlines of the

more numerous, large, unstained type I ganglion cells (arrows) are

shown. Scale bar = 20 pm.

Fig. I. Photomicrograph of a surface preparation of a decalcified

segment of the osseous spiral lamina in the basal turn of the cochlea.

The small. round AChEstained cells (clear arrowheads) represent a

small. but distinct population of spiral ganglion cells. The fine

central (c) and peripheral (p) processes of some cells are in focus.

The larger, unstained ganglion cells are not evident (see Fig. 2). The stained bundles running through or spiraling within the ganglion

represent efferent fibers (black arrowheads). Scale bar = 20 pm.

et al. (1986). Tissues were first washed in 0.1 M phos- phate buffer three times, five minutes each. Next, they were treated with 0.1% HzO, for 30 minutes to de- stroy endogenous peroxidase and reduce background staining. This was followed by washing in 0.1 M phos- phate buffer, three times, five minutes each, and then treating with 1% Triton X 100 for one hour. After another wash in phosphate buffer (three times, 5 min- utes each) the tissue was incubated for 30 minutes in a solution containing 35 FM acetylthiocholine iodide, 5 PM K,Fe(CN),, 20 PM CuSO,, and 50 PM sodium citrate in 0.1 M maleate buffer, pH 6.0. FolIowing another series of washes, with five changes of 50 mM Tris-HCl, pH 8.2, five minutes each, the tissue was incubated for five minutes in a solution containing 0.03% 3’3’ diaminobenzidine and 0.3% nickel ammo- nium sulfate in 50 mM Tris-HCl, pH 8.2. The tissue was then incubated in the same solution with 0.003%

H,O, for 10 minutes. Finally, the tissue was washed with three changes of 5 mM Tris-HCI, pH 8.2, five minutes each and then dehydrated with a graded ethanol series, cleared with xylene, and coverslipped with DPX. Consistent reactions were obtained when the solutions containing the tissue were constantly, vigorously agitated.

To establish that AChE-staining in the OHC region was not restricted to the single large efferent terminals, the crossed olivocochlear bundle (COCB) was sec- tioned in the floor of the fourth ventricle. This was accomplished by stereotaxically placing a tungsten elec- trode through a small hole in the dorsal surface of the skull. The electrode was lowered to the predetermined level of the COCB and then the animal’s head was moved anteriorly and posteriorly over a distance of a few mm. This produced a fine midline lesion which could easily be seen in frozen sections. The COCB was transected in seven cases and survival times after tran- section ranged from five days (one animal) to 2-3

weeks (six animals). In all cases the success of the transections was also evaluated by examining surface preparations of the cochlear segments. In the lesioned animals there was an obvious reduction in the number of efferent fibers and their terminals on OHCs. Where the efferent terminals degenerated the afferent termi- nals could be easily visualized.

Staining in the absence of substrate was used as a control. We did not use any of the highly toxic chemi- cals that would have allowed us to distin~Llish AChE from other esterases.

The results of this study are based on observations on 11 animals (22 cochleae) where the AChE-staining was of good quality for the large efferent terminals at the bases of the outer hair cells. For the type II ganglion cells the staining was classified as ‘intense’ in four cochleae (e.g. as shown in Fig. 1) and ‘good’ in the remaining 18.

The care and use of the animals reported on in this study were approved by the IACUC (Institutional Ani-

Fig. 3. The peripheral processes of type II ganglion cells within the organ of Corti. In this specimen the efferent terminals have degenerated. The

fine AC’hE-stained processes <clear arrows) run basally on the inner surface of unstained outer phalangeal ceils (of Deiters). Branches (arrows)

arise from these spiraling fibers to course toward OHCs where terminals CT) are evident. Each branch arises from a dilated segment of the nerve

fiber. The regions marked T consists of S-7 indjvidual afferent fibers and thus do not represent the size OF a single ending. Scale bar = IO ,um.

ma1 Care and Use Committee) at The University of North Carolina at Chapel Hill, Animal Assurance Number A3410-01.

3. Results

When surface preparations of the AChE-stained material were examined, it was evident that a small population of the ganglion cells was selectively stained (Figs. 1 and 2). Th e stained cells were smaller than adjacent unstained ganglion cells and the processes of these small cells could be traced centrally and periph- erally. The central processes spiraled within the trunk of the VIIIth nerve and could be followed into the cochlear nucleus where they were obscured by a dark AChE-positive region. In the osseous spiral lamina there was a striking contrast between large, darkly stained efferent nerve bundles and the fine peripheral processes of the stained ganglion cells (Fig. 1). The small peripheral processes could be traced through the osseous spiral lamina to the habenula perforata. The fibers were faintly stained in the floor of the tunnel of Corti and first space of Nuel. In the organ of Corti

AChE-positive fibers were especially cvldent where they contributed to the outer spiral plexus on the modiolar side of the three rows of the outer phalangeal (Deiters’) cells (Fig. 3). In the outer spiral plexus the branches arose from local dilatations that occurred at regular intervals along the spiral fibers. As the branches ascended toward the outer hair cells they joined other fibers, often forming a very distinct arching bundle that projected toward the hair cell base. Prominent ‘cn passant’ swellings and terminals were evident. The terminals were most clearly seen in the organ of Corti after the COCB had been transected and some of the large AChE-positive efferent terminals had degener- ated (Figs. 3 and 4). The individual afferent terminals were small ( < 2 pm) but since there were often several close together, the reaction product appeared much larger than the normal size of a single ending.

4. Discussion

In this report we have interpreted the AChE-stained ganglion cells and processes as type II afferents. This is justified on the basis of their population size, soma

Fig. ‘ I. AChE staining of type 11 terminals at the base of outer hair cells (row three) in an animal in which the COCB was transected three weeks

prior to being sacrificed. Some of the large, single, normal (uncrossed) efferent endings are marked by an E; the dark staining structures in the

foreg round are out-of-focus efferent terminals of rows one and two. Where the OHC efferent terminals have degenerated the reaction product is

small and represents the terminals of the type II ganglion cells (clear arrows). Some AChE-positive outer spiral fibers (black arrows) are also

evide nt in this micrograph and an individual branch can be followed to a terminal (topmost arrow). Scale bar = 5 pm.

size, course, terminal size and distribution. The cell bodies that were AChE-positive in the mustached bat were typically smaller than the other ganglion cells and they were much fewer in number (Figs. 1 and 21. In all mammals studied to date type II ganglion cells consti- tute only a small percentage (5-U%) of the total population of ganglion cells and they are usually smaller in size (see Kiang et al., 1982; Spoendlin, 1981: Schwartz, 1986; Berglund and Ryugo, 1986). In addi- tion, the peripheral processes of the type I1 ganglion cells are distinctive in that they travel basally on the floor of the tunnel of Corti and first space of Nuel; they eventually form the outer spiral plexus on the inner surface of the phalangeal cells. This is exactly the course observed for the AChE-stained fibers in the mustached bat. The peripheral processes of type I ganglion cells, by contrast, terminate on inner hair cells and thus, they do not enter the tunnel of Corti or contribute to the outer spiral plexus (Spoendlin 1979; Kiang et al., 1982). Previous studies on the OHC efferent endings in the mustached bat have shown that efferent terminals are characteristically much larger than the afferent terminals (Fig. 41 (Bishop and Hen- son, lY88; Xie at al., 1993). The fact that the spiral fibers did not degenerate after COCB transection pro- vides an additional indication that the AChE-positive fibers were not olivocochlear efferents. In addition, we have studied the course and distribution of efferent fibers by the iontophoretic injection of Phaseohds WI-

gmis leucoagglutinin (PHA-Ll into the regions contain- ing efferent neurons in the brainstem of mice and bats (Wilson et al., lYY1; unpublished data); in no instance did any of more than 300 labeled medial efferent fibers reach the OHCs by traveling in the outer spiral plexus. Thus, we are confident in concluding that the labeled fibers were afferent and not efferent.

The AChEstaining of spiral ganglion cells has been noted by a number of investigators in a wide range of mammals (Firbas, 1972; Ishii et al., 1967; Iurato et al., 1974, 1975; Stopp and Comis, 1979). The variability of AChEstaining in the ganglion cells and their processes in different mammals has been a source of confusion (see Iurato et al., 1974) and in our opinion none of the hypotheses that have been advanced seem to account for the variations in staining in different species. One of Iurato’s suggestions for differences in AChE-stain- ing among mammals was the degree of penetration of the soma by reacting substrates; those ganglion cells with myelinated sheaths (type I) would not be as read- ily penetrated and thus would show little or no reaction product. If this were the case, it would be expected that the unmyelinated, type II cell bodies and their unmyelinated processes might be well-stained in most mammals, whereas the myelinated type I ganglion cells would not. This is clearly not the case; in published micrographs of the cochlea of other mammals labeling

of most spiral ganglion cells is evident. In addition, it should be noted that we have used the AChE-staining procedure on other bats (Epresicus ,ftr.scusf and other mammals (rats and gerbils, unpublished data) and there was no selective labeling of the type 11 cells and pro- cesses as we have described for the mustached bat.

The presence of AChEpositive processes in the core of the VIIIth nerve and the continuity of these processes with the stained ganglion ceils (Fig. 2) is interesting. Hozawa and Kimura (1YYO) reported that type II neurons demonstrate AChE activity; on this basis they suggested that the AChE-positive fibers in the simian VIIIth nerve trunk, previously believed to be of efferent origin, are the central processes of type II ganglion cells. Our data show direct continuity of the cells and processes (Fig. 2) and support this conclusion, but we also know from PHA-L, studies (Wilson et al.. 1091) that some efferent fibers reach the cochlea by traveling in the core of the cochlear nerve and these should be AChE-positive.

In our preparations we were also able to follow the fine AChE-positive fibers from the ganglion cells to the periphery. Stopp and Comis (1979) reported AChE- positive fibers traveling along the floor of the tunnel of Corti in the guinea pig and they observed that scction- ing the crossed and uncrossed efferent fibers did not cause them to degenerate; they suggested that the fibers in the floor of the tunnel might bc cholincrgic afferents. Although they considered this an ‘unusual c~~nlbination’, it is now clear that AChE-p~~sitivc fibers are present in other sensory neural systems. such as the retina (Appleyard, 1992; Brandon, 1987; Cauna and Naik, 1963: Hutchins and Hollyfield, 19X7; Pourcho and Osman, 1986). Although Ishii et al. (1967) sug- gested that this enzyme may participate in the conduc- tion of impulses. its function in sensory and neural systems remains highly speculative. In addition to its well known roles in neural functions, AChE has the capacity to affect certain membrane conductances, to enhance excitatory amino acid transmission and hydro- lyze peptides (Appleyard, 19921. It should also be noted that AChE should be active in cells that synapse with cholinergic neurons and studies indicate that there arc synapses between cholinergic efferent fibers and type II ganglion cells and their processes (Hozawa and Kimura, lY90).

Although we, and other investig~~t~~rs. have referred to the reaction product in ganglion cells and proccsscs as AChE-positive, very specific criteria arc needed to establish that the stained fibers contain acetylcholine and that they arc in fact part of a cholincrgic system, i.e. one that uses acetylcholine as a neurotransmittcr. In the past ten years, immun~~cytochcmical studies with choline acetyltransferase (CHAT), the ACh synthesiz- ing enzyme, have been combined with AChE studios, but this also has produced results that are difficult to

interpret. Cell bodies and processes in other sensory ganglia, such as those of the spinal nerve (see Malatovi et al., 1985), vagus nerve (Palouzier et al., 1987) and retina (Pourcho and Osman, 1986) show positive AChE and ChAT reaction products but there is considerable interspecies variation just as there is in the spiral ganglion. It is of further interest that neurophysiologi- cal and neuropharmacological studies have failed to prove a cholinergic or cholinoreceptive function of primary afferent terminals although ACh is clearly present in many ganglion cells (Nishi, 1974; Karczmar et al., 1980). A general emerging theme is that neither ACh or ChAT alone in normal adult animals have important roles in sensory ganglion cells and their processes, but under certain conditions, e.g.. lesions and regeneration or during development, these en- zymes may play a significant, yet unknown function (MalatovB et al., 1985; Pourcho and Osman, 1986). Judging from the intensity of the ganglion cell staining in our preparations, in both normal and lesioned ani- mals, there is no evidence that the lesions placed in the brain stem had any effect on the AChE-positive reac- tion in the type II fibers.

Acknowledgements

This work was supported by NIH grant NIDCD DC001 14.

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