Proc. NatL Acad. Sci. USA Vol. 78, No. 2, pp. 1255-1259, February 1981 Neurobiology

Enkephalin-like immunoreactivity of olivocochlear nerve fibers in ofguinea pig and cat (opioid/cholinergic neurons/organ ofCorti/immunofluorescence/efferent ) JORGEN FEX AND RICHARD A. ALTSCHULER Laboratory ofNeuro-otolaryngology, National Institute ofNeurological and Communicative Disorders and Stroke, National Institutes ofHealth, Bethesda, Maryland 20205 Communicated by William D. Neff, October 17, 1980

ABSTRACT The distribution ofenkephalin-like immunoreac- ents may cross at the tunnel floor (17). Large synaptic terminals tivity in the cochlea ofthe guinea pig and cat was studied. Indirect on outer hair cells are endings ofupper tunnel crossing fibers (9, immunofluorescence immunohistochemistry using antisera gen- 12, 16) and are efferent (18, 19). Smaller efferent endings form erated against a methionine enkephalin-bovine thyroglobulin con- jugate was applied to surface preparations of the synapses with auditory nerve dendrites at the outer and cryostat sections of the whole of the cochlea. In the cochlear bases (20, 21). The efferent innervation is maximal in the first osseous spiral lamina, immunofluorescence was localized to un- and second turns ofthe cochleaand diminishes towards the apex myelinated fibers ofthe intraganglionic spiral bundle. In the organ (22, 23). The large efferent endings on outer hair cells are totally of Corti, immunofluorescence was localized to a small number of (22) or subtotally (23) absent in the most apical part ofthe organ fibers at inner hair cells, the inner spiral bundle, and tunnel spiral ofCorti. bundle, to tunnel crossing fibers at the level ofthe tunnel floor, to an occasional spiral outer fiber, and to the synaptic region ofouter The efferent innervation of the organ of Corti was divided hair cells in the three rows of the basal turn of the cochlea. Less into crossed and uncrossed olivocochlear fibers, corresponding immunofluorescence was found in this region as one progressed to cells of origin that project to the contralateral or ipsilateral towards the apex, with none seen at the apex. At the most apical cochlea (24-26). Recently a case has been made for a different region the inner spiral bundle became patchy and the tunnel spiral dichotomy ofthe olivocochlear neurons (27-29). One system of bundle developed arcades. There was no immunofluorescence olivocochlear neurons is characterized by relatively small cell found in cells, in auditory nerve fibers, or in the hair cells ofthe organ ofCorti. The findings were the same in cat bodies, located mostly in the lateral superior olivary region. as in guinea pig, the latter being studied in more detail. It was con- These cells give rise to both crossed and uncrossed thin, perhaps cluded that efferent, olivocochlear neurons ofthe cochlea, synaps- unmyelinated, axons that with cells and auditory nerve ing predominantly with primary auditory nerve fibers from the in- dendrites predominantly in the region of inner hair cells (29). ner sensory cells or with the sensory cells, contain enkephalin-like The other system has larger cell bodies located mostly in the immunoreactivity. Also, the findings indicate that endings of oli- medial superior olivary region and . These cells vocochlear neurons that synapse predominantly with outer hair cells contain enkephalin-like immunoreactivity. It has previously send both crossed and uncrossed, perhaps myelinated, axons been shown that ofivocochlear neurons are likely to be cholinergic. that synapse predominantly with large endings on outer hair cells (29). Methionine enkephalin and leucine enkephalin are opioid pen- The functions of the cochlear efferents are unknown. There tapeptides originally isolated from the whole brain (1, 2). Im- is strong evidence that there are inhibitory olivocochlear neu- munohistochemical mapping of enkephalin-like immunoreac- rons (30, 31), cholinergic olivocochlear neurons (32-35), and tivity in the central nervous system was done (3-5), and it was olivocochlear neurons that form part of an auditory feedback noted that in certain regions enkephalin-like immunoreactivity loop (36, 37). It is not known ifthese categories completely over- and opiate receptors had a similar distribution (3, 5). A much lap or ifthere is a correspondence to the two systems ofmyelin- entertained working hypothesis is that enkephalins may act as ated and unmyelinated olivocochlear neurons. It is likely, how- or modulate the action of neurotransmit- ever, that neurons ofboth systems contain acetylcholinesterase ters (6). (23, 38, 39). In the organ ofCorti, each inner hair cell synapses with about In the present study, using immunohistochemical tech- 20 auditory nerve boutons from about 90% of the total spiral niques, we have found enkephalin-like immunoreactivity in oh- ganglion cell population (7-9). The outer hair cells make rela- vocochlear efferent nerve fibers and in synaptic regions in the tively few synapses with auditory nerve boutons from about 10% organ ofCorti and in unmyelinated nerve fibers ofthe intragan- of the spiral ganglion cells (7-9). The auditory receptor organ glionic spiral bundle ofthe cochlea. also receives efferent, centrifugal innervation from the brain- stem, through olivocochlear neurons. In the cochlea, myelin- MATERIALS AND METHODS ated and unmyelinated olivocochlear fibers spiral in the fascicles ofthe intraganglionic bundle (10, 11) and then fan out to the or- Antiserum. Antibodies to methionine enkephalin were gen- gan of Corti. In the organ of Corti, such fibers form the inner erated in female New Zealand White rabbits by using methio- spiral bundle and the tunnel spiral bundle (12-14). Inner hair nine enkephalin (Boehringer Mannheim) coupled with glutar- cells and auditory nerve dendrites under inner hair cells receive aldehyde (Polysciences 70%, EM grade) to bovine thyroglobulin efferent innervation from the inner spiral bundle (15). The up- (Sigma type I), following procedures described in detail else- per tunnel crossing fibers are efferent (9, 16), and other effer- where (40, 41). Tissue Preparation. NIH strain guinea pigs ofboth sexes, 14 The publication costs ofthis article were defrayed in part by page charge young (85-145 g) and 28 mature (275-325 g), were used, mature payment. This article must therefore be hereby marked "advertise- animals for surface preparations and young animals for surface inent" in accordance with 18 U. S. C. §1734 solely to indicate this fact. preparations and cryostat sections. Cats were also examined, 3 1255 Downloaded by guest on September 26, 2021 1256 Neurobiology: Fex and Altschuler Proc. Natl. Acad. Sci. USA 78 (1981) mature (2-2.4 kg) and 1 young (0.9 kg). Guinea pigs were anes- nine or leucine enkephalin, the latter of much less intensity. thetized with chloral hydrate and perfused through the heart Antiserum to methionine enkaphalin was therefore used in sub- with 0.1 M sodium cacodylate buffer, pH 7.2, at 40C followed sequent experiments and the term "enkephalin" is used in de- by 4% (wt/vol) paraformaldehyde in 0.1 M sodium cacodylate scribing immunoreactivity. Structures were considered to ex- buffer at 40C. The temporal bones were removed, the cochleae hibit enkephalin-like immunoreactivity if they showed were exposed, and the inner ears were perfused with fixative immunofluorescent staining with enkephalin antisera and no through the round window. The cochleae were then rinsed. staining when the antiserum was absorbed with enkephalin Phosphate-buffered saline, pH 7.2, was used for all rinses. The (Fig. 1J). The fluorescence that was seen after such absorption bony shell and vascular stria ofeach cochlea were removed un- was considered nonspecific. Nonspecific fluorescence varied in der phosphate-buffered saline and the remaining cochlear spiral distribution and intensity between preparations and structures. with the organ of Corti was used for surface preparations or The tectorial membrane showed strong nonspecific fluores- cryostat sections. Cats were handled similarly to guinea pigs but cence. The basilar membrane often showed nonspecific fluores- were anesthetized with Nembutal. cence of variable intensity. The use of preimmune serum and Immunohistochemical Staining. Modifications of the indi- antisera against f3-endorphin and 13-lipotropin gave no specific rect immunofluorescence technique ofCoons (42) were used on immunofluorescence. The antiserum supplied by R. Elde gave both preparations. For surface preparations the cochlear spiral the same results as the antibody we generated, as did antiserum was incubated in a Beem capsule with 250 .1A of antiserum absorbed with thyroglobulin. against methionine enkephalin diluted 1:20. Phosphate-buff- A substance having immunoreactive sites identical to enke- ered saline with 0.3% Triton X-100 was used for all dilutions. phalin's could give the same immunocytochemical visualization The incubation proceeded for 25 min at 370C. A 1:200 dilution (43). For this reason the term "enkephalin-like immunoreactiv- with a 16-hr incubation at 40C was used interchangeably. After ity" was used. rinse, the second incubation was carried out with 250 A.l offlu- Methodological Considerations. In this study, the surface orescein isothiocyanate-labeled swine antiserum to rabbit im- preparations ofthe organ ofCorti gave excellent information on munoglobulin (Bio-Rad) diluted 1:10, for 25 min at 370C, fol- the distribution of the immunofluorescence of the longitudi- lowed by a rinse. Dissection ofthe cochlear spiral then yielded nally running inner spiral bundle and tunnel spiral bundle and 1- to 2-mm-long surface preparations consisting of segments of also on the course and spacing ofradial nerve fibers crossing the the organ ofCorti and the osseous spiral lamina. Removing the tunnel ofCorti. Cryostat cross sections gave complementary in- tectorial membrane was necessary for obtaining good results formation, for localizing enkephalin-like immunoreactivity to with the surface preparations. Segments composing all of the the synaptic region of hair cells and for visualizing parts of the cochlear spiral were examined in three guinea pigs and one cat; cochlea not accessible to inspection in surface preparations. representative segments were examined in all other animals. It has been recognized that there are species differences in Segments were viewed with a Zeiss photomicroscope under the distribution ofolivocochlear nerve fibers and auditory nerve epifluorescent illumination. The organ of Corti was optically fibers between the guinea pig and the cat cochlea (8, 9, 12). No dissected under normal light optics for localization of the fluo- major difference in the distribution of enkephalin-like immu- rescence (Fig. 1C) with the condensor unfocused for increased noreactivity between the guinea pig and cat cochlea was found contrast. during the course ofthe present study, which was focused on the For cryostat sections the cochlear spiral was placed for 30 min guinea pig cochlea. It should be recalled that the guinea pig in phosphate-buffered saline with 6% sucrose (wt/vol) and cochlea has slightly more than four full turns, whereas the cat freeze mounted. Ten-micrometer cryostat sections were cochlea has only three. The results as given for the guinea pig mounted, kept at room temperature for 15 min, and then cov- can thus not be immediately applied to the cat cochlea. ered with phosphate-buffered saline containing 10% (vol/vol) Guinea Pig. Many intraganglionic spiral bundles were iden- normal swine serum (Cappel Laboratories, Cochranville, PA) tified and all showed immunofluorescence (Fig. LA). In cross for 20 min. The serum was removed and 60 ,Al of methionine sections of the intraganglionic spiral bundles and at high mag- enkephalin antiserum diluted 1:20 was placed over each section nification, myelinated efferent fibers could be identified as for incubation at 37°C for 25 min. A 1:200 dilution with a 16-hr small nonfluorescent rings. Immunofluorescence was found in incubation at 4°C was used interchangeably. After a rinse, 60 ,ul between these rings, never in the rings. It was thus concluded offluorescein isothiocyanate-labeled swine antiserum to rabbit that the enkephalin-like immunoreactivity of the intragan- immunoglobulin, diluted 1:10, was applied at 37°C for 25 min. glionic spiral bundle was located in unmyelinated fibers. A rinse followed, and sections were mounted in phosphate-buff- Spiral ganglion cells (Fig. LA) and myelinated auditory nerve ered saline/glycerol (1:3, vol/vol). fibers showed no enkephalin-like immunoreactivity.It has been Specificity Studies. Specificity controls (Fig. 1J) were ob- suggested (44) but not established that there are unmyelinated tained by incubation of surface preparations and cryostat sec- axons of the auditory nerve corresponding to the central exten- tions with antiserum that had been absorbed with 100 ,ug of sions of the 5-10% ofthe spiral ganglion cells called type 2 cells. methionine enkephalin per 50 Al of undiluted antiserum. In- No immunofluorescence was found that could be attributed to cubations were also made with preimmune serum and anti- such unmyelinated fibers. serum absorbed with 200 pg of bovine thyroglobulin per 50 ,ul Enkephalin-like immunoreactivity was found in the inner of undiluted antiserum. Additional antiserum to methionine spiral bundle and tunnel spiral bundle and in radial fibers cours- enkephalin kindly provided by Robert Elde and previously ing between the two spiral bundles (Fig. 1 B, D, E, and F). The characterized (40) was used in confirmational studies on both immunofluorescence of these structures was strongest in the preparations, as were antisera against 3lipotropin, ,3-endor- second and third turn, usually seen in surface preparations as phin, and leucine enkephalin (Immuno Nuclear, Stillwater, two intensely fluorescent bands with interconnecting beaded MN). fibers. At the apex (Fig. 1G) the immunofluorescence of the in- ner spiral bundle was broken up in patches and the tunnel spiral RESULTS bundle showed arcade formations. Specificity. In early surface preparations the same pattern of No enkephalin-like immunoreactivity was seen in inner hair immunofluorescence was found with antisera to either methio- cells. Often, in cross sections, one or two fibers were found Downloaded by guest on September 26, 2021 Neurobiology: Fex and Altschuler Proc. Natl. Acad. Sci. USA 78 (1981) 1257

FIG. 1. Fluorescence micrographs ofguinea pig cochleae, incubated with antisera to methionine enkephalin (A-I) or antiserum absorbed with methionine enkephalin (J). (Bar = 20 pm.) (A) Cryostat section showing immunofluorescence in fascicles ofthe intraganglionic spiral bundle and no fluorescence in spiral ganglion cells. (B) Surface preparation, mature animal, second turn, showing immunofluorescent inner spiral bundle (ISB) and tunnel spiral bundle (TSB). (C) Lightmicrograph ofB at slightly different focus. (D,E, andF) Different focuses ofsurface preparation fromyoung animal, fourth turn, showing immunofluorescent ISB, TSB, and crossing fibers (cf). (G) Surface preparation from mature animal showing immuno- fluorescent fiber bundles at apex. (H) Cryostat section, second turn, showing immunofluorescence in ISB, TSB, and the synaptic region ofouter hair cells (arrowheads). (I) Cryostat section, third turn, showing immunofluorescence in ISB, cf, and the synaptic regions ofthe first row of outer hair cells. (J) Cryostat section, second turn, incubated with antiserum to methionine enkephalin absorbed with methionine enkephalin, showingno spe- cific immunofluorescence. Downloaded by guest on September 26, 2021 1258 Neurobiology: Fex and Altschuler Proc. Natl. Acad. Sci. USA 78 (1981) munofluorescence was found to disappear from the third and second rows (Fig. 11). At the most apical part of the organ of Corti, no immunofluorescence was seen in the region of the outer hair cells. Cat. Enkephalin-like immunoreactivity was found in the same structures in the cat cochlea as in the guinea pig and in no other structures. In surface preparations, however, the inner spiral bundle was seen to fluoresce only in the third turn ofthe cochlea. This may have been due to masking ofthe fluorescence by the osseous spiral lamina, because in cryostat sections the in- ner spiral bundle showed fluorescence in all turns. Immunoflu- orescence was not found in any upper tunnel crossing fibers and in only very fewfibers crossing the tunnel floor. As in the guinea pig (Fig. 2A), enkephalin-like immunoreactivity was found in patches at the synaptic region ofouter hair-cells (Fig. 2B). At the apex, no immunofluorescence was found in the outer hair cell region.

DISCUSSION All structures and regions in which enkephalin-like immuno- reactivity was found contain olivocochlear fibers or endings of such fibers. The finding ofenkephalin-like immunoreactivity in the inner spiral bundle and tunnel spiral bundle leads to the conclusion that the efferent system predominantly innervating the innerhair cell region is enkephalin-like immunoreactive. All tunnel spiral fibers are fibers of olivocochlear neurons (9, 14, 16); the tunnel spiral bundle and inner spiral bundle most likely form part ofthe same efferent system (9, 16); and the inner spiral bundle provides the inner hair cells and auditory nerve den- drites under inner hair cells with efferent innervation (15). The patches ofenkephalin-like immunoreactivity in the syn- aptic region ofouter hair cells, which decrease from first to sec- ond to third row ofouter hair cells (Fig. 2) and from base to apex (Fig. 1 H and I), correspond in distribution to the large efferent endings on outer hair cells as described (20, 22, 23). This indi- FIG. 2. Fluorescence micrographs of surface preparations of the cates that the olivocochlear system predominantly innervating cochlea, showing immunofluorescent patches in the synaptic region of outer hair cells contains enkephalin-like immunoreactivity. The outer hair cells. (Bar = 20 ,um.) (A) Guinea pig organ ofCorti, mature small and relatively few auditory nerve synapseswith outer hair animal, second turn. (B) Cat organ ofCorti, young animal, first turn. cells (9, 12, 20) would be unlikely to give rise to the relatively large fluorescent patches that were found in outer hair cell syn- coursing from the inner spiral bundle towards an inner hair cell, aptic regions in both the guinea pig and cat (Fig. 2), particularly perhaps ending on or touching the cell. This corresponds well to in basal turns. It is not possible, however, to resolve synaptic the distribution ofthe relatively few synapses that efferents may issues at the light microscopic level, and therefore immunoelec- form with inner hair cells in the guinea pig (15). tron microscopic studies will be necessary to determine the pre- No immunofluorescence was seen in the upper tunnel cross- cise location and relationships ofimmunoreactive terminals. ingfibers. These could be-easily identified, particularly in lower The present finding of enkephalin-like immunoreactivity in turns. On the other hand, in surface preparations (Fig. iF) and the intraganglionic spiral bundle (Fig. 1A) can be due to the un- in cryostat sections (Fig. 11), fluorescent thin beaded fibers myelinated olivocochlear fibers known (10, 11) to travel in the were found that turned down from the region ofthe inner spiral bundle. The superior cervical ganglion, which has been shown bundle, or the tunnel spiral bundle, towards the floor ofthe tun- to contain cells with enkephalin-like immunoreactivity (45), nel to disappear from view halfway across the tunnel. These sends unmyelinated fibers to the bundle (11) and may also con- fluorescent fibers were seen mostly in the third and fourth turns tribute to the immunofluorescence seen in the bundle. These and were irregularly spaced in surface preparations. No such fibers terminate at the lips ofthe osseous spiral lamina (11) and fibers were found in the most apical part of the organ of Corti. cannot contribute to any immunoreactivity in theorgan ofCorti. Rarely, a fluorescent fiber could be seen to cross the tunnel at Tunnel crossing fibers showing enkephalin-like immunoflu- its floor and then run as an outer spiral fiber, ending in a series orescence crossed at the floorofthe tunnel. Previous studies re- of bright patches in the synaptic region at outer hair cell bases. ported evidence for (17) and against (9) the presence ofefferent Sometimes such a series of fluorescent patches was seen in an fibers at this level of the tunnel. Two types of outer hair cells upper turn with fluorescent segments of outer spiral fibers, have been described (20), corresponding to the distribution of without a corresponding tunnel crossing fiber being identified. efferent endings. Type B cells, with mostly small efferent end- The synaptic regions of the outer hair cells showed discrete ings, were seen in the outer row ofhair cells in the second turn patches of enkephalin-like immunoreactivity in all three rows and in the outer two rows in the third and fourth turns (20). Con- at the first and second turns (Figs. 1H and 2A). The immunoflu- ceivably, the immunofluorescent low tunnel crossing fibers, orescence decreased from the first row to the third (Fig. 1H) also seen predominantly in the third and fourth turns, repre- and from the second turn to the~apex. In the third turn, the im- sented efferents leading to type B outer hair cells. The distri- Downloaded by guest on September 26, 2021 Neurobiology: Fex and Altschuler Proc. Natd Acad. Sci. USA 78 (1981) 1259

bution of these fluorescent fibers thus did not match the dis- 12. Engstrom, H., Ades, H. W. & Andersson, A. (1966) in Structural tribution of the large immunofluorescent patches found in Pattern of the Organ of Corti (Williams & Wilkins, Baltimore, the outer hair cell synaptic region, predominantly in the more MD). 13. Wright, C. G. (1975) Acta Otolaryngol. 80, 220-229. basal turns. Upper tunnel crossing fibers were not immuno- 14. Wright, C. G. & Preston, R. E. (1975) Acta Otolaryngol 80, fluorescent. 335-342. The present study has given evidence that hair cells, and den- 15. Smith, C. A. (1961) Ann. Otol Rhinoi Laryngot 70, 504-528. drites, cell bodies, and axons ofthe auditory nerve in the cochlea 16. Wright, C. G. & Preston, R. E. (1973) Brain Res. 58, 37-59. do not show enkephalin-like immunoreactivity. 17. Stopp, P. E. & Comis, S. D. (1978) Neuroscience 3, 1197-1206. Finally, it has been shown previously that the efferent olivo- 18. Iurato, S. (1962) Exp. 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