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The innervation of outer hair cells: 3D reconstruction from TEM serial sections in the Japanese macaque
Hearing Research 135 (1999) 29^38
The innervation of outer hair cells: 3D reconstruction from TEM serial sections in the Japanese macaque Michiya Sato a , Miriam M. Henson b , O.W. Henson Jr. c , David W. Smith
d;
*
a
Department of Otolaryngology-Head and Neck Surgery, National Defense Medical College, Saitama 359, Japan Division of Otolaryngology/Head and Neck Surgery, The University of North Carolina, Chapel Hill, NC, USA c Department of Cell Biology and Anatomy, The University of North Carolina, Chapel Hill, NC, USA Hearing Research Laboratories, Division of Otolaryngology-Head and Neck Surgery, Box 3550, Duke University Medical Center, Durham, NC 27710, USA b
d
Received 10 December 1998; received in revised form 20 April 1999; accepted 5 May 1999
Abstract Transmission electron micrographs from serial sections were obtained from the neural pole of outer hair cells (OHCs) in the Japanese macaque (Macaca fuscata) and reconstructions of nerve terminals were made using computer software. Data are based on observations of six cells in the basal turn, eight in the middle turn and four in the apex. In general, the number of afferent (type II) terminals on each OHC increased from base to apex, and for a given turn, the numbers appeared unrelated to OHC row. On the other hand, the number of efferent terminals was greater in the middle turn than in other areas, and the number decreased from row 1 to row 3. Reciprocal synapses increased in frequency from the upper basal turn apicalward. The total number of terminals synapsing on an individual OHC increased from base to apex by nearly 100%. Three-dimensional reconstructions showed that nerve fibers terminating on basal and middle turn OHCs ascended directly from sub-OHC regions to synapse on the subnuclear regions of the OHC. In contrast, apical turn fibers ran horizontally at the level of the subnuclear region and the terminals appeared as en passant swellings along a single fiber. Although physiological data are wanting for the macaque, the anatomical findings suggest that functional differences may exist along the length of the cochlea. ß 1999 Elsevier Science B.V. All rights reserved. Key words: Outer hair cell; Synapse; Organ of Corti; Japanese macaque; Three-dimensional reconstruction; Innervation
1. Introduction In recent years, there has been a growing appreciation of the role of the medial olivocochlear (MOC) e¡erent system in modulating the mechanical behavior of outer hair cells (OHCs), and subsequently the response properties of the auditory system as a whole (see recent review by Guinan, 1997). MOC activity, whether a result of electrical or contralateral acoustic stimulation, is capable of altering the vibratory response of the cochlear partition (cf. Mountain, 1980 ;
* Corresponding author. Tel.: +1 (919) 681-6379; Fax: +1 (919) 684-5160; E-mail: [email protected]
Dolan and Nuttall, 1994 ; Henson et al., 1995), threshold and tuning properties of single auditory a¡erent ¢bers (cf. Winslow and Sachs, 1987; Warren and Liberman, 1989), the amplitude and latency of compound action potentials (Liberman, 1989), and the amplitude and frequency characteristics of both spontaneous and evoked otoacoustic emissions (Mott et al., 1989 ; Collet et al., 1990; Puel and Rebillard, 1990). It is now generally accepted that these e¡ects, in both human and non-human animal models, are mediated by MOC-induced changes in OHC shape and/or contractility. Although several species, including the guinea pig, mustached bat, cat, non-human primate and human, have served as subjects in both anatomical and physiological studies of the MOC system, there are limited data indicating links between physiological response characteristics and underlying anatomical structural
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specializations. For example, in some species, there are well-documented changes in the number and distribution of terminals at the base of OHCs with cochlear position, such as row-by-row and base to apex di¡erences (Nadol, 1988; Liberman et al., 1990; Sato et al., 1997). In general, the number and/or size of e¡erent terminals/OHC is greatest in ¢rst row OHCs and in frequency regions of maximum sensitivity (Nadol, 1988 ; Liberman et al., 1990; Xie et al., 1993). In contrast, type II a¡erent terminals appear to be more plentiful in the apex than in the base (Nadol, 1988; Liberman et al., 1990). The magnitude of MOC-induced changes in cochlear responses is frequency-dependent and has been correlated with the number and distribution of MOC terminals along the cochlear partition (Liberman, 1988 ; Warren and Liberman, 1989). In the guinea pig, Brown (1988) has shown that the spontaneous discharge rate of single MOC e¡erent ¢bers is frequency-dependent, and peaks in ¢bers with characteristic frequencies in the range of 1.0^10.0 kHz, corresponding to the area of greatest MOC innervation. Because there are also apparent ultra-structural variations in MOC synapses on OHCs with place in the cochlea (Nadol, 1988; Sato et al., 1997), it is likely that the absolute number and physical distribution of e¡erent terminals within the organ of Corti are not the only factors in£uencing OHC behavior under MOC stimulation (Guinan and Gi¡ord, 1988; Liberman et al., 1990). For example, supranuclear e¡erent terminals have been reported on OHCs in the middle and apical turns of the guinea pig (Bredberg, 1977; Hashimoto and Kimura, 1988), the cat (Liberman et al., 1990) and monkey (Nakai and Igarashi, 1974; Sato et al., 1997). Reciprocal synapses, which have structural characteristics of both a¡erent and e¡erents terminals, are evident in the macaque (Sato et al., 1997) and man (Nadol, 1983, 1984) and are more plentiful in the apical than the basal end of the cochlea. The present project is an extension of our earlier work on the Japanese macaque and focuses on changes in the course, distribution and arrangement of the afferent and e¡erent ¢bers and terminals at the base of OHCs. Three-dimensional reconstructions were made of the neural pole of individual OHCs as a means of illustrating di¡erences in terminal morphology at the base of outer hair cells as a function of location within the cochlea.
2. Methods 2.1. Animals The temporal bones from a 10-year-old Japanese macaque, obtained as a cull from the Oregon Regional Primate Center, were used in this study. This monkey had no history of ear disease and was involved in studies of free-ranging behavior. The care and use of the animal in this research was approved by the Duke University Institutional Animal Care and Use Committee. 2.2. Tissue harvesting and processing The animal was anesthetized with sodium pentobarbital, and the head was perfused via carotid catheterization with modi¢ed Bouin's solution (4% paraformaldehyde, 0.5% glutaraldehyde and 0.2% picric acid in 0.15 M sodium phosphate bu¡er, pH 7.4). The temporal bones were removed and immersed in the same ¢xative for 48 h. After the mastoid was removed, and the otic capsule was thinned with a dental burr, the tissue was rinsed in 0.15 M phosphate bu¡er and decalci¢ed in 0.1 M disodium ethylenediamine tetraacetic acid (EDTA) with 2% glutaraldehyde in 0.1 M phosphate bu¡er, pH 7.4. Decalci¢cation required approximately 2 months ; the solution was constantly agitated and the EDTA was changed daily. Following decalci¢cation, the right cochlea was used for light microscopy, and the left was prepared for electron microscopy. There were no apparent abnormalities of the tympanic membrane, ossicles or temporal bone and a light microscopic examination of the organ of Corti revealed a normal-appearing complement of inner and outer hair cells. The cochlea for light microscopy was dehydrated through a graded series of alcohols and embedded in celloidin. To reconstruct in two dimensions, following the method of Schuknecht (1957), serial sections were made in the horizontal plane (parallel to the £oor of the middle cranial fossa) at a thickness of 10 Wm. The sections were stained with hematoxylin and eosin. The cochlea for electron microscopy was cut into halves, washed in 0.15 M phosphate bu¡er, post-¢xed in 1% osmium tetroxide in 0.15 M phosphate bu¡er for 1 h, rinsed in distilled water and stained en bloc with 2% aqueous uranyl acetate for 1 h. The specimens were
C Fig. 1. Reconstructions of OHCs and related nerve terminals from the basal, middle and apical turns (left, middle and right columns, respectively). The top row presents a view of each OHC from the side (100³, as measured from the extreme basal/axial view), and subsequent rows show progressive 30³ tilts of the hair cells until the basal or axial view is reached (0³). The OHC is shown in blue, e¡erent terminals in red and a¡erent terminals in yellow.
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then dehydrated through increasing concentrations of ethanol, followed by two changes of propylene oxide, in¢ltrated and embedded in Polybed 812 (Polysciences, Inc., Warrington, PA). 2.3. Serial sections and 3D reconstructions Thick sections were stained with 1% toluidine blue and used for orientation; thin sections from the same blocks were then stained with 4% uranyl acetate and 0.4% lead citrate. Serial thin sections (90 nm) were cut in the radial plane from each cochlear turn. For the serial section series, ¢ve di¡erent segments were selected. OHCs were studied from two locations in each of the basal and middle turns and one location from the apical turn. In each location, the nerve endings were counted from either one or two hair cells from each row. Locations I and II were from the basal turn, approximately 34% and 41% along the length of the cochlea from the basal end; locations III and IV were from the middle turn, approximately 63% and 78% from the basal end; and location V was from the apex, approximately 98% along the length of the cochlea. The innervation patterns of 18 OHCs were examined : three each from locations I and II; ¢ve from location III; three from location IV; four from location V. These sections were examined and photographed with a Zeiss EM 10A transmission electron microscope (TEM) at 60 kV accelerating voltage. Each nerve ending observed at the base of a studied OHC was classi¢ed as non-vesiculated (a¡erent, type II), vesiculated (e¡erent) or reciprocal (a terminal that contained both e¡erent- and a¡erent-like synaptic specializations within a single nerve ending) (Nadol, 1984 ; Sato et al., 1997). Morphological criteria for each terminal type were de¢ned as detailed in Macaca fuscata by Sato et al. (1997). Brie£y, an a¡erent, non-vesiculated ending was characterized by the presence of presynaptic and postsynaptic membrane thickenings and, in most instances, a presynaptic body surrounded by synaptic vesicles in the outer hair cell side of the synapse. An e¡erent ending was characterized by the presence of many presynaptic clear- and dense-core vesicles within the nerve terminal and a subsynaptic cisterna within the OHC. Following classi¢cation, the outlines of cells and nerve endings below the OHC nucleus in photomicrographs were traced on a digitizing tablet. `Stacks' were created and OHCs and nerves were segmented using the
public domain program NIH Image (written by Wayne Rasband at the US National Institutes of Health and available on the Internet via anonymous FTP at zippy.nimh.nih.gov) on a Power Macintosh 6500. Three-dimensional reconstructions of the segmented structures were created from the stacks using VoxelView/ULTRA 2.1.2 software on a Silicon Graphics Power series IRIS 4D/310VGX computer. Sections were aligned using the cuticular plate and the nuclei of outer hair cells and Deiters' cells as reference points. 3. Results 3.1. Types and numbers of terminals Table 1 presents a summary of the quantitative ¢ndings from this study. In general, the total number of terminals at the base of the OHCs nearly doubled from the base to apical turn and appeared unrelated to OHC row. The number of non-vesiculated, a¡erent endings increased by more than two-fold from base to apex, from 3^6/OHC in the lower basal turn to 13 in the apical turn. There was no consistent di¡erence in the number of a¡erent endings across OHC rows. In a previous report we noted that reciprocal synapses were present in each cochlear turn of M. fuscata (Sato et al., 1997). The present study extends that ¢nding by demonstrating that the number of these synapses is highest in the apex. Although only a small number of OHCs were studied in detail, there appears to be relatively more reciprocal endings on 2nd and 3rd row OHCs, than on row 1. Vesiculated, e¡erent nerve endings, in contrast, were most plentiful in the middle turn of the cochlea where there were 5^12 per OHC. The number of e¡erent terminals decreased both toward the base and, most severely, toward the apical turn. The occurrence of vesiculated endings decreased from the 1st to the 3rd row. 3.2. Three-dimensional reconstructions Fig. 1 shows three-dimensional reconstructions of the neural pole of three OHCs from the basal, middle and apical turns, respectively. The top row presents a view of each OHC from the side (100³, as measured from the extreme basal/axial view), and subsequent rows show progressive 30³ tilts of the hair cells until the basal or axial view is reached (0³).
C Fig. 2. Reconstructions of OHCs and related nerve terminals from the basal, middle and apical turns (left, middle and right columns, respectively). In the top row each cell is shown in a slightly tilted, lateral view and in the lower rows with progressive 90³ rotations. The OHC is shown in blue, e¡erent terminals in red and a¡erent terminals in yellow.
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Fig. 3. Micrograph and partial reconstruction of an OHC and synapse from the apical part of the cochlea. The reconstruction shown in B has been terminated at approximately the same level as the micrograph to illustrate the neural pole at this point in the reconstruction. The OHC is shown in blue, e¡erent terminals in red and a¡erent terminals in yellow. Clear synaptic specializations can be seen in the TEM while bifurcations and the course of the nerve are best appreciated in the reconstruction. A supranuclear ¢ber can be seen ascending along the side of the OHC. Bar = 0.5 Wm. C
From these reconstructions, the a¡erent and e¡erent nerve branches terminating on receptors in the basal and middle turns can be seen to approach the OHCs from directly below, arising from ¢bers in or adjacent to the outer spiral bundle (OSB). For basal turn OHCs, the a¡erent terminals have a ring-shaped con¢guration around the base of the hair cell (Fig. 1, left column, bottom row). The much larger e¡erent bouton terminates on the OHC at a level beginning at the level of the a¡erent terminals, and extends up to a level near the bottom of the nucleus of the cell. A ring of a¡erent boutons can also be seen in the reconstruction of the middle turn OHC (Fig. 1, middle column, bottom row), but is somewhat displaced by the four large e¡erent terminals. A¡erent and e¡erent neurons associated with apical turn OHCs, however, di¡er in that they have a horizontal orientation at the level of the hair cell base, as if approaching from adjacent OHCs (Fig. 1, right column). These horizontally running ¢bers have en passant
terminals on the OHCs. Previous studies in our laboratories have shown that these terminals make axosomatic synapses with the OHCs, as well as axodendritic synapses between a¡erent and e¡erent ¢bers (Sato et al., 1997). A ¢ber can also be seen to be ascending along the wall of the OHC, to subsequently form a supranuclear terminal with this OHC, just below the level of the reticular lamina. Supranuclear terminals in the Japanese macaque were observed in all three cochlear turns (Sato et al., 1997). Fig. 2 shows axial rotations for the reconstructed OHCs shown in Fig. 1. In the top row each cell is shown in a slightly tilted, lateral view and in the lower rows with progressive 90³ rotations. These views again demonstrate the relative change in innervation with place. They also reveal two major di¡erences in the nerve ¢ber terminal arrangement between basal and middle turn OHCs as compared with apical OHCs (i.e., the vertical versus the horizontal direction from which the ¢bers approach the neural pole of the
Table 1 Nerve endings on OHCs by organ of Corti location Location I. Lower basal turn 1st row OHC 2nd row OHC 3rd row OHC II. Upper basal turn 1st row OHC 2nd row OHC 3rd row OHC III. Lower middle turn 1st row OHC 2nd row OHC A B 3rd row OHC A B IV. Upper middle turn 1st row OHC 2nd row OHC 3rd row OHC V. Apical turn 1st row OHC 2nd row OHC A B 3rd row OHC
Number of reciprocal endings/OHC
Number of a¡erent endings/OHC
Number of e¡erent endings/OHC
Number of both a¡erent and e¡erent endings/OHC
0 0 0
3 6 4
2 2 2
5 8 6
0 0 1
8 7 6
3 2 1
11 9 7
0
5
6
11
0 1
7 8
4 3
11 11
0 1
9 6
0 1
9 7
0 1 0
5 12 7
4 1 0
9 13 7
0
7
5
12
5 4 4
13 13 13
0 1 0
13 14 13
Counts are from a total of 18 OHCs studied by TEM serial section.
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OHC) and discrete single terminal swellings vs. elongated ¢bers with en passant terminals. The top panel in Fig. 3 shows a TEM micrograph of the base of an apical turn OHC, at a point where an a¡erent ¢ber becomes enlarged and forms several synapses on the same OHC. By following several subsequent serial sections it was evident that this swelling divided and formed two separate a¡erent ¢bers, which subsequently coursed individually within a bundle of a¡erent and e¡erent ¢bers (see Sato et al., 1997, Fig. 5b for details). The bottom panel in Fig. 3 shows a partial 3-D reconstruction of this cell and illustrates the value of such a reconstruction for direct visualization of the course of individual nerve ¢bers to, and around, the receptor cells. This pattern can also be seen in the full 3-D reconstruction of this cell (Fig. 1, right column, top three rows, tilts 100³, 70³, and 40³ ; Fig. 3, right column, top two rows, rotations 0³, and 90³). (Quicktime movies of the 3-D reconstructions are available on CDs as teaching and research instruments for the cost of production. Interested persons should contact the corresponding author for information.) 4. Discussion To study the distribution and arrangement of nerve terminals on the base of cochlear outer hair cells, TEM serial sections were made through 18 OHCs from the basal, middle and apical turns of the cochlea from a Japanese macaque. The neural pole of several OHCs were then reconstructed from the serial sections. As examples, one reconstructed OHC was shown from each cochlear turn. Several of our ¢ndings are in general agreement with previous studies in other species (cf. Warr et al., 1986; Nadol, 1988), including: (1) for a given cochlear location the number of e¡erent terminals decreases from row 1 to row 3; (2) the number of a¡erent terminals is fairly consistent across the three rows of OHCs; (3) the total number of a¡erent and e¡erent nerve ¢bers associated with each cell increases nearly two-fold from base to apex; and (4) the population of e¡erent terminals is greatest in the middle turn, as compared with the basal and apical turns. Comparisons of 3-D reconstructions across the three cochlear turns, however, suggest the present ¢ndings di¡er in at least one signi¢cant way from data reported from previous studies ; that is, a¡erent and e¡erent ¢bers that synapse on OHCs in the basal and middle turns in the Japanese macaque cochlea arise from areas directly beneath the OHC, approach in a direction parallel to the long axis of the hair cell, and terminate in the subnuclear regions of an individual OHC. On the other hand, for apical turn OHCs, both a¡erent and
e¡erent ¢bers approach from the side, perpendicular to the long axis of the OHC, and have en passant terminals suggesting that each ¢ber synapses on multiple, adjacent OHCs. In the apex, the OSB appears to be placed much closer to, if not surrounding the lower portions of the OHC. These ¢ndings highlight the value of 3-D reconstructions as a tool for studying the complex innervation pattern of the cochlea. The number and distribution of a¡erent and e¡erent ¢bers synapsing with OHCs along the Japanese macaque organ of Corti is comparable to most other species studied to date (guinea pig, Smith and Sjo«strand, 1961 ; Takasaka and Shinkawa, 1987; cat, Spoendlin and Gacek, 1963 ; Guinan et al., 1984; Liberman et al., 1990). The ¢ne structure of the terminals synapsing on the OHCs in M. fuscata has also been shown to share many characteristic of other species, but, perhaps, has more features in common with humans than do other species (Sato et al., 1997). The typical anatomical view of an OHC shows a¡erent and e¡erent ¢bers approaching the neural pole of the OHC in a plane parallel to the long axis of the hair cell. Each branch arising from a ¢ber in the OSB ascends to terminate on a single OHC. The arrangement has been illustrated in previous studies on other species (cf. Spoendlin and Gacek, 1963 ; Spoendlin, 1969; Warr et al., 1986 ; Bishop and Henson, 1986). This is the same basic pattern found in the basal and middle turns of the cochlea of M. fuscata. In the apex, however, each ¢ber branch from the OSB appears to directly contact a consecutive series of receptor cells. Here, in contrast to other areas, the nerve impulses must pass along ¢bers in direct contact with adjacent receptor cells and nerves. In more basal areas nerve interactions could still occur but the receptor base and its environment would not a¡ect these interactions. The classical view of the innervation of the OHC does not hold in the apical end of the organ of Corti. In the apex, all terminals on individual OHCs, both a¡erent and e¡erent, pass in a plane orthogonal to the hair cell at the level of neural pole. While we did not directly observe ¢bers connecting adjacent OHCs, this arrangement would provide for a more e¡ective pathway for interactions between adjacent apical receptor cells. Regardless of whether or not adjacent OHCs interact in the apex, the di¡erences in terminal morphology are suggestive of a physiological di¡erence across cochlear turns. Studies in other species, however, provide su¤cient evidence that apical nerve ¢bers do synapse on multiple, adjacent outer hair cells (Spoendlin and Gacek, 1963; Spoendlin, 1969). In the cat (Spoendlin and Gacek, 1963) and guinea pig (Ades and Engstro«m, 1974), both a¡erent and e¡erent ¢bers spiral a short distance basalward after crossing the tunnel of Corti, before
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branching and rising to synapse on multiple OHCs. While this branching is much more widespread in the case of a¡erents, Spoendlin (1966) has estimated there are 40 000 e¡erent terminals in the cat cochlea, which arise from some 500 neurons. The functional consequences of the di¡erences observed in basal and middle vs. apical turn innervation is unclear. Other investigators have demonstrated that e¡erent-mediated e¡ects are frequency/place-dependent, and are related to di¡erences in e¡erent innervation patterns. For example, in the cat, the number of e¡erent terminals per OHC along the cochlear partition correlates well with the magnitude of MOC-induced suppression across frequency (Guinan and Gi¡ord, 1988 ; Liberman et al., 1990). Liberman (1988) has, furthermore, shown that the physiological response of single MOC ¢bers demonstrates clear basal-apical di¡erences in sound-evoked discharge patterns, where apical MOC e¡erents are relatively more binaurally responsive than are high-frequency ¢bers. A direct interaction between adjacent receptors and/ or their neurons is well known in some sensory systems, such as the eye. Here a direct interaction has been shown to modify nerve impulses and potentially sharpen perception, even at the very beginning of the sensory pathway. There are no known physiological data to suggest such an interaction in the organ of Corti, but the di¡erence in arrangement of the ¢bers in and around the neural pole in the apex vs. other areas suggests a basis for di¡erences in mode of interaction. In the apex, the a¡erent and e¡erent ¢bers constitute a bundle of longitudinally running ¢bers that are in contact with the OHC base; Sato et al. (1997), in the same preparations, showed evidence of synaptic connectivity among these ¢bers. In other, more basal areas, the synapses among the ¢bers also occur but they are much further removed from the neural pole of the OHC and this could constitute a more indirect, less e¡ective pathway for interactions among OHCs. Keeping with the tonotopic organization of the basilar membrane, this arrangement also demonstrates that a relatively greater convergence of receptor activity is present in the apex. Three-dimensional reconstruction studies do not directly address these functional di¡erences, though they o¡er important insights into the complex structure and organization of the cochlea. Acknowledgements The authors are greatly indebted to Dr. C. Robert Bagnell and Ms. Victoria J. Madden for their superb technical skills, their assistance in the preparation and sectioning of the tissue, and with the TEM evaluation
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of the sections. We would also like to thank Thomas Hazel and William Presson for considerable work on the 3-D reconstructions. This work was supported by Grants DC 001692 (D.W.S.) and DC 00114 (M.M.H. and O.W.H) from the National Institute for Deafness and Other Communication Disorders. Tissue was obtained from animals supported, in part, by NIH Grant RR 00163 to the Oregon Regional Primate Research Center.
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Nadol, J.B., Jr., 1983. Serial section reconstruction of the neural poles of hair cells in the human organ of Corti. II. Outer hair cells. Laryngoscope 93, 780^791. Nadol, J.B., Jr., 1984. Incidence of reciprocal synapses on outer hair cells of the human organ of Corti. Ann. Otol. Rhinol. Laryngol. 93, 247^250. Nadol, J.B., Jr., 1988. Comparative anatomy of the cochlea and auditory nerve in mammals. Hear. Res. 34, 253^266. Nakai, Y., Igarashi, M., 1974. Distribution of the crossed olivo-cochlear bundle terminals in the squirrel monkey cochlea. Acta Otolaryngol. 77, 393^404. Puel, J.-L., Rebillard, G., 1990. E¡ects of contralateral sound stimulation on the distortion product 2F1-F2: Evidence that the medial e¡erent system is involved. J. Acoust. Soc. Am. 87, 1630^ 1635. Sato, M., Henson, M.M., Smith, D.W., 1997. Synaptic specializations associated with the outer hair cells of the Japanese macaque. Hear. Res. 108, 46^54. Schuknecht, H.F., 1957. Techniques for study of cochlear function and pathology in experimental animals. Arch. Otolaryngol. Head Neck Surg. 58, 377^397. Smith, C.A., Sjo«strand, F.S., 1961. A synaptic structure in the hair cells of the guinea pig cochlea. J. Ultrastruct. Res. 5, 185^192. Spoendlin, H., 1966. The Organization of the Cochlear Receptor. Karger, Basel.
Spoendlin, H., 1969. Innervation patterns in the organ of Corti of the cat. Acta Otolaryngol. 67, 239^254. Spoendlin, H., Gacek, R.R., 1963. Electronmicroscopic study of the e¡erent and a¡erent innervation of the organ of Corti in the cat. Ann. Otol. Rhinol. Laryngol. 72, 660^687. Takasaka, T., Shinkawa, H., 1987. Serial section reconstruction of the guinea pig outer hair cells as studied with a high-voltage electron microscope and a computer-graphic display. Acta Otolaryngol. (Stockh.) 435 (Suppl.), 7^20. Warr, W.B., Guinan, Jr., J.J., White, J.S., 1986. Organization of the e¡erent ¢bers: The lateral and medial olivocochlear systems. In: Altschuler, R.A., Bobbin, R.P., Ho¡man, D.W. (Eds.), Neurobiology of Hearing: The Cochlea. Raven Press, New York, pp. 333^ 348. Warren, E.H., III, Liberman, M.C., 1989. E¡ects of contralateral sound on auditory nerve responses. II. Dependence on stimulus variables. Hear. Res. 37, 105^122. Winslow, R.L., Sachs, M.B., 1987. E¡ect of electrical stimulation of the crossed olivocochlear bundle on auditory nerve response to tones in noise. J. Neurophysiol. 57, 1002^1021. Xie, D.H., Henson, M.M., Bishop, A.L., Henson, O.W., Jr., 1993. E¡erent terminals in the cochlea of the mustached bat: Quantitative data. Hear. Res. 44, 81^90.
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The innervation of outer hair cells: 3D reconstruction from TEM serial sections in the Japanese macaque Michiya Sato a , Miriam M. Henson b , O.W. Henson Jr. c , David W. Smith
d;
*
a
Department of Otolaryngology-Head and Neck Surgery, National Defense Medical College, Saitama 359, Japan Division of Otolaryngology/Head and Neck Surgery, The University of North Carolina, Chapel Hill, NC, USA c Department of Cell Biology and Anatomy, The University of North Carolina, Chapel Hill, NC, USA Hearing Research Laboratories, Division of Otolaryngology-Head and Neck Surgery, Box 3550, Duke University Medical Center, Durham, NC 27710, USA b
d
Received 10 December 1998; received in revised form 20 April 1999; accepted 5 May 1999
Abstract Transmission electron micrographs from serial sections were obtained from the neural pole of outer hair cells (OHCs) in the Japanese macaque (Macaca fuscata) and reconstructions of nerve terminals were made using computer software. Data are based on observations of six cells in the basal turn, eight in the middle turn and four in the apex. In general, the number of afferent (type II) terminals on each OHC increased from base to apex, and for a given turn, the numbers appeared unrelated to OHC row. On the other hand, the number of efferent terminals was greater in the middle turn than in other areas, and the number decreased from row 1 to row 3. Reciprocal synapses increased in frequency from the upper basal turn apicalward. The total number of terminals synapsing on an individual OHC increased from base to apex by nearly 100%. Three-dimensional reconstructions showed that nerve fibers terminating on basal and middle turn OHCs ascended directly from sub-OHC regions to synapse on the subnuclear regions of the OHC. In contrast, apical turn fibers ran horizontally at the level of the subnuclear region and the terminals appeared as en passant swellings along a single fiber. Although physiological data are wanting for the macaque, the anatomical findings suggest that functional differences may exist along the length of the cochlea. ß 1999 Elsevier Science B.V. All rights reserved. Key words: Outer hair cell; Synapse; Organ of Corti; Japanese macaque; Three-dimensional reconstruction; Innervation
1. Introduction In recent years, there has been a growing appreciation of the role of the medial olivocochlear (MOC) e¡erent system in modulating the mechanical behavior of outer hair cells (OHCs), and subsequently the response properties of the auditory system as a whole (see recent review by Guinan, 1997). MOC activity, whether a result of electrical or contralateral acoustic stimulation, is capable of altering the vibratory response of the cochlear partition (cf. Mountain, 1980 ;
* Corresponding author. Tel.: +1 (919) 681-6379; Fax: +1 (919) 684-5160; E-mail: [email protected]
Dolan and Nuttall, 1994 ; Henson et al., 1995), threshold and tuning properties of single auditory a¡erent ¢bers (cf. Winslow and Sachs, 1987; Warren and Liberman, 1989), the amplitude and latency of compound action potentials (Liberman, 1989), and the amplitude and frequency characteristics of both spontaneous and evoked otoacoustic emissions (Mott et al., 1989 ; Collet et al., 1990; Puel and Rebillard, 1990). It is now generally accepted that these e¡ects, in both human and non-human animal models, are mediated by MOC-induced changes in OHC shape and/or contractility. Although several species, including the guinea pig, mustached bat, cat, non-human primate and human, have served as subjects in both anatomical and physiological studies of the MOC system, there are limited data indicating links between physiological response characteristics and underlying anatomical structural
0378-5955 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 9 ) 0 0 0 8 6 - 6
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specializations. For example, in some species, there are well-documented changes in the number and distribution of terminals at the base of OHCs with cochlear position, such as row-by-row and base to apex di¡erences (Nadol, 1988; Liberman et al., 1990; Sato et al., 1997). In general, the number and/or size of e¡erent terminals/OHC is greatest in ¢rst row OHCs and in frequency regions of maximum sensitivity (Nadol, 1988 ; Liberman et al., 1990; Xie et al., 1993). In contrast, type II a¡erent terminals appear to be more plentiful in the apex than in the base (Nadol, 1988; Liberman et al., 1990). The magnitude of MOC-induced changes in cochlear responses is frequency-dependent and has been correlated with the number and distribution of MOC terminals along the cochlear partition (Liberman, 1988 ; Warren and Liberman, 1989). In the guinea pig, Brown (1988) has shown that the spontaneous discharge rate of single MOC e¡erent ¢bers is frequency-dependent, and peaks in ¢bers with characteristic frequencies in the range of 1.0^10.0 kHz, corresponding to the area of greatest MOC innervation. Because there are also apparent ultra-structural variations in MOC synapses on OHCs with place in the cochlea (Nadol, 1988; Sato et al., 1997), it is likely that the absolute number and physical distribution of e¡erent terminals within the organ of Corti are not the only factors in£uencing OHC behavior under MOC stimulation (Guinan and Gi¡ord, 1988; Liberman et al., 1990). For example, supranuclear e¡erent terminals have been reported on OHCs in the middle and apical turns of the guinea pig (Bredberg, 1977; Hashimoto and Kimura, 1988), the cat (Liberman et al., 1990) and monkey (Nakai and Igarashi, 1974; Sato et al., 1997). Reciprocal synapses, which have structural characteristics of both a¡erent and e¡erents terminals, are evident in the macaque (Sato et al., 1997) and man (Nadol, 1983, 1984) and are more plentiful in the apical than the basal end of the cochlea. The present project is an extension of our earlier work on the Japanese macaque and focuses on changes in the course, distribution and arrangement of the afferent and e¡erent ¢bers and terminals at the base of OHCs. Three-dimensional reconstructions were made of the neural pole of individual OHCs as a means of illustrating di¡erences in terminal morphology at the base of outer hair cells as a function of location within the cochlea.
2. Methods 2.1. Animals The temporal bones from a 10-year-old Japanese macaque, obtained as a cull from the Oregon Regional Primate Center, were used in this study. This monkey had no history of ear disease and was involved in studies of free-ranging behavior. The care and use of the animal in this research was approved by the Duke University Institutional Animal Care and Use Committee. 2.2. Tissue harvesting and processing The animal was anesthetized with sodium pentobarbital, and the head was perfused via carotid catheterization with modi¢ed Bouin's solution (4% paraformaldehyde, 0.5% glutaraldehyde and 0.2% picric acid in 0.15 M sodium phosphate bu¡er, pH 7.4). The temporal bones were removed and immersed in the same ¢xative for 48 h. After the mastoid was removed, and the otic capsule was thinned with a dental burr, the tissue was rinsed in 0.15 M phosphate bu¡er and decalci¢ed in 0.1 M disodium ethylenediamine tetraacetic acid (EDTA) with 2% glutaraldehyde in 0.1 M phosphate bu¡er, pH 7.4. Decalci¢cation required approximately 2 months ; the solution was constantly agitated and the EDTA was changed daily. Following decalci¢cation, the right cochlea was used for light microscopy, and the left was prepared for electron microscopy. There were no apparent abnormalities of the tympanic membrane, ossicles or temporal bone and a light microscopic examination of the organ of Corti revealed a normal-appearing complement of inner and outer hair cells. The cochlea for light microscopy was dehydrated through a graded series of alcohols and embedded in celloidin. To reconstruct in two dimensions, following the method of Schuknecht (1957), serial sections were made in the horizontal plane (parallel to the £oor of the middle cranial fossa) at a thickness of 10 Wm. The sections were stained with hematoxylin and eosin. The cochlea for electron microscopy was cut into halves, washed in 0.15 M phosphate bu¡er, post-¢xed in 1% osmium tetroxide in 0.15 M phosphate bu¡er for 1 h, rinsed in distilled water and stained en bloc with 2% aqueous uranyl acetate for 1 h. The specimens were
C Fig. 1. Reconstructions of OHCs and related nerve terminals from the basal, middle and apical turns (left, middle and right columns, respectively). The top row presents a view of each OHC from the side (100³, as measured from the extreme basal/axial view), and subsequent rows show progressive 30³ tilts of the hair cells until the basal or axial view is reached (0³). The OHC is shown in blue, e¡erent terminals in red and a¡erent terminals in yellow.
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then dehydrated through increasing concentrations of ethanol, followed by two changes of propylene oxide, in¢ltrated and embedded in Polybed 812 (Polysciences, Inc., Warrington, PA). 2.3. Serial sections and 3D reconstructions Thick sections were stained with 1% toluidine blue and used for orientation; thin sections from the same blocks were then stained with 4% uranyl acetate and 0.4% lead citrate. Serial thin sections (90 nm) were cut in the radial plane from each cochlear turn. For the serial section series, ¢ve di¡erent segments were selected. OHCs were studied from two locations in each of the basal and middle turns and one location from the apical turn. In each location, the nerve endings were counted from either one or two hair cells from each row. Locations I and II were from the basal turn, approximately 34% and 41% along the length of the cochlea from the basal end; locations III and IV were from the middle turn, approximately 63% and 78% from the basal end; and location V was from the apex, approximately 98% along the length of the cochlea. The innervation patterns of 18 OHCs were examined : three each from locations I and II; ¢ve from location III; three from location IV; four from location V. These sections were examined and photographed with a Zeiss EM 10A transmission electron microscope (TEM) at 60 kV accelerating voltage. Each nerve ending observed at the base of a studied OHC was classi¢ed as non-vesiculated (a¡erent, type II), vesiculated (e¡erent) or reciprocal (a terminal that contained both e¡erent- and a¡erent-like synaptic specializations within a single nerve ending) (Nadol, 1984 ; Sato et al., 1997). Morphological criteria for each terminal type were de¢ned as detailed in Macaca fuscata by Sato et al. (1997). Brie£y, an a¡erent, non-vesiculated ending was characterized by the presence of presynaptic and postsynaptic membrane thickenings and, in most instances, a presynaptic body surrounded by synaptic vesicles in the outer hair cell side of the synapse. An e¡erent ending was characterized by the presence of many presynaptic clear- and dense-core vesicles within the nerve terminal and a subsynaptic cisterna within the OHC. Following classi¢cation, the outlines of cells and nerve endings below the OHC nucleus in photomicrographs were traced on a digitizing tablet. `Stacks' were created and OHCs and nerves were segmented using the
public domain program NIH Image (written by Wayne Rasband at the US National Institutes of Health and available on the Internet via anonymous FTP at zippy.nimh.nih.gov) on a Power Macintosh 6500. Three-dimensional reconstructions of the segmented structures were created from the stacks using VoxelView/ULTRA 2.1.2 software on a Silicon Graphics Power series IRIS 4D/310VGX computer. Sections were aligned using the cuticular plate and the nuclei of outer hair cells and Deiters' cells as reference points. 3. Results 3.1. Types and numbers of terminals Table 1 presents a summary of the quantitative ¢ndings from this study. In general, the total number of terminals at the base of the OHCs nearly doubled from the base to apical turn and appeared unrelated to OHC row. The number of non-vesiculated, a¡erent endings increased by more than two-fold from base to apex, from 3^6/OHC in the lower basal turn to 13 in the apical turn. There was no consistent di¡erence in the number of a¡erent endings across OHC rows. In a previous report we noted that reciprocal synapses were present in each cochlear turn of M. fuscata (Sato et al., 1997). The present study extends that ¢nding by demonstrating that the number of these synapses is highest in the apex. Although only a small number of OHCs were studied in detail, there appears to be relatively more reciprocal endings on 2nd and 3rd row OHCs, than on row 1. Vesiculated, e¡erent nerve endings, in contrast, were most plentiful in the middle turn of the cochlea where there were 5^12 per OHC. The number of e¡erent terminals decreased both toward the base and, most severely, toward the apical turn. The occurrence of vesiculated endings decreased from the 1st to the 3rd row. 3.2. Three-dimensional reconstructions Fig. 1 shows three-dimensional reconstructions of the neural pole of three OHCs from the basal, middle and apical turns, respectively. The top row presents a view of each OHC from the side (100³, as measured from the extreme basal/axial view), and subsequent rows show progressive 30³ tilts of the hair cells until the basal or axial view is reached (0³).
C Fig. 2. Reconstructions of OHCs and related nerve terminals from the basal, middle and apical turns (left, middle and right columns, respectively). In the top row each cell is shown in a slightly tilted, lateral view and in the lower rows with progressive 90³ rotations. The OHC is shown in blue, e¡erent terminals in red and a¡erent terminals in yellow.
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Fig. 3. Micrograph and partial reconstruction of an OHC and synapse from the apical part of the cochlea. The reconstruction shown in B has been terminated at approximately the same level as the micrograph to illustrate the neural pole at this point in the reconstruction. The OHC is shown in blue, e¡erent terminals in red and a¡erent terminals in yellow. Clear synaptic specializations can be seen in the TEM while bifurcations and the course of the nerve are best appreciated in the reconstruction. A supranuclear ¢ber can be seen ascending along the side of the OHC. Bar = 0.5 Wm. C
From these reconstructions, the a¡erent and e¡erent nerve branches terminating on receptors in the basal and middle turns can be seen to approach the OHCs from directly below, arising from ¢bers in or adjacent to the outer spiral bundle (OSB). For basal turn OHCs, the a¡erent terminals have a ring-shaped con¢guration around the base of the hair cell (Fig. 1, left column, bottom row). The much larger e¡erent bouton terminates on the OHC at a level beginning at the level of the a¡erent terminals, and extends up to a level near the bottom of the nucleus of the cell. A ring of a¡erent boutons can also be seen in the reconstruction of the middle turn OHC (Fig. 1, middle column, bottom row), but is somewhat displaced by the four large e¡erent terminals. A¡erent and e¡erent neurons associated with apical turn OHCs, however, di¡er in that they have a horizontal orientation at the level of the hair cell base, as if approaching from adjacent OHCs (Fig. 1, right column). These horizontally running ¢bers have en passant
terminals on the OHCs. Previous studies in our laboratories have shown that these terminals make axosomatic synapses with the OHCs, as well as axodendritic synapses between a¡erent and e¡erent ¢bers (Sato et al., 1997). A ¢ber can also be seen to be ascending along the wall of the OHC, to subsequently form a supranuclear terminal with this OHC, just below the level of the reticular lamina. Supranuclear terminals in the Japanese macaque were observed in all three cochlear turns (Sato et al., 1997). Fig. 2 shows axial rotations for the reconstructed OHCs shown in Fig. 1. In the top row each cell is shown in a slightly tilted, lateral view and in the lower rows with progressive 90³ rotations. These views again demonstrate the relative change in innervation with place. They also reveal two major di¡erences in the nerve ¢ber terminal arrangement between basal and middle turn OHCs as compared with apical OHCs (i.e., the vertical versus the horizontal direction from which the ¢bers approach the neural pole of the
Table 1 Nerve endings on OHCs by organ of Corti location Location I. Lower basal turn 1st row OHC 2nd row OHC 3rd row OHC II. Upper basal turn 1st row OHC 2nd row OHC 3rd row OHC III. Lower middle turn 1st row OHC 2nd row OHC A B 3rd row OHC A B IV. Upper middle turn 1st row OHC 2nd row OHC 3rd row OHC V. Apical turn 1st row OHC 2nd row OHC A B 3rd row OHC
Number of reciprocal endings/OHC
Number of a¡erent endings/OHC
Number of e¡erent endings/OHC
Number of both a¡erent and e¡erent endings/OHC
0 0 0
3 6 4
2 2 2
5 8 6
0 0 1
8 7 6
3 2 1
11 9 7
0
5
6
11
0 1
7 8
4 3
11 11
0 1
9 6
0 1
9 7
0 1 0
5 12 7
4 1 0
9 13 7
0
7
5
12
5 4 4
13 13 13
0 1 0
13 14 13
Counts are from a total of 18 OHCs studied by TEM serial section.
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OHC) and discrete single terminal swellings vs. elongated ¢bers with en passant terminals. The top panel in Fig. 3 shows a TEM micrograph of the base of an apical turn OHC, at a point where an a¡erent ¢ber becomes enlarged and forms several synapses on the same OHC. By following several subsequent serial sections it was evident that this swelling divided and formed two separate a¡erent ¢bers, which subsequently coursed individually within a bundle of a¡erent and e¡erent ¢bers (see Sato et al., 1997, Fig. 5b for details). The bottom panel in Fig. 3 shows a partial 3-D reconstruction of this cell and illustrates the value of such a reconstruction for direct visualization of the course of individual nerve ¢bers to, and around, the receptor cells. This pattern can also be seen in the full 3-D reconstruction of this cell (Fig. 1, right column, top three rows, tilts 100³, 70³, and 40³ ; Fig. 3, right column, top two rows, rotations 0³, and 90³). (Quicktime movies of the 3-D reconstructions are available on CDs as teaching and research instruments for the cost of production. Interested persons should contact the corresponding author for information.) 4. Discussion To study the distribution and arrangement of nerve terminals on the base of cochlear outer hair cells, TEM serial sections were made through 18 OHCs from the basal, middle and apical turns of the cochlea from a Japanese macaque. The neural pole of several OHCs were then reconstructed from the serial sections. As examples, one reconstructed OHC was shown from each cochlear turn. Several of our ¢ndings are in general agreement with previous studies in other species (cf. Warr et al., 1986; Nadol, 1988), including: (1) for a given cochlear location the number of e¡erent terminals decreases from row 1 to row 3; (2) the number of a¡erent terminals is fairly consistent across the three rows of OHCs; (3) the total number of a¡erent and e¡erent nerve ¢bers associated with each cell increases nearly two-fold from base to apex; and (4) the population of e¡erent terminals is greatest in the middle turn, as compared with the basal and apical turns. Comparisons of 3-D reconstructions across the three cochlear turns, however, suggest the present ¢ndings di¡er in at least one signi¢cant way from data reported from previous studies ; that is, a¡erent and e¡erent ¢bers that synapse on OHCs in the basal and middle turns in the Japanese macaque cochlea arise from areas directly beneath the OHC, approach in a direction parallel to the long axis of the hair cell, and terminate in the subnuclear regions of an individual OHC. On the other hand, for apical turn OHCs, both a¡erent and
e¡erent ¢bers approach from the side, perpendicular to the long axis of the OHC, and have en passant terminals suggesting that each ¢ber synapses on multiple, adjacent OHCs. In the apex, the OSB appears to be placed much closer to, if not surrounding the lower portions of the OHC. These ¢ndings highlight the value of 3-D reconstructions as a tool for studying the complex innervation pattern of the cochlea. The number and distribution of a¡erent and e¡erent ¢bers synapsing with OHCs along the Japanese macaque organ of Corti is comparable to most other species studied to date (guinea pig, Smith and Sjo«strand, 1961 ; Takasaka and Shinkawa, 1987; cat, Spoendlin and Gacek, 1963 ; Guinan et al., 1984; Liberman et al., 1990). The ¢ne structure of the terminals synapsing on the OHCs in M. fuscata has also been shown to share many characteristic of other species, but, perhaps, has more features in common with humans than do other species (Sato et al., 1997). The typical anatomical view of an OHC shows a¡erent and e¡erent ¢bers approaching the neural pole of the OHC in a plane parallel to the long axis of the hair cell. Each branch arising from a ¢ber in the OSB ascends to terminate on a single OHC. The arrangement has been illustrated in previous studies on other species (cf. Spoendlin and Gacek, 1963 ; Spoendlin, 1969; Warr et al., 1986 ; Bishop and Henson, 1986). This is the same basic pattern found in the basal and middle turns of the cochlea of M. fuscata. In the apex, however, each ¢ber branch from the OSB appears to directly contact a consecutive series of receptor cells. Here, in contrast to other areas, the nerve impulses must pass along ¢bers in direct contact with adjacent receptor cells and nerves. In more basal areas nerve interactions could still occur but the receptor base and its environment would not a¡ect these interactions. The classical view of the innervation of the OHC does not hold in the apical end of the organ of Corti. In the apex, all terminals on individual OHCs, both a¡erent and e¡erent, pass in a plane orthogonal to the hair cell at the level of neural pole. While we did not directly observe ¢bers connecting adjacent OHCs, this arrangement would provide for a more e¡ective pathway for interactions between adjacent apical receptor cells. Regardless of whether or not adjacent OHCs interact in the apex, the di¡erences in terminal morphology are suggestive of a physiological di¡erence across cochlear turns. Studies in other species, however, provide su¤cient evidence that apical nerve ¢bers do synapse on multiple, adjacent outer hair cells (Spoendlin and Gacek, 1963; Spoendlin, 1969). In the cat (Spoendlin and Gacek, 1963) and guinea pig (Ades and Engstro«m, 1974), both a¡erent and e¡erent ¢bers spiral a short distance basalward after crossing the tunnel of Corti, before
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branching and rising to synapse on multiple OHCs. While this branching is much more widespread in the case of a¡erents, Spoendlin (1966) has estimated there are 40 000 e¡erent terminals in the cat cochlea, which arise from some 500 neurons. The functional consequences of the di¡erences observed in basal and middle vs. apical turn innervation is unclear. Other investigators have demonstrated that e¡erent-mediated e¡ects are frequency/place-dependent, and are related to di¡erences in e¡erent innervation patterns. For example, in the cat, the number of e¡erent terminals per OHC along the cochlear partition correlates well with the magnitude of MOC-induced suppression across frequency (Guinan and Gi¡ord, 1988 ; Liberman et al., 1990). Liberman (1988) has, furthermore, shown that the physiological response of single MOC ¢bers demonstrates clear basal-apical di¡erences in sound-evoked discharge patterns, where apical MOC e¡erents are relatively more binaurally responsive than are high-frequency ¢bers. A direct interaction between adjacent receptors and/ or their neurons is well known in some sensory systems, such as the eye. Here a direct interaction has been shown to modify nerve impulses and potentially sharpen perception, even at the very beginning of the sensory pathway. There are no known physiological data to suggest such an interaction in the organ of Corti, but the di¡erence in arrangement of the ¢bers in and around the neural pole in the apex vs. other areas suggests a basis for di¡erences in mode of interaction. In the apex, the a¡erent and e¡erent ¢bers constitute a bundle of longitudinally running ¢bers that are in contact with the OHC base; Sato et al. (1997), in the same preparations, showed evidence of synaptic connectivity among these ¢bers. In other, more basal areas, the synapses among the ¢bers also occur but they are much further removed from the neural pole of the OHC and this could constitute a more indirect, less e¡ective pathway for interactions among OHCs. Keeping with the tonotopic organization of the basilar membrane, this arrangement also demonstrates that a relatively greater convergence of receptor activity is present in the apex. Three-dimensional reconstruction studies do not directly address these functional di¡erences, though they o¡er important insights into the complex structure and organization of the cochlea. Acknowledgements The authors are greatly indebted to Dr. C. Robert Bagnell and Ms. Victoria J. Madden for their superb technical skills, their assistance in the preparation and sectioning of the tissue, and with the TEM evaluation
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of the sections. We would also like to thank Thomas Hazel and William Presson for considerable work on the 3-D reconstructions. This work was supported by Grants DC 001692 (D.W.S.) and DC 00114 (M.M.H. and O.W.H) from the National Institute for Deafness and Other Communication Disorders. Tissue was obtained from animals supported, in part, by NIH Grant RR 00163 to the Oregon Regional Primate Research Center.
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