The course and distribution of medial efferent fibers in the cochlea of the mustached bat

ItMIln¢. ELSEVIER

Hearing Research 102 (1996) 99 115

The course and distribution of medial efferent fibers in the cochlea of the mustached bat M.M. Henson a,,, D.-H. Xie b, R.H. Wynne c, J.L. Wilson d, O.W. Henson Jr. b Division of OtolaryngologylHead and Neck Surgery, Department of Surgery, University of North Carolina, Chapel Hill, NC 27599, USA b Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, NC 27599, USA Environmental Remote Sensing Center, University of Wiseonsin, Madison, WI 53706, USA d Department of OtolaryngologylHead and Neck Surgery, University of Indiana, Indianapolis, IN 46202, USA

Received 29 January 1996; revised 13 August 1996; accepted 27 August 1996

Abstract The course and distribution of medial olivocochlear (MOC) nerve fibers were studied in the cochlea of the mustached bat. This animal is of interest because of the very sharp tuning of the ear and fine frequency resolution in small frequency bands near 60 and 90 kHz. The MOC fibers arise from about 400 cells in the dorsomedial periolivary (DMPO) nucleus and they are distributed to approximately 4500 outer hair cells (OHCs), resulting in an average OHC unit size of 11.25. Individual fibers appear to have a small number of branches and each branch entering the tunnel of Corti terminates on a patch of OHCs. The patch size is typically 1-3 OHCs with the smallest average patch sizes in the regions tuned to 60 and 90 kHz. The majority of the MOC terminals are derived from the contralateral DMPO. Contralateral vs. ipsilateral projecting fibers are not preferentially distributed within any of the three rows of OHCs or within specific regions throughout most of the cochlea. It can be concluded that the main differences between the mustached bat's MOC system and that of most other mammals are: (1) origin from a single nucleus; (2) relatively small sizes of the patches; (3) a single terminal on each OHC; (4) a gradient in the size of the terminals but not in the number of terminals from row to row or from base to apex. Keywords: Cochlea; Olivocochlear; Bat; Efferent; PHA-L; AChE

I. Introduction T h e o l i v o c o c h l e a r n e u r o n s a s s o c i a t e d with the m a m m a l i a n i n n e r e a r are a r r a n g e d in l a t e r a l ( L O C ) a n d m e d i a l ( M O C ) g r o u p s . M O C nuclei o c c u r in the b r a i n stem where they are closely a s s o c i a t e d with fibers f r o m the c o c h l e a r nuclei. T h e l o n g d e n d r i t e s o f the i n d i v i d u a l M O C n e u r o n s are a s s o c i a t e d with d e s c e n d i n g fibers f r o m the inferior colliculus a n d , b a s e d on these a n a t o m ical r e l a t i o n s h i p s , M O C n e u r o n s are t h o u g h t to be a m o n g the best i n f o r m e d a u d i t o r y cells in the b r a i n stem ( W a r r , 1992). T h e a x o n s give off c o l l a t e r a l s to

* Corresponding author. Present address: Department of Cell Biology and Anatomy, Taylor Hall, CB #7090, Chapel Hill, NC 27599, USA. Tel.: +1 (919) 966-3801; Fax: +1 (919) 966-1856; E-mail: [email protected]

the c o c h l e a r nucleus b u t their m a i n s y n a p t i c targets a p p e a r to be the o u t e r h a i r cells ( O H C s ) . T h e o r g a n i z a t i o n o f the o l i v o c o c h l e a r system into l a t e r a l a n d m e d i a l s u b d i v i s i o n s was initially b a s e d on studies o f the cat b y W a r r (1975) a n d has since been d e s c r i b e d in m a n y o t h e r m a m m a l s , including a variety o f r o d e n t s , p r i m a t e s a n d b a t s ( W a r r a n d G u i n a n , 1979; W h i t e a n d W a r r , 1983; R o b e r t s o n , 1985; L i b e r m a n a n d Brown, 1986; T h o m p s o n a n d T h o m p s o n , 1986; A s c h o f f a n d O s t w a l d , 1987; B i s h o p a n d H e n s o n , 1987; Brown, 1987, 1989; L i b e r m a n et al., 1990). Interest in b a t s centers on a c o u s t i c specializations for echol o c a t i o n a n d the high s o u n d levels to which their ears are subjected d u r i n g e c h o l o c a t i o n a n d when the b a t s are in their n a t u r a l cave roosts. A m o n g the m o s t acoustically specialized b a t s are those t h a t D o p p l e r - s h i f t c o m p e n s a t e , i.e., a d j u s t the c o n s t a n t frequency c o m p o -

0378-5955/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved PH S 0 3 7 8 - 5 9 5 5 ( 9 6 ) 0 0 1 53-0

100

M.M, Henson et al./Hearing Research 102 (1996) 99 115

nent of their pulses to compensate for small changes in echo frequency induced by motion (Schnitzler, 1970a,b). There are two primary examples of this type of echolocation: the horseshoe bats (Rhinolophidae) of the old world and the neotropical mustached bats, Pteronotus parnellii. These species have specialized ("foveal") regions of the cochlea where the neurons are sharply tuned to narrow bands that correspond to the constant frequency (CF) components in their biosonar signals. Other regions of the cochlea process broadband, frequency modulated (FM) components (K6ssl and Vater, 1985; Zook and Leake, 1989). in spite of the similarities in the biosonar signals and acoustic environment of the rhinolophids and the mustached bats, it is well established that an MOC system is completely absent in the rhinolophids but well developed in the mustached bat (Bruns and Schmieszek, 1980; Bishop and Henson, 1987, 1988; Vater et al., 1992; Xie et al., 1993). The mustached bat's cochlea has two and one quarter turns plus a hook region. Frequency maps of the mustached bat's cochlea (K6ssl and Vater, 1985; Zook and Leake, 1989) indicate that the regions that process the CF biosonar signal components can be recognized on the basis of the size and density of myelinated nerve fiber trunks. As shown in Fig. 1, there are two distinct densely innervated regions. One of these occurs in the hook and beginning of the basal turn. Most of this region processes the bat's third harmonic, ca. 90 kHz CF component and it has been called the proximal densely innervated region (PDI) (Henson and Henson, 1991). Approximately 45-60% along the length of the duct there is a distal densely innervated (DDI) region which processes the bat's ca. 60 kHz, second harmonic CF. A large sparsely innervated (SI-1) region separates the two densely innervated areas and at the apical end of the SI-I region is a straight region (SR). Frequency maps indicate that the SI-1 and SR respond to frequencies not included in the bat's sonar signals. Other designated regions of the cochlea include a second turn and an apical quarter turn (apex). In the apical 25% of the cochlea there are two peaks in innervation density. The first is somewhat variable but seems to correspond to the ca. 30 kHz, first harmonic CF processing region. The second probably processes sounds below 20 kHz and some of the frequencies included in the bat's communication calls. The intervening sparsely innervated regions have been called SI-2 and SI-3. The purpose of this study was to examine the MOC input to the outer hair cells in these different regions and to correlate the anatomical findings with recent physiological observations. In the mustached bat it has been shown that there are finely graded, MOC induced changes (suppression) in cochlear mechanics when the contralateral ear is stimulated with broadband noise or natural bat sounds (Henson et al., 1995; Xie

and Henson, 1996). Data indicate that robust suppression may also accompany some types of vocalizations (Goldberg and Henson, 1996; Henson et al., 1996). Specifically, in this study we have: (1) examined the course and distribution of the MOC fibers in the sharply vs. broadly tuned regions; (2) compared the distribution of ipsilaterally and contralaterally projecting fibers; (3) determined the number of OHCs innervated by single tunnel crossing fibers and calculated the size of putative O H C " m o t o r " units in different areas; and (4) compared the organization of the system in the mustached bat with other species.

2. Methods

2.1. Animals used The animals used in this study were Jamaican mustached bats, Pteronotus parnellii parnellii. Permission to collect the animals for research was approved by the Natural Resources Conservation Authority and Ministry of Agriculture, Division of Veterinary Services of Jamaica. Their care and use were approved by the Institutional Animal Care and Use Committee at The University of North Carolina at Chapel Hill, Animal Assurance Number A3410-01.

2.2. Techniques Three techniques were used to study the MOC nerves: staining of surface preparations for acetylcholinesterase (ACHE) in isolated cochlear turns, labeling single fibers with the retrograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) and transmission electron microscopy (TEM) to examine fiber diameters and ultrastructural features. TEM results dealing with MOC fibers and terminals on OHCs of the mustached bat have previously been published (Bishop and Henson, 1988; Xie et al., 1993) and only a few additional comments will be made in this report. For harvesting the tissue, animals were deeply anesthetized with methoxyflurane (Metofane, PitmanMoore, Inc.) and killed by decapitation. The cochleae were rapidly removed and placed in fixative; the stapes and round window membrane were removed to allow better penetration of fixative. Tissue to be stained for AChE was washed in 0.9% NaC1 and placed in cold (4°C) fixative for 8 12 h. The fixative consisted of 4% paraformaldehyde, 0.5% glutaraldehyde, and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4. After fixation, cochleae were decalcified in 0. l M E D T A in 0,1 M phosphate buffer, pH 7.4 for 4 days in the cold. The E D T A solution was changed every 24 h. The decalcified cochleae were then cut into 4 segments (hook, basal turn, second turn and apex),

M.M. Henson et al./Hearing Research 102 (1996) 9~115

stained for acetylcholinesterase and viewed as whole m o u n t surface preparations. In several preparations, 50 ~tm serial sections of frozen AChE-stained brain stem and cochleae were made. These were especially valuable for tracing the efferent bundle in the vestibular root of the V I I I t h nerve, and into the intraganglionic spiral bundle. The staining procedures were carried out at r o o m temperature and followed the method of Tago et al. (1986), as modified and described by Xie et al. (1993, 1994). To study the distribution of crossed vs. uncrossed fibers, the crossed olivocochlear bundle (COCB) was sectioned in the floor of the fourth ventricle. This was accomplished by stereotaxically placing a sharpened tungsten electrode through a small hole in the dorsal surface of the skull near the COCB and then moving the electrode anteriorly and posteriorly over a distance of a few millimeters. This produced fine midline lesions which could easily be identified in coronal sections through the brain stem. The COCB was transected in seven animals and survival times after transection ranged from 5 days (one bat) to 2 3 weeks (six bats). Cochleae were removed, fixed, decalcified and stained as described above. The brain was placed in the same fixative and after 2 4 h was put into fresh fixative containing 30% sucrose; it remained in this solution in the cold overnight. The brain was then frozen and serially sectioned on a freezing microtome at 50 ktm. Sections were stained according to the procedure described for the cochleae, mounted on gelatin subbed slides and dried overnight. Finally, sections were counter-stained with cresyl violet, dehydrated, coverslipped with D P X and viewed with a Nikon Biophot microscope. Lesions could be accurately m a p p e d relative to the known position of the COCB and other structures in the brain stem. The effect of/the lesions was assessed by quantification of A C h E positive terminals in the two ears. The results with A C h E are based on observations of 11 normal animals (22 cochleae) and 7 animals in which the COCB was sectioned. Eleven mustached bats were used to inject P H A - L into the brain stem to label single fibers and terminals. The animals were anesthetized with Metofane and the head was firmly fixed in a custom-made head holder. The hair over the dorsal surface of the skull was removed with a chemical hair remover cream (Neet; Reckitt and Colman, Wayne, NJ) and the skin was cleaned with 95% ethanol. A midline incision through the scalp was made, the underlying portion of the temporalis muscle removed, and the exposed skull was cleaned so that the underlying inferior colliculi and other landmarks could be seen through the thin bone. A small hole was drilled near the posterior border of the right inferior colliculus for insertion of a glass micropipette through the cerebellum and into the brain stem.

101

The methods used for injection of PHA-L, tissue fixation and processing for identification of labeled cells and fibers have been described by Wilson et al. (1991). Both the AChE-stained and PHAL-labeled tissue were examined as surface preparations. Drawings were made with the aid of a drawing tube on a Nikon Biophot or an Olympus BH-2 microscope. Cochleae to be examined in the T E M were fixed in 2% paraformaldehyde, 2.5% glutaraldehyde and 2% sucrose in 0.15 M Karlsson and Schultz phosphate buffer, p H 7.4. Tissue was fixed at room temperature overnight and then placed in 0.1 M E D T A in phosphate buffer and decalcified over a period of 4 days. The tissue was dehydrated through a graded series of alcohols and then a graded series of propylene oxide and Polybed 812 to 100% resin. Cochleae were left in 100% resin overnight and embedded in fresh resin and polymerized at 60 ° for 24-48 h. Sections were cut at 70 nm on an L K B Ultratome V and stained with aqueous uranyl acetate and lead citrate. Tissue was examined on a Zeiss 10A T E M at 60 kV.

3. Results

3.1. General comments on labeling and A C h E staining When the superior olivary complex was iontophoretically injected with P H A - L on one side, labeled efferent fibers were observed in both the contralateral and ipsilateral cochleae. Most traceable fibers were in the large SI-1 and adjacent P D I regions of the basal turn. The number of labeled fibers varied greatly from specimen to specimen. Individual fibers in the osseous spiral lamina were often prominent (Fig. 2) but most were difficult to trace and when two labeled fibers crossed it was often not possible to determine which branch belonged to which fiber unless they were markedly different in size. Therefore, comments on branching patterns are limited to a few unambiguous cases where only a few fibers were labeled and where the labeling appeared complete. Labeled fibers in the tunnel of Corti and spaces of Nuel were usually easier to trace and terminals and en passant swellings were almost always darkly stained and prominent (Fig. 3). In some cases there was evidence of incomplete filling, i.e., a single fiber sometimes led to a well-filled terminal but an adjacent O H C terminal, although visible, was poorly labeled. Although the P H A - L labeling m a y have been incomplete in some cases, the data obtained with A C h E staining corroborated the P H A - L observations and conclusions. Individual AChE-stained fibers were especially evident and easy to follow after COCB transections reduced the population of M O C fibers and terminals up to 82% (see below).

M. M. Henson et al. / Hearing Research 102 (1996) 99 115

102

A

1.0

BASAL

TURN

MM

SECOND TURN

400

B

DDI

300

PDI

¢3

200

Z

0 r,l

Z

100

SI-I

0

20

40

60

BASILAR MEMBRANE

80

100

L E N G T H , ~g

Fig. 1. The regional organization of the cochlea based on the density of nerve fibers. A shows micrographs of surface preparations of the large basal turn (minus hook) and a smaller segment showing some of the second turn; the myelinated nerve fibers are darkly stained with Sudan black B. Quantitative changes in nerve fiber density are displayed in B. The PDI and DDI (proximal and distal densely innervated) regions are separated by a prominent SI-1 region. Apical to the DDI are sparsely innervated (SI-2 and SI-3) areas that lie to either side of a somewhat variable "apical densely innervated" region. The latter probably corresponds to the area that encodes the relatively weak, ca. 30 kHz, first harmonic CF of the bat's pulse; the prominent DD! is a region which responds to the ca. 60 kHz, second harmonic, and the PDI region is stimulated by the ca. 90 kHz, third harmonic CF. The apical portions of each densely innervated region represent areas that process FM components attached to the initial and terminal part of the CF. The SI regions appear to represent areas which process frequency bands not included in the bat's sonar signals. The straight region (SR) is a part of the SI-1 region where the spiral ligament is greatly enlarged and appears to distort the normal curvature of the cochlear duct. In the graph, the densities are shown from the base (0%) to the apex (100%). (Figure from Henson and Henson (1991), Hearing Research, with permission. Nerve density graph originally redrawn from Zook and Leake (1989).)

3.2. The course o f efferent fibers in the osseous spiral lamina B a s e d o n o b s e r v a t i o n s o f serial s e c t i o n s a n d A C h E s t a i n i n g , t h e g r e a t m a j o r i t y , if n o t all, M O C fibers r e a c h t h e i n t e r n a l a c o u s t i c m e a t u s as p a r t o f t h e v e s t i b u l a r r o o t o f t h e V I I I t h n e r v e ; t h e y l e a v e t h e v e s t i b u l a r divis i o n a d j a c e n t to t h e s a c c u l a r g a n g l i o n (as O o r t ' s a n a s t o m o s i s ) a n d t h e n j o i n a l a r g e n e r v e t r u n k t h a t is dist r i b u t e d to t h e h o o k a n d P D I r e g i o n s . A s the e f f e r e n t

fiber b u n d l e e n t e r s t h e o s s e o u s spiral l a m i n a it f o r m s t h e i n t r a g a n g l i o n i c spiral b u n d l e ( I G S B ) . A s s h o w n in Fig. 4, this p r o m i n e n t b u n d l e d o e s n o t j o i n t h e spiral g a n g l i o n in R o s e n t h a l ' s c a n a l until t h e m i d d l e o f t h e b a s a l t u r n (SI-1 region). F i b e r s t h a t l e a v e t h e I G S B g e n e r a l l y a c c o m p a n y a f f e r e n t fibers b u t m a n y s m a l l seco n d a r y spiral b u n d l e s (plexuses) arise f r o m t h e s e nerves. N o P H A - L - l a b e l e d M O C fibers w e r e f o u n d in t h e t r u n k o f the c o c h l e a r nerve. S o m e A C h E - s t a i n e d fibers w e r e seen in t h e n e r v e t r u n k b u t t h e m a j o r i t y o f t h e s e

M.M. Henson et al./Hearing Research 102 (1996) 99 115

103

the secondary spiral bundles was consistently about 100 gm from the edge of the osseous spiral lamina. Peripheral to this, the efferent fibers were typically straight and radially oriented; they accompanied the afferent fibers directly to the foramina nervosa (habenula perforata).

3.3. Fiber classification

Fig. 2. Micrograph of PHA-L-labeled MOC fibers in the osseous spiral lamina. Two fibers with markedly different diameters are shown. The smaller fiber is marked by arrows. Bar = 5 I.tm.

could be traced to type II ganglion cells and they clearly represented the central processes of these neurons (see Xie et al., 1994). In the apical part of the cochlea the ganglion cells are in the internal acoustic meatus rather than Rosenthal's canal. MOC fibers reach the osseous spiral lamina in the apex as extensions of one or two of the secondary bundles that represent extensions of the IGSB (Fig. 4). The position and appearance of the IGSB was similar in all preparations, but the patterns formed by the subsidiary spiral bundles varied from individual to individual. These bundles were poorly developed in the SI-1 region and prominent apical to the D D ! (Fig. 4). Throughout the cochlea the most distal of

The nerve fibers in the mustached bat's ear had the same basic characteristics as fibers described in a variety of other mammals. In AChE-stained surface preparations the intraganglionic spiral bundle had numerous, well-stained fibers. These could be easily traced to secondary spiral bundles and onward to the foramina nervosa, tunnel of Corti and OHCs. About 75-80% of the fibers with these characteristics were not seen after the COCB was sectioned in the midline of the floor of the fourth ventricle, and they were therefore of contralateral origin. All PHA-L-labeled fibers that projected to the ipsilateral cochlea and OHCs were classified as ipsilateral, uncrossed MOC fibers, while those distributed to the contralateral OHCs were designated as crossed, MOC fibers. Within the intraganglionic spiral bundle and osseous spiral lamina there were some very small AChE-stained fibers (see below) that could only be seen with a 100 × oil immersion objective. They were difficult or impossible to follow for any distance but a few of the larger ones could be traced to the region of the inner spiral bundle and inner hair cells. Fibers with these characteristics were classified as LOC fibers. Because of their small size, they did not contribute significantly to the

B

Fig. 3. PHA-L-labeled fibers and terminals in the region of the OHCs. In A, the characteristic appearance of single, large terminals at the base of 5 0 H C s is shown. In B the fibers leading to the OHCs are well labeled and each fiber has a number of en passant swellings; arrows mark the terminals. Bars = 5 gm.

M.M. Henson et al./Hearing Research 102 (1996) 99 115

104

SI-1

SR

.

IGSB

CF ' ,

-~ _ J . - ~ 1 ~ v u

SG

~

PDI

ISB DDI

ISB SB

DI

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0

l"

/

Fig. 4. Drawing of AChE-stained efferent fibers in a surface preparation of the mustached bat's cochlea. Regions identified in Fig. 1 which are also marked in this figure, include the PDI, SI-I, SR, and DDI. The apical densely innervated region (DI) is also shown. Features illustrated are the spiral ganglion (SG), intraganglionic spiral bundle (IGSB), secondary spiral bundles (plexuses) within the osseous spiral lamina (SB), and the radially oriented efferent fibers (white arrows) that extend from the secondary bundles to the region of the inner spiral bundle (ISB). Fiber representation peripheral to the ISB has not been included. The AChE-stained fibers marked CF are the central processes of OHC (type II) ganglion cells. The asterisk indicates a break in the preparation. Bar=100 gm.

c h a n g e s in n e r v e fiber d e n s i t y t h a t w e r e a p p a r e n t in s u r f a c e p r e p a r a t i o n s . S o m e A C h E - s t a i n e d fibers c o u l d be t r a c e d to A C h E - s t a i n e d g a n g l i o n cells. T h e s e f o r m e d

a s m a l l p a r t o f the n e u r o n a l p o p u l a t i o n a n d w e r e identified as O H C a f f e r e n t fibers ( t y p e II a f f e r e n t s ) ; t h e y f o r m e d p a r a l l e l r o w s o f fibers t h a t w e r e closely a p p l i e d

105

M.M. Henson et aL /Hearing Research 102 (1996) 99-115

III'IMIIII III IIIIIII Ill IIIIlflll III IIIIU III IPli II Ull illP .3!JIJUIJUE!L

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d

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.

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.

MOC FIBER DIAMETER Fig. 5. Histogram of myelinated MOC fiber diameters (in gm) based on measurements of cross-sectional profiles in TEM micrographs through the initial segment of the intraganglionic spiral bundle. Diameters do not include the myelin sheath.

to the floor a n d walls o f the t u n n e l o f Corti a n d spaces o f Nuel. A d j a c e n t to the spaces of N u e l they f o r m e d rows o f o u t e r spiral fibers; b r a n c h i n g s only occurred a l o n g the m o d i o l a r side of the Deiters' cells. There did n o t seem to be m o r e o f these O H C afferent fibers or g a n g l i o n cells in the densely i n n e r v a t e d region b u t a detailed analysis was n o t made. The type I ( I H C ) afterents did n o t stain with A C h E a n d they, like the type II afferents, were never labeled with P H A - L injected into the region of the D M P O .

A

3.4. F i b e r n u m b e r s a n d d i a m e t e r s

T E M sections t h r o u g h the I G S B showed the presence o f b o t h m y e l i n a t e d a n d u n m y e l i n a t e d fibers. The u n m y e l i n a t e d fibers were thin, with diameters typically in the 0.25-0.40 g m range ( m e a n = 0 . 3 2 g m ; N = 30 fibers). T h e i r location, small size a n d u n m y e l i n ated n a t u r e are consistent with L O C fibers described in other m a m m a l s (see Brown, 1987; W a r r , 1992). As in other m a m m a l s most, if n o t all, o f the thicker,

B

3

•

Fig. 6. Examples of branching patterns of PHA-L-labeled efferent fibers. In A, note that each branch has a single OHC terminal. Also note the hook-shaped part leading to the terminals; this shape is due to the recurrent course of the fibers in the long neural conduit. The fibers first contact the slanting Deiters' cell body at a point that is more lateral than the hair cell base; as the fiber ascends in the conduit it courses medially and upward. In B, the continuity of the daughter branches with the main fiber could not be traced but only one labeled fiber was seen to enter the area. These drawings illustrate the maximum (6) and minimum (1) patch sizes encountered. ISB = inner spiral bundle; 1, 2 and 3 indicate the rows of OHCs. Bars= 100 gm.

106

M.M. Henson et al./Hearing Research 102 (1996) 99-115

Fig. 7. Cochlear segments stained for ACHE. A shows a segment of the large basal turn with the junction between the PDI and adjacent SI-1 region indicated by the large arrow. Note the difference in size of the spiral ganglion (S) and radiating nerve trunks in the two regions. In the densely innervated region the AChE-stained efferent fibers can be distinguished within the lighter unstained nerve tissue. The AChE positive band (O) marks the position of the MOC terminals on the three rows of OHCs; the AChE positive band (I) marks the position where MOC fibers enter the region beneath the IHCs and where one or two prominent en passant swellings occur. The difference in density of efferent fibers in the sparsely and densely innervated regions can be appreciated by differences in the population and staining of straight radial fibers leading to the foramina nervosa (arrowhead) and by the change in density of the band of en passant swellings that occur just peripheral to these openings. This band is shown in greater detail in B. Examples of the prominent en passant swellings are marked with small arrows. The continuity of the swellings with tunnel crossing fibers is evident. The continuity of the tunnel crossing fibers with the OHC terminals is not evident because of changes in the focal plane. Bar in A = 100 gm; in B = 20 gm.

m y e l i n a t e d fibers in t h e I G S B b e l o n g to t h e M O C syst e m . U p to 300 fibers w e r e c o u n t e d in c r o s s - s e c t i o n s t h r o u g h t h e I G S B n e a r its e n t r y i n t o t h e o s s e o u s spiral l a m i n a . T h i s is a b o u t 7 7 % o f the n u m b e r o f M O C n e u r o n s e s t i m a t e d to o c c u r in the b r a i n s t e m ( B i s h o p a n d H e n s o n , 1987). M a n y fibers l e a v e the b u n d l e as it

Table 1 Average patch size as a function of regional difference in myelinated afferent nerve fiber densities Average patch size/100 gm Hook PDI SI-1 SR DDI SI-2 Second turn Apex

2.0 1.3 2.1 2.3 1.4 1.9 2.1 2.8

e n t e r s t h e o s s e o u s spiral l a m i n a to s u p p l y the o u t e r h a i r cells in the h o o k a n d P D I r e g i o n . T h e e x c l u s i o n o f these fibers in o u r c o u n t s p r o b a b l y a c c o u n t s f o r t h e d i s c r e p a n c y in t h e n u m b e r o f D M P O n e u r o n s a n d t h e n u m b e r o f m y e l i n a t e d fibers c o u n t e d in t h e I G S B . Fig. 2 s h o w s a n e x a m p l e o f P H A - L - l a b e l e d M O C fibers o f m a r k e d l y d i f f e r e n t d i a m e t e r s . T h e A C H E s t a i n e d m a t e r i a l also s h o w e d t h a t the fibers v a r i e d in size w h e r e t h e y c r o s s e d t h e t u n n e l o f C o r t i . T h i c k a n d t h i n fibers w e r e d i s t r i b u t e d to O H C s in all o f the t h r e e r o w s . I n g e n e r a l , t h e t h i n n e s t fibers i n n e r v a t e d 1 2 O H C s a n d the t h i c k e s t fibers w e r e d i s t r i b u t e d to 3 - 4 O H C s . A f t e r C O C B lesions, p o p u l a t i o n s o f t h i c k a n d t h i n fibers w e r e m a i n t a i n e d in e a c h ear. T h u s , as the P H A - L studies also i n d i c a t e d , M O C fibers o f d i f f e r e n t d i a m e t e r s arise f r o m b o t h the c o n t r a l a t e r a l a n d ipsilateral D M P O . A h i s t o g r a m b a s e d o n T E M s e c t i o n s t h r o u g h the m i d p a r t o f I G S B ( N = 1 6 3 fibers; o n e a n i m a l ) s h o w e d

107

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Fig. 8. Graph showing the population density of AChE-stained efferent fibers (A) and OHC (B) per 100 ~tm span. A shows the density of MOC fibers entering the tunnel of Corti (see Fig. 10) in 3 normal animals vs. 2 animals with COCB transected. The data in B were obtained from the same 3 normal animals. Note the increase in density of fibers in the foveal regions, PDI and DDI.

a range of axon diameters (not including myelin sheath) from 0.15 g m to 1.35 ~tm with a mean of 0.61 g m (Fig. 5). Thus, the mean diameter of the M O C fibers is about twice that of the L O C fibers.

3.5. Branching patterns and span length The branching of individual M O C fibers occurred in the IGSB, the peripheral secondary bundles, the region of the inner spiral bundle (ISB), the tunnel of Corti and as terminal arborizations in the spaces of Nuel a m o n g the OHCs. Fig. 6 shows two of five examples where PHA-L-labeled fibers could be followed throughout most of the osseous spiral lamina and to the OHCs.

In all cases, each branch had terminal aborizations that led to five or fewer O H C s ; fibers terminating on a single O H C were commonly found and in one case (Fig. 6A), each main branch terminated on a single OHC. For a given fiber, there was no preference for a particular O H C row. Two other fibers, although incompletely filled, gave off several branches over a distance of about 2.0 m m indicating that long span lengths do occur. In the five cases where fibers could be traced and where labeling appeared complete, the span lengths ranged from 150 ~tm to 450 g m ; thus span length appears to be highly variable. The number of branches in the OSL ranged from 2 to 5. It is possible that these fibers had branches prior to or within the osseous spiral lamina, but in general, where m a n y fibers were labeled with

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Fig. 10. Drawings of AChE-stained M O C fibers and terminals in a normal bat (A) vs. one with COCB transection (B) (12 day survival). Each drawing shows the fibers and terminals within a 150 g m longitudinal span of the organ of Corti. In A, the individual M O C fibers entering the tunnel of Corti can be counted (arrows) prior to the point where they usually join adjacent fibers to form entwined bundles. In this illustration 36 nerve fibers are distributed to 66 OHCs. Here the continuity of fibers with terminals a m o n g the O H C rows has been omitted because of the complexity and difficulty in following superimposed structures. In B, only a few normal M O C terminals remain and the individual fibers can be followed to most of the endings. The uncrossed terminals associated with the O H C s are relatively large and black. Small deposits of A C h E stain are visible in relation to all other OHCs; these deposits represent O H C afferent terminals (see Xie et al., 1994).

PHA-L, almost all had branches that arose peripheral to the IGSB. Within the OSL the number of branches observed was in general agreement with the number expected, i.e., based on an estimate of 1500 foramina nervosa in the mustached bat's cochlea and 400 M O C fibers, the average number of branches would be 3.75. In general, AChE-stained fibers were evenly spaced near the edge of the OSL (see Fig. 7B). This suggests that most foramina nervosa contain a single M O C fiber.

3.6. The density of MOC fibers One of the most striking characteristics of the ACHEstained surface preparations was the high density of M O C fibers leading from the secondary spiral bundles to the O H C s in P D I and D D ! regions. In some specimens a third area of dense innervation occurred more apically (Fig. 4). As shown in Figs. 4 and 7, the junctions between regions of high and low efferent fiber

density were distinct and corresponded to the changes in density of the I H C (type I) afferent fibers. Although type II afferents were also AChE positive, they were not associated with the secondary spiraling bundles and there was no indication of marked changes in type II innervation density at the sparse-dense junctions. Thus innervation density changes are due primarily, if not entirely, to the M O C fiber population. This is further shown by the absence of density peaks in AChE-stained material after COCB lesions. Fig. 8A illustrates changes in fiber density as a function of cochlear distance in normal animals vs. COCB lesioned animals; here the different regions based on afferent nerve density are also identified. O H C counts (Fig. 8B) showed some, but not a complete, correspondence between the density of the M O C fibers and the O H C s when measured in the same specimens. The absence of density peaks after COCB transection suggests that the normal increase in fibers in the densely innervated regions might be due to contralateral input;

M.M. Henson et al./Hearing Research 102 (1996) 99-115

109

Fig. 11. TEM micrograph showing prominent, vesicle-filled swellings in the region below the IHCs. These swellings (arrows) are interposed among the mitochondria-rich IHC afferent fibers and according to their size and position, appear to represent the large en passant swellings of MOC fibers. This section is just superior to the level of the ISB. IP = inner pillar cell. Bar = 1 ~tm.

however, a 25% change in the small number of remaining fibers, roughly the maximum percentage change between sparse and dense regions, would represent only 1.25 fibers in the small uncrossed population and this would not result in a significant peak on the graphs. 3. 7. O H C unit size

The data for fiber density suggest that the MOC "unit size" (the number of OHCs innervated by a single MOC fiber) might be smaller in the densely innervated foveal regions than in other regions. Calculation of unit size is, however, dependent on knowing how many branches each fiber has and how many hair cells are contacted by each branch. This information is available

for only a few fibers, such as those shown in Fig. 6. Here one fiber has a unit size of 6 and the other 15. Other traceable fibers had unit sizes of 4, 5 and 6. We estimate an average unit size of 11.25; this is calculated by dividing the estimated number of MOC neurons in the D M P O (400) (Bishop and Henson, 1987) into the number of OHCs in the cochlea (4500). 3.8. Patch size

Although data for unit size are scant, patch size could be determined for a large number of fibers in many different areas. Patch size is defined as the number of OHCs innervated by a single upper tunnel crossing fiber. One method of measuring patch size is to

110

M. M. Henson et al. / Hearing Research 102 (1996) 99 115

count the number of PHA-L-labeled terminals associated with a labeled upper tunnel fiber. Patch size determined by this technique was always less than 4 and usually only 1 or 2, as shown in the histogram in Fig. 9. Another way of calculating patch size is to count the number of AChE-stained efferent fibers entering the tunnel of Corti over a distance of 100 ~tm and dividing this into the number of OHC terminals within this span; this gives the average patch size. This was done in three normal cochleae and in two others after COCB transection; Fig. 10 shows drawings of the fibers and terminals seen under these conditions. In the cochleae of COCB transected animals 72-82% of the fibers and terminals degenerated and thus the individual patches formed by ipsilateral projecting fibers could be seen (Fig. 10B). Although the tunnel crossing fibers enter the tunnel of Corti as separate fibers, they join up to five adjacent fibers to form entwined bundles; the number of fibers entering the tunnel could be determined with little ambiguity (Fig. 10A). The values obtained for the average patch size/100 ~m span were similar to those determined by tracing PHA-L-labeled fibers. Table 1 shows the average patch size for different regions of the cochlea. The values which correspond to those at the approximate center of each region were derived from the data on fiber and O H C density shown in Fig. 8. Note that the smallest average patch sizes occur in the foveal regions (PDI and DDI) where the density of myelinated afferent nerves is the highest (Fig. 1). No significant difference in patch size was found between crossed and uncrossed fibers in any region in either the AChE or P H A - L materal. 3.9. The distribution o f crossed and uncrossed fibers to O H C rows

The distribution of crossed vs. uncrossed fibers was studied by counting the number of PHA-L-labeled terminals in the ears of four animals where the iontophoretic injections were clearly limited to one side and where labeling was extensive throughout the cochlea. Table 2 provides data for each row of OHCs and for the cochlea as a whole. In three of the four bats shown

in Table 2, nearly 80% of the terminals were from crossed fibers; the mean for the four was 76.9%. These data show no pronounced difference in distribution of the terminals of the crossed vs. uncrossed fibers among the three rows of OHCs. In addition to studying the row by row distribution of the crossed and uncrossed MOC fibers we also examined the distribution in different regions of the cochlea. No differences were found in the hook, PDI, SI-1 or DDI regions (average crossed= 80%+2.5%). Percentage values for the second turn and apex also averaged about 80 but the variation was high ( + 20%). 3.10. Additional notes on M O C fibers

An interesting feature of most MOC fibers was the presence of one or two, somewhat irregular-shaped en passant swellings in the region of the ISB. The long straight fibers leading to the foramina nervosa usually lacked en passant swellings but as soon as the fibers passed through the foramina the swellings were conspicuous. These are largely responsible for the dark band of AChE staining at the peripheral end of the straight fibers (Fig. 7). T E M micrographs showed single, large swellings filled with synaptic vesicles and interposed among the IHC afferent fibers as the latter ascended toward the IHCs (Fig. 11). An important question is whether they represent the dilated segments of MOC fibers or are associated with LOC fibers. In T E M micrographs through the region of the ISB, many small, vessicle-filled swellings were observed, in addition to large, single swellings. It is clear from the AChE staining that the MOC fibers do not have numerous small dilations or terminals in the regions of the ISB. Although the large vessicle-filled swellings seen in TEM probably belong to the MOC system, verification will be dependent on T E M serial sections showing the continuity of tunnel crossing fibers with these swellings. Whether or not these swellings have synaptic relations with other fibers in the area will also require further study. Another point of interest is that although MOC fibers usually extended straight through the region of the ISB, it was not unusual for fibers to spiral

Table 2 The distribution of PHA-L-labeled terminals in the ipsilateral and contralateral ears of four preparations with the heaviest labeling Animal no. (cochlea)

OHC row 1 (%)

OHC row 2 (%)

OHC row 3 (%)

N

Ave (%)

3 3 6 6 7 7 8 8

25 34 44 29 45 36 16 30

41 34 30 35 33 30 45 40

33 31 26 36 21 34 39 30

36 152 66 265 43 218 60 115

21 79 19.9 80.1 17.5 82.5 34 66

(ipsilateral) (contralateral) (ipsilateral) (contralateral) (ipsilateral) (contralateral) (ipsilateral) (contralateral)

Total no. terminals, 995; mean ("/, crossed), 76.9.

M.M. Henson et al. / Hearing Research 102 (1996) 9 ~ 1 1 5

apically or basally within the ISB before passing into the tunnel of Corti. The spiral distance traveled was usually only over the distance of one or two inner hair cells. Some fibers branched in the area of the ISB. En passant swellings were also a characteristic feature of MOC fibers crossing the tunnel of Corti and they occurred on the terminal arborizations (see Fig. 2). The en passant swellings on the terminal arborizations were often close to the OHCs but T E M serial sections showed that en passant swellings did not contact the OHCs. In order to reach the neural pole of the OHC, an MOC fiber must pass through a neural conduit in the cup-shaped part of Deiters' cell. This conduit is relatively long and because of the pronounced slant of the OHCs and Deiters' cells toward the modiolus, the terminal part of each MOC fiber often appeared to be hook-shaped in surface preparations (Fig. 6). T E M serial sections showed that each neural conduit contains only one efferent fiber and from 4 to 8 afferent fibers. In the mustached bat, the deep Deiters' cup encloses a substantial part of the OHC. N o efferent fibers were observed to ascend on the outside of the cup to reach high up on the sides of the OHC, nor were MOC fibers observed to synapse with any of the O H C afferent fibers.

4. Discussion

4.1. Synopsis of the M O C system in the mustached bat This study, with other publications from our laboratory (Bishop and Henson, 1987, 1988; Xie et al., 1993), provide a fairly complete picture of the MOC system in the mustached bat. The important CNS features include the origin of the fibers exclusively from about 400 neurons in the DMPO, the occurrence of ipsilaterally and contralaterally projecting fibers and the joining of MOC and LOC fibers to form an olivocochlear bundle which extends into the vestibular division of the VIIIth nerve. About 77% of the fibers are involved in the formation of the COCB which lies close to the floor of the fourth ventricle. Peripherally, the efferent fiber bundle leaves the vestibular division just distal to the saccular ganglion and then enters the osseous spiral lamina as the IGSB. Fibers leaving this bundle radiate directly to the organ of Corti or they form spiral fascicles from which radially oriented fibers arise. The latter pass through the foramina nervosa to reach the region of the ISB. Here they have prominent vesicle-filled en passant swellings. The peripheral extensions of these fibers cross the tunnel of Corti as upper tunnel crossing fibers or bundles. The individual fibers may be distributed to a single OHC, or arborize into 2-6 terminal branches which innervate a patch of OHCs. Each terminal branch accompanies about 6 (4-8) O H C afferent fibers

111

through a long neural conduit in the base of the cupshaped portion of Deiters' cell (Bishop and Henson, 1988). At the base of the OHC, there is almost always only one, large, centrally located terminal that is surrounded by a ring of afferent fibers. In the mustached bat, the average O H C unit size is estimated as 11.25 and the patch size is 6 or fewer and often only 1-3. The average patch size is smallest in the "foveal regions" that process the 60 and 90 kHz constant frequency components of the biosonar signals. The MOC innervation is constant from O H C row to row and base to apex but the terminals show a size gradient, with the largest average size occurring in the first row and the smallest in the third; the average size is greater in the foveal regions than in adjacent areas. The distribution of crossed and uncrossed fibers is similar throughout the cochlea and from O H C row to row, but the apex appears to have a higher percentage of uncrossed fibers. 4.2. Comparison with other mammals Many of the features of the mustached bat's MOC system are basically similar to those described for common laboratory mammals (cats, guinea pigs, rats, mice, monkeys and humans). Some of the most notable differences between the mustached bat and these other mammals are: (1) the brain stem origin from a single nucleus; (2) the lack of a row by row or base to apex gradient in the number of terminals on the OHCs; (3) a small O H C unit or patch size; and (4) the lack of marked differences in fiber distributions between rows and in apical vs. basal cochlear segments. Data on the brain stem origins of MOC fibers in the mustached bat and other species and the lack of base to apex and row to row gradients in the distribution of terminals have been discussed in previous works from our laboratory and will not be reviewed here. One of the main findings of the present study was the marked increase in the number of MOC fibers in the foveal regions compared with adjacent regions (Fig. 3, Figs. 6 and 7). This increase is probably related to the small O H C unit size, and most certainly to small patch sizes. Because of the small number of adequately labeled, traceable fibers, it is not possible to relate unit size to specific frequency processing regions. We estimated an average unit size of 11.25 by dividing the number of OHCs by the maximum number of neurons labeled by the retrograde transport of HRP. The O H C unit sizes, determined for five labeled fibers, ranged from 16 to 4. Other mammals for which some data on unit size are available include the guinea pig, gerbil, mouse and cat. In guinea pigs (Robertson, 1984; Robertson and Gummer, 1985; Brown, 1987, 1989) and cats (Liberman and Brown, 1986) MOC fibers innervate at least 15 and up to 100 OHC. Brown (1989) recon-

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M. M. Henson et al. / Hearing Research 102 (1996) 99-115

structed 6 fibers and found an average unit size of 36 with a range of 15-61. In cats, MOC efferents seem to innervate even more OHCs over a broader span (Liberman and Brown, 1986) and the patch sizes are large. In the laboratory mouse (Wilson et al., 1991) and postnatal gerbil (Simmons et al., 1990) patch sizes appear to be small (1-5) and not significantly different than the mustached bat. In the latter, however, it seems clear that many upper tunnel crossing fibers innervate only 1 - 3 0 H C s . Although patch size may be small in mice and gerbils, the OHCs in these animals are generally innervated by several MOC terminals, rather than the single terminal seen in the mustached bat. The only other mammal where a single terminal appears to be common is the chinchilla, but even in this species the OHCs may have 2-3 terminals (Iurato et al., 1978). In guinea pigs, 6-15 efferent terminals are commonly associated with a single O H C and multiple terminals are also characteristic of cats and primates (Saito, 1980; Hashimoto and Kimura, 1987; Liberman et al., 1990). Potentially, the presence of a single, large MOC terminal and small unit sizes translates into fine control of efferent induced changes in the mechanical properties of the inner ear; most notable would be control of the damping characteristics of the cochlear partition as recently demonstrated by Henson et al. (1995). In the mustached bat, finely graded suppression of damping has been demonstrated in response to changes in the level of broadband noise presented to the contralateral ear. In other mammals graded MOC induced effects can also be demonstrated, but whether the gradation is finer in one species than another is not known. Many different functions can be assigned to the mechanical control provided by MOC activity as they relate to signal perception. Some of the main effects demonstrated for other mammals seem to be: (1) antimasking or improvement of signal detection in noise (Dewson, 1967, 1968; Nieder and Nieder, 1970; Winslow and Sachs, 1987; Dolan and Nuttall, 1988; Kawase and Liberman, 1993; Kawase et al., 1993); (2) giving the system or segments of the system a mechanical bias that can be constantly adjusted according to the background (e.g. Siegel and Kim, 1982; LePage, 1987, 1989, 1990); and similarly, setting the gain for sensitive amplification mechanisms (Davis, 1983; Sziklai and Dallos, 1993). Protection of the delicate ear from overload, temporary threshold shifts and mechanical trauma have also been advocated; the literature is extensive and often controversial (see for example, Cody and Johnstone, 1982; Handrock and Zeisberg, 1982; Rajan and Johnstone, 1983, 1988, 1989; Guinan and Gifford, 1988a,b,c; Puel et al., 1988; Warren and Liberman, 1989a,b; Rajan, 1990; Liberman, 1991; Patuzzi and Thompson, 1991 ; Pujol, 1994; Liberman and Gao, 1995; Reiter and Liberman, 1995). It is interesting to note that the MOC reflex in the

mustached bat is as fast as the middle ear muscle reflex and preliminary data indicate that phasic suppression, probably MOC induced, occurs during vocalization of communication sounds (Goldberg and Henson, 1996). The MOC system also appears to be tonically active when mustached bats are in their noisy roosts and during sustained periods of echolocation (Xie and Henson, 1996). The system could play a role similar to the middle ear muscles in terms of controlling energy levels reaching the receptors. The middle ear muscles contract prior to pulse emission, become maximally contracted when the pulse is being emitted, and relax within a few milliseconds after the end of the pulse (Henson and Henson, 1972; Suga and Jen, 1975). In this way, the ear is kept in a sensitive state for the perception of echoes and the muscles can function as a volume control or as a means of controlling the echolocative depth of field (Henson, 1965; Simmons and Kick, 1984). One potential advantage of MOC-induced suppression over that provided by middle ear muscle contractions is a more precise control of suppressive effects over limited frequency bands and it is the small size of the O H C units in the bat that could make this effect especially selective. The average smaller patch size in the foveal regions compared with the sparsely innervated regions suggests smaller unit sizes and this hypothetically translates into finer mechanical control in those regions concerned with fine frequency analysis, i.e. the regions associated with Doppler-shift compensation and motion detection. It should be noted, however, that to date, phasic changes of cochlear damping with vocalizations have been reported only for broadband communication sounds. In other mammals, studies have revealed differences in the distribution of MOC fibers in basal vs. apical regions of the cochlea. One common finding is that after sectioning the COCB, the MOC fibers and terminals in basal regions show much greater degeneration than in apical turns (Iurato et al., 1978; Nakai and Igarashi, 1974). This was also the case in the mustached bat (Fig. 8). On the basis of these and other differences, it has been suggested that the crossed and uncrossed MOC fibers might have different functions and that uncrossed fibers do not simply represent a less numerous mirror image of the crossed system (Robertson et al., 1987). In the mustached bat, with a single ending on each OHC from base to apex, it would be difficult to reach such a conclusion. Some of the other differences reported to occur in apical regions of several species (cats, guinea pigs, chinchillas and monkeys) include the presence of efferent fibers that climb the sides of the OHC to terminate above the level of the nucleus, ramification of the efferent fibers among the supporting cells of Deiters and Henson, and contacts and synaptic relationships with the dendrites of O H C afferent fibers (Wright and

M.M. Henson et al./Hearing Research 102 (1996) 99-115

Preston, 1973; Nakai and Igarashi, 1974; Iurato et al., 1978; Stopp and Comis, 1979; Liberman and Brown, 1986; Liberman et al., 1990). In the human, Nadol and Burgess (1994) observed supranuclear efferent nerve endings on outer hair cells and Deiters' cells throughout the cochlea. None of these features were observed in the mustached bat. The more stable pattern in the bat may be related to the origin of MOC fibers from a single nucleus and/or the fact that the apex of the bat's cochlea processes relatively high frequency sounds compared with other species (K6ssl and Vater, 1985; Zook and Leake, 1989). Although no synaptic contacts between MOC fibers and the O H C afferent dendrites were observed, we were impressed by the large en passant swellings in the region of the ISB (Fig. 11). These swellings were seen as direct extensions of the MOC fibers as they entered the region and peripheral extensions could be followed into the tunnel of Corti (Fig. 7B). These features, and the fact that most degenerated after sectioning the COCB, indicate that they are derived from the crossed MOC fibers and are not components of the numerous ipsilateral LOC fibers which also invade the region of the ISB. Further studies of serial T E M sections are needed to determine if these swellings have synaptic relationships with other fibers. Some investigators have noted that synapses between M O C and radial afferent fibers occur (Iurato et al., 1978; Liberman et al., 1990); others have looked for, but not found, them (Brown, 1989), or have described relationships that meet some but not all of the criteria for synapses (White, 1986). The functional significance of such contacts is not known, but the size and position relative to the afferent fibers suggest that they may have an important function.

Acknowledgments We wish to acknowledge the technical assistance of Arthur Keating. This work was supported by N I H Grant N I D C D DC 00114.

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