Hearing Research, 31 (1987) 175-182

Elsevier

175

HRR 01005

The efferent cochlear projections of the superior olivary complex in the mustached bat

A.L. Bishop * and 0-W. Henson, Jr. Department of Anatomy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. U.S.A

(Received 23 June 1987; accepted 30 August 1987)

Following the placement of horseradish peroxidase in the scala tympani, labeled neurons were found in the ipsilateral interstitial

nucleus (INT) and throughout the ipsilateral and contralateral dorsomedial periolivary nuclei (DMPO). The neurons in the INT were

morphologically distinct from those in the DMPO. The INT neurons formed a thin shell over the lateral superior olivary nucleus

(LSO) and their dendrites extended into the body and hilar region. The DMPO neurons had long, tapering dendrites that extended in

every direction. Data indicate that the crossed fibers in the floor of the ventricle arise entirely from the DMPO while uncrossed

olivocochlear fibers originate in the INT and DMPO. It was estimated that 75% of the efferent fibers arise from the INT and 25%

from the DMPO. Approximately 70% of the efferent neurons in each DMPO project to the contralateral cochlea via the crossed

olivocochlear bundle. The number of olivocochlear neurons associated with each ear was determined to be approximately 1585. This

number is similar to that found in cats and guinea pigs, but the number of neurons per unit length of the basilar membrane is

considerably higher in the mustached bat than in other species examined to date. The compact, restricted locations of the neurons in

the INT and DMPO in the mustached bat are different from those described for most other mammals and the arrangement in the

mustached bat offers advantages over other species for future anatomical and physiological studies.

Brainstem: Auditory system; Cochlear efferent; Superior olivary complex; Olivocochlear neuron: Bat

Introduction

The auditory system of the mustached bat has been the subject of numerous anatomical and physiological studies. The interest in this system in Pteronotus parnellii stems from its remarkable Doppler-shift compensation sonar system (see Schnitzler, 1970a, b; Kobler et al., 1985). Specific interest in the olivocochlear neurons follows from

the observation that in P. parnellii there is a single efferent nerve ending on each outer hair cell (Bi- shop and Henson, 1987). Other Doppler-shift

compensating bats (Rhinolophw and Hippo- sideros) are unusual in that they have no efferent

Correspondence to: O.W. Henson, Jr., Department of Anatomy,

Swing Building 217H, The University of North Carolina,

Chapel Hill, NC 27599, U.S.A.

* Present address: Glaxo Inc., New Product Development

Division, Five Moore Drive, Research Triangle Park, NC

27709, U.S.A.

fibers to the outer hair cells of the cochlea (Bruns and Schmieszek, 1980; Bishop and Henson, 1987).

The purpose of this report is to describe the origins of the efferent olivocochlear system in the mustached bat and to compare the organization of the system in this species with that known for other mammals. In this study we will show that

the efferent system of Pteronotus parnellii resem- bles that of most other mammals, yet it has some attributes that make it well suited for further anatomical and physiological studies to explore the role of the efferent system in biosonar imaging as well as hearing in general.

A preliminary report dealing with the results of this study was presented at the Third Interna-

tional Animal Sonar Systems Symposium in Helsingor, Denmark (Bishop and Henson, 1987).

Materials and Methods

The animals used in this study were Pteronotw p. parnellii from Jamaica, W.I. In order to identify

0378-5955/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

176

the origins of crossed and uncrossed olivocochlear neurons, horseradish peroxidase (Type VI, Sigma Chemical Co., St. Louis, MO) was placed unilater- ally in the Scala tympani of five bats. The animals

were first anesthetized with Metofane (meth- oxyflurane; Pitman-Moore, Washington Crossing, NJ). They were placed in a custom-made device in which the head was firmly held while the teeth

were pressed into a dental impression plate. The round window was exposed and incised via a

lateral surgical approach. The introduction of horseradish peroxidase

(HRP) into the cochlea was made by one of two methods. In the first method, perilymph was blotted from the scala tympani with a small piece of cotton. Approximately 2.0 pl of a 40% solution (by weight) of HRP in sterile 0.9% saline was then injected into the Scala tympani with a 25 ~1 syringe. In the second method, a small pellet was made by mixing HRP with a few microliters of sterile saline and allowing the mixture to dry. The pellet, which

weighed approximately 1 mg and had a diameter

of about 1 mm, was placed in the Scala tympani with fine forceps or on the moistened end of an etched tungsten rod. In both procedures, the round window was sealed with bone wax and the skin sutured with silk. Survival times ranged from 24 to 54 h, during which time the animals were kept in a temperature and humidity controlled recovery area. All surgical procedures were performed un-

der sterile conditions. In order to identify the origins of crossed

olivocochlear neurons and the course of their axons in the brainstem, HRP was placed on the cut ends of fibers belonging to the crossed olivocochlear bundle (COCB) in the floor of the fourth ventricle. To expose the COCB, the part of the skull overly- ing the cerebellum was cut with a scalpel and the thin bone was reflected posteriorly as a flap. The

vermis of the cerebellum was then aspirated with a glass pipette attached to a vacuum pump. The COCB was cut by making a shallow incision about 2 mm long in the midline between the facial colliculi. HRP crystals picked up on the moistened end of a finely etched tungsten rod were placed directly on the tissue at the lesion site. The bone flap was then placed back in its normal position, bone wax was applied, and the skin sutured with silk. These studies were carried out on four

animals. Survival times ranged from 8 to 22 11. The brains were prepared for HRP histochem-

istry by anesthetizing the animals with Metofane and perfusing them with a solution of 1.25% sodium nitrite and 0.25% polyvinylpyrrolidone in

0.1 M phosphate buffer (pH 7.4) followed by 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The brain was removed and stored overnight

in a 30% solution of sucrose in phosphate buffer. Sections of the brainstem were cut transversely at

a thickness of 40 pm on a freezing microtome and reacted with 3,3’,5,5’-tetramethyl benzidine (Sigma

Chemical Co.) according to the method of Mesu- lam (1978). The sections were then mounted on glass slides, counterstained with neutral red, covered with a coverslip, and examined by light microscopy.

Drawings of brainstem sections were made with the use of a drawing tube attached to a micro- scope and the locations of labeled cells were placed on the drawings. To minimize the possibility of

counting a neuron twice, labeled cells were in- cluded in counts only if it appeared that the

majority of the perikaryon was within the section. Nissl and myelin stains were used on serial

sections of the brainstem so that the organization of the superior olivary complex could be under- stood. Descriptions of the brainstem nuclei by Zook (1979) and Zook and Casseday (1982a, b)

were used to classify different brainstem nuclei and the nomenclature adopted by them is used in this report.

Results

Following placement of HRP in the cochlea, labeled olivocochlear neurons were localized in two regions of the superior olivary complex (SOC) (Fig. 1). Labeled neurons were found in the ipsi-

lateral interstitial nucleus (INT) and ipsilaterally and contralaterally in the dorsomedial periolivary nucleus (DMPO). Labeled neurons in the INT were densely packed and formed a ‘shell’ lying just outside the lateral superior olivary nucleus (LSO). No labeled neurons were found within the LSO, but labeled dendrites extended into the body and hilar region. The labeled neurons bordering the LSO were arranged in different positions de- pending on the level of section; at caudal levels

Fig. 1. Drawings of cross-sections through the brainstem of

Preronorus parnellii parnelh showing nuclei of the superior

olivary complex and the localization of label in the INT and

DMPO after the injection of HRP into the right cochlea. Note

that the label is restricted to the ipsilateral INT and the

ipsilateral and contralateral DMPO. The most caudal section

in the series is A, and the most rostra1 is D. CN, cochlear

nucleus; DMPO, dorsomedial periolivary nucleus; INT, inter-

stitial nucleus; LSO, lateral superior olive; MNTB, medial

nucleus of trapezoid body; MSO, media1 superior olive.

they were situated at the dorsolateral margin of

the LSO; at more rostra1 levels they were along its dorsal and dorsomedial boundary: at the most rostra1 level, labeled neurons were medial and ventromedial to the LSO (Fig. 1).

The Iabeled neurons in the DMPO were found throughout the rostrocaudal extent of the nucleus. These cells were densely packed and most numer- ous in the caudal portion of the nucleus; the density of labeled cells progressively decreased at more rostra1 levels (Fig. 1).

Labeled neurons in the INT differed from those in the DMPO (Fig. 2). INT neurons were fusiform with their long axes oriented transversely and parallel to the adjacent margin of the LSO. No

Fig. 2. Profiles of HRP labeled cells and processes in the

DMPO (A) and INT (B). These illustrations show the morpho-

logical differences in the shape and orientation of the cells and

their processes. The DMPO cells are multipolar with dendrites

extending in all directions; the INT cells are fusiform with

their long axes oriented transversely.

apparent differences in size or shape were oh- served between cells labeled in different regions of

the INT. All neurons in the INT appeared to be labeled when HRP was placed in the cochlea. By contrast, labeled cells of DMPO were larger than the labeled cells of the INT and they had long tapering dendrites which extended in every direc- tion.

When HRP was applied to the cut fibers along the floor of the fourth ventricle, the background labeling varied according to the size of the lesion. In two animals with extensive lesions, large ovoid cells were labeled in the LSO on each side along with fibers and cells of the reticular formation. The ovoid, LSO cells were presumably labeled when their contralaterally projecting. ascending

Fig. 3. Summary of the distribution of olivocochlear neurons determined by injection of HRP into the cochlea and by application of HRP to the cut ends of COCB fibers. DMPO, dorsomedial periolivary nucleus; MSO, medial superior olivary nucleus; INT, interstitial nucleus; LSO, lateral superior olivary

nucleus.

axons were included in the deep cuts. Cells in the DMPO, however, were labeled in all animals with a consistent pattern They were clustered in caudal portions of the DMPO and their size and shape resembled cells labeled in the DMPO following co&ear apportion of HRP. The dense layer of labeled INT neurons, clearly demonstrated with deposits of HRP in the cochlea, was not seen in

any of the cases where HRP was applied only to the crossing fibers.

Fig. 3 is a summary of the distribution of olivocochlear neurons determined from both methods of labeling. The numbers in different loci are based on the experiment giving the most com- plete reaction follo~g unilateral placement of HRP in the cochlea. The distribution and per- centages of neurons in other cases were compara- ble. The total number of labeled neurons in the most complete case was 1585. Of these, 117 (8%) were in the ipsilateral DMPO and 272 (17%) in the contralateral DMPO. Thus, 389 (25%) of the olivocochlear (OC) neurons were in the DMPO. The remaining 1196 (75%) were in the ipsiiateral INT. Of the OC fibers arising from the DMPO, 70% cross in the floor of the fourth ventricle.

The course of COCB fibers in the brainstem distal to the decussation in the floor of the fourth ventricle is shown by the labeled processes filled with HRP reaction product distal to a midline injury (Fig. 4). These fibers pass between the descending root of the Vth nerve and the cochlear nucleus before exiting from the brainstem with

Pig. 4. The course of the COCB within the brainstem of Pteronotusparnellii. The fibers were labeled by placing HRP on the cut ends of the COCB fibers in the floor of the fourth ventricle. L, midline lesion; CN, cochlear nucleus; COCB, labeled COCB fibers.

179

fibers of the vestibular division of the VIIIth nerve.

Discussion

We have shown that the injection of HRP into one cochlea results in the labeling of cells in the contralateral and ipsilateral DMPO and in the ipsilateral INT. On the basis of these observations

we conclude that all auditory OC fibers in P.

parnellii arise from neurons within these two

nuclei. From the labeling pattern it is clear that each INT projects to the ipsilateral ear while each

DMPO projects to both the contralateral and ipsilateral ear. It is possible that some neurons might innervate both ears, but double labeling studies on other animals (guinea pig and squirrel monkey) indicate that only a small percentage of OC neurons do this (Robertson and Gummer, 1985; Thompson and Thompson, 1986; Robertson et al., 1987; Cole et al., 1987).

The application of HRP to cut fibers of the COCB in the floor of the fourth ventricle resulted in the labeling of neurons in the right and left DMPO, but not in either INT. These findings, as

well as the labeling patterns seen with cochlear injections, confirm that the COCB arises exclu- sively from DMPO contributions and that the INT gives rise only to uncrossed fibers. Since the DMPO appears to be composed largely, if not entirely, of efferent neurons (see below) and the labeled COCB cells were restricted mostly to the caudal portions of the DMPO, it appears that the DMPO neurons which give rise to uncrossed fibers may be segregated from those associated with

crossed fibers. The labeled neurons in the INT and DMPO of

P. parne~l~j have the same anatomical features (size, shape and dendritic organization) as the two major groups of OC neurons described for cats,

guinea pigs and rats (Warr, 1975, 1980; Altschuler et al., 1983; Guinan et al., 1983; White and Warr, 1983). Thus, in P. parneilii, like other mammals

studied to date, the OC neurons can be arranged in two major groups (medial and lateral) depend- ing not only on their size and shape but also on their location within the SOC. Evidence strongly indicates that medial group efferents in mammals are distributed to the outer hair cells while lateral

group efferents terminate in relation to the affer- ent fibers associated with inner hair cells (Iurato, 1974; Warr and Guinan, 1979; Guinan et al., 1984). One of the most interesting aspects of the OC system in P. parnetlii is that the lateral group efferents are found exclusively in the INT and the medial group exclusively in the DMPO.

If all fibers arising from the DMPO of P.

parndii project to the outer hair cells (OHCs) it must be concluded that some OHCs are in- fluenced by contralateral DMPO cells while others

are under the control of ipsilateral DMPO cells. This follows from the observation that each OHC

in P. Farne~~i~ has only one efferent nerve ending (Bishop and Henson, 1987). Such a conclusion could not easily be reached by studies on other animals because there are multiple efferent termi- nals on each OHC and it would be difficult to demonstrate whether all of the terminals were from the same or different neurons.

Comparative data for mammalian auditory efferent systems are shown in Tables I, II and III.

Although medial and lateral groups have been found in almost all mammals investigated the

location of the neurons within the SOC is so variable that a common, general mammalian pat- tern of efferent centers is difficult to describe. In

Table I, for example, note that single neural centers for the medial and lateral group efferent neurons occur in the white rat and in P. ~~rnel~~i while at least five nuclear groups house efferent neurons in

the cat, squirrel monkey and guinea pig; in all species except P. parnellii the ventral nucleus of the trapezoid body (VNTB) is a medial group efferent center. In addition, the DMPO is a source of medial group efferents in the cat, guinea pig and P. par~el~~i, but not in the rat or the squirrel

monkey. In P. parneilii and in the cat, the lateral group

fibers arise from cells that form a shell over the LSO; although we have followed the terminology of Zook and Casseday (1982a, b) in calling this nucleus the INT. there is little question that this represents what other investigators have some- times called peri-LSO neurons. In the cat and bat the cells of the lateral group have similar shapes and the dendrites extend into the LSO. In the rat, by contrast, cells with similar features are scattered among the cells of the LSO (White and Warr,

TABLE I

REPORTED BRAINSTEM ORIGINS FOR MEDIAL AND

LATERAL GROUP EFFERENT NEURONS 1N MAM-

MALS (CAT, WHITE RAT, GUINEA PIG [GP]. SQUIR-

REL MONKEY [SMJ AND BAT f P. parneliii J)

Abbreviations: DMPO, dorsomedial periolivary nucleus;

MNTB, medial nucleus of the trapezoid body; VNTB, ventral

nucleus of the trapezoid body; LNTB, lateral nucleus of the

trapezoid body; VNLL, ventral nucleus of the lateral lemnis-

GUS; LSO, lateral superior olive; PERI-LSO, neurons bordering

the LSO, = INT of this report or neurons between MS0 and

LSO, and lateral to LSO, monkey. MSOD and SOCM refer to

nuclei that are dorsal to the medial superior olivary nucleus

and medial to the superior olivary complex. Note that medial

and lateral group neurons have been found in all species, but

in no two species are the efferent origins identical. The data

shown in this table, as well as in Tables If and III have been

taken from, or calculated from, data published for the cat

(Warr. 1978; Warr et al., 1982) rat (White and Warr, 1983).

guinea pig (Robertson, 1985) and squirrel monkey (Thompson

and Thompson, 1986).

Cat Rat GP Bat SM

Medial group

(1) DMPO X X X

(2) MNTB X X

(3) VNTB X X X X

(4) LNTB X

(5) VNLL X

(6) MSOD X

(8) SOCM X

Lateral group

(I) PERILS0 X X X X X

(2) LSO X

1983); in the guinea pig they are both around and within the LSO. Thus, in animals so far examined, the cells and dendritic processes of lateral group neurons are closely associated with the LSO and it seems likely that LSO neurons may govern the activity of these efferent neurons.

The organization of efferent centers in P. parnellii is similar to that observed in the rat by White and Warr (1983); in both species the COCB contains only medial group efferent fibers. This is unlike the cat and monkey in which the COCB contains axons from both medial and lateral group neurons. Therefore, in the rat and in P. parnellii

we anticipate that experiments which manipulate the crossing fibers in the floor of the fourth ventricle will affect only medial group efferents to OHCs. In all species, however, medial group effer- ents project to both cochleae so that any manipu-

lation will affect only a certain percentage of medial group cells that project to the contralateral ear. This varies from 70% in P. parneki to 58% in the squirrel monkey (Table II).

In the albino rat, White and Warr (1983) noted that both of the nuclei cont~~ng medial and lateral group efferents contained other auditory neurons; this is also true of other species studied to date, In P. parnellii, however, the lateral and medial group neurons are very densely packed, the cell bodies are isolated from adjacent nuclei and it seems likely that both the DMPO and the INT are almost, if not entirely, purely efferent centers. The close packing of the cells in these nuclei is not only apparent from our HRP labeled material but also from acetylcholinesterase (CHAT antibody) staining (Fitzpatrick et al., 1987).

Counts of labeled cells in P. parneilii gave an estimated maximum of 1585 neurons projecting to each ear. Of these, 75% reside in the INT and 25% in the ipsilateral and contralateral DMPO. Since the accuracy of such counts is dependent on the number of fibers which transport the label, we must address the question of whether all efferent fibers transported HRP back to the cell bodies. Additional studies in our laboratory have shown that all of the efferent neurons in P. parneflii are cholinergic, and that each DMPO contains ap- proximately 400 cholinergic positive neurons (Fitzpatrick and Henson, Unpubl.). Since the number of HRP labeled cells in the right and left DMPO, after application of HRP to one ear, was 389 we are confident that the label injected into the cochlea was taken up by most, if not all, of the medial group efferent fibers. The density of the labeled cells in the INT was high and similar to that observed in tissue studied by immuno- cytochemical (CHAT) techniques; this suggests that the majority of the lateral group efferent cell bodies were also labeled.

The results of this study support the conclusion of other investigators who have found that more than 60% of olivocochlear neurons project to the ipsilateral cochlea (Table II). It is clear, however, that the percentage of neurons projecting to the ipsilateral cochlea is significantly higher in P. parnellii (83%) than has been determined for other species (cat, 67%; rat, 68%; guinea pig, 61%; squir- rel monkey, 66%). This high percentage is due to

181

TABLE II

COMPARISON OF TOTAL (MAXIMUM) NUMBER OF AUDITORY OC NEURONS, THE PERCENTAGES OF NEURONS

IN THE MEDIAL AND LATERAL CLASSES AND THE PERCENTAGES THAT PROJECT TO THE CONTRALATERAL

AND IPSILATERAL EAR

Bat = P~rranotus p. pamellii. Data references are same as shown in Table I.

Bat Cat Rat Guinea

pig

Squirrel

monkey

Total number of

auditory OC neurons

Percent of OC neurons

in lateral class

Percent of OC neurons

that project to ipsi-

lateral ear

Percent of lateral OC

neurons that project

to ipsilateral ear

Percent of medial OC

neurons that project

to ipsilateral ear

1585 1230 480 1375 880

75% 60% 50% 38% 75%

83% 67% 68% 61% 66%.

100% 92% 100% 99% 80%

30% 31% 33% 39% 42%

the large population of cells in the INT (75% of (Table III). Comparisons of the numbers of neu- the OC fibers). rons in each efferent class are shown in Table II.

Tables II and III show maximum values calcu- lated for the number of efferent neurons in differ- ent species. In these cases all data were obtained by the use of retrograde transport techniques. It is interesting to note that the total number of neu- rons and the percentage of neurons in the medial vs lateral classes vary considerably. The total number of OC cells is similar in the cat (1230), guinea pig (1375) and P. parneffii (1585) but the number of efferent neurons per millimeter of basilar membrane length is much higher in P. parnelfii than in any of the other mammals studied

It is obvious that much more data are needed for the comparison of efferent auditory systems in different mammals, but there is already a signifi- cant amount of data indicating that there are marked differences in the organization and struc- ture of the system, even in closely related species. The complete absence of medial group efferents in ~inolop~dae (Bruns and Schmieszek, 1980; Bishop and Henson, 1987) is particularly note- worthy since the acoustic demands in these bats are probably similar to those of P. parnellii; all of these species use Doppler compensation sonar.

TABLE III

RATIOS OF EFFERENT NEURONS TO BASILAR MEMB~NE LENGTH

Note the high N/BML and LN/BML ratios in P. parnellii compared to other species. Data references are same as cited in Table 1.

Species

Pteronotus

pan&iii Cat

Rat

Guinea pig Squirrel

monkey

Basilar

membrane

length

(BML)

I4mm

23

10

19

20

Number

efferent

neurons

(N)

1585

1230

480

1375

880

Ratio

I\i,‘BML

113

53

48

12

44

Number

lateral

neurons

(LN)

1189

720

240

522

660

Ratio

LN,‘BML

85

31

24

27

33

Number

medial

neurons

(MN)

396

510

240

852

220

Ratio

MN/BML

28

22

24

45

11

182

In summary, the efferent auditory system of P. parnellii seems especially well suited for further investigations: (1) there is a relatively large num- ber of efferent neurons, especially lateral group efferents; (2) only medial group efferent fibers form the COCB; (3) the medial group and the lateral group neuronal cell bodies are each con- fined to a single nucleus; and (4) there are few, if any, additional auditory neurons included in the two efferent nuclei. Further studies are currently being directed toward establishing the hair cell connections of the crossed vs uncrossed fibers of the DMPO within the cochlea, and determining the activity of the efferent system in association with biosonar signal emission and echo detection and analysis.

Acknowledgements

This work was supported by USPHS grants NS 12445 and NS 19031. We wish to thank Miriam Henson, Art Keating and Doug Fitzpatrick for technical help during these experiments and for their comments on this manuscript.

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