THE JOURNAL OF COMPARATIVE NEUROLOGY 201:25-49 (1981)

The Connections of the Inferior Colliculus and the Organization of the Brainstem Auditory System in the Greater Horseshoe Bat (Rhinolophus ferrumequinum) HERMANN SCHWEIZER Arbeitskreis Neuro- und Rezeptorphyszologte, Fachbereich Biologie (Zoologre), J W Goethe Unzuersttaet, D 6000 FrankfurtlM Federal Republic of Germany

ABSTRACT The connections of the inferior colliculus, the mammalian mid- brain auditory center, were determined in the greater horseshoe bat (Rhinolophus ferrumequinum), using the horseradish peroxidase method. In order to localize the auditory centers of this bat, brains were investigated with the aid of cell and fiber-stained material. The results show that most auditory centers are highly developed in this echo- locating bat. However, the organization of the central auditory system does not generally differ from the mammalian scheme. This holds also for the organization of the superior olivary complex where a well-developed medial superior olivary nucleus was found. In addition to the ventral and dorsal nuclei of the lateral lemniscus a third well-developed nucleus has been defined which projects ipsi- laterally to the inferior colliculus and which was called the intermediate nucleus of the lateral leminiscus. All nuclei of the central auditory pathway project ipsi-, contra-, or bilaterally to the central nucleus of the inferior colliculus with the exception of the medial nucleus of the trapezoid body and the medial geniculate body. The tonotopic organization of these projections and their possible functions are discussed in context with neurophysiological investigations.

Insectivorous bats are mammals that hunt are the higher auditory centers, the medial at night with the help of an active sonar sys- geniculate body, and the auditory cortex? tem. They emit ultrasonic calls and extract The most detailed anatomical study of the from the returning echoes information about auditory system in bats is the early work of their environment and their prey with the aid Poljak ('26a,b) in Rhinolophus ferrumequinum of a highly developed auditory system. Since and Nyctulus noctulu. Poljak concluded that the discovery of this specialized orientation the organization of the central auditory path- system in bats many neurophysiological ex- way in these bats does not differ significantly periments have been performed on acoustic from that of other mammals. However, his processing in the central auditory system of studies of fiber connections were done only these animals. The neurophysiological data with myelin-stained material. show that the auditory system of echolocating In contrast to Poljak's findings, Harrison and bats is highly specialized and adapted to their Irving ('661, Irving and Harrison ('671, and specific orientation behavior (e.g., Neuweiler, Masterton and Diamond ('67) emphasized '80; Pollack, '80; Suga and O'Neill, '80). some differences in the central auditory system Surprisingly, only a few anatomical data of bats, especially concerning the organization exist for the central auditory system in bats of the superior olivary complex. They advo- (for review see Henson, '70). Most anatomical cated that bats do not have a medial superior investigations show that some of the auditory olivary nucleus. Therefore it was generally centers are highly developed. But, two main agreed that the organization of the auditory questions arise from earlier investigations: system of echolocating bats differed from that How is the auditory pathway organized up to of other mammals, and not until the auditory the inferior colliculus and how well-developed pathways of bats were recently investigated

0021-996'718112011-0025$07.00 @ 1981 ALAN R. LISS, INC. 26 H. SCHWEIZER with modern neuroanatomical tracing tech- 40-60-pm-thick frontal sections. The sections niques (Schweizer, '78; Schweizer and Radtke, were collected in cold 0.1 M phosphate buffer '80; Zook, '79; Zook and Casseday, '78; Zook (pH 7.2) and were processed the same day for and Casseday, '80) did this concept begin to the HRP reaction. change. Three different procedures for HRP reaction This paper presents data on the structure of were used: The DAB method (Graham and the lower auditory centers and the connections Karnovsky, '66), the DAB cobalt chloride of the inferior colliculus of the greater horse- method (Adams, '771, and the TMB method shoe bat, Rhinolophus ferrumequinum, and (Mesulam, '78). After washing, the sections tries to clarify the question of whether there were mounted on gelatinized slides, air dried, are fundamental differences in the anatomical counterstained in cresylviolet (for DAB-re- organization of the central auditory system of acted sections) or in a 194 acidified neutral red echolocating bats in comparison to that of solution (for TMB reacted sections), dehy- other mammals. drated in graded ethanol, cleared in xylene, and coverslipped. METHODS The sections were examined with a Zeiss In order to examine the cytoarchitecture of photomicroscope under brightfield or darkfield the auditory centers of the greater horseshoe illumination or between crossed polarizers (for bat, six brains were fixed in formalin, embed- the TMB method) (Illing and Waessle, '79). ded in paraplast or celloidin, and cut in the Drawings were made from frontal sections to three standard planes in 20-km-thick sections. show the cytoarchitecture of auditory nuclei Sections were stained with cresylviolet for and to show the spatial distribution of labeled cells, with hematoxyline for myelinated fibers, cells. or with cresylviolet and luxol fast blue for a combined cell and fiber stain. For visualization of unmyelinated fibers, some sections were A bbreviatrons processed according to a modified Bodian silver impregnation method (Ziesmer, '52). ALP0 Anterolateral periolivary nucleus AVCN Anteroventral cochlear nuclues The connections of the inferior colliculus BIC Brachium of the inferior colliculus were studied with the horseradish peroxidase CGM Central pray matter (HRP)method. The animals were anesthetized CN Cochlear nucleus by intraperitoneal injection of nembutal DCN Dorsal cochlear nucleus DNLL Dorsal nucleus of the lateral lemniscus (2.5-3.0 mg/100 gm body weight) and placed DPO Dorsal periolivary nucleus in a head holder. The skin and muscles over 1c lnfenor colliculus the midbrain were removed and a small hole ICC Central nucleus of the infenor colliculus was drilled into the skull. HRP (Sigma type VI ICP Pericentral nucleus of the inferior colliculus ICX External nucleus of the interior colliculus or HRP from Serva) was injected into different INLL Intermediate nucleus of the lateral lemniscus parts of the IC either by pressure or ionto- LL Lateral lemniscus phoretically. In five cases HRP was applied in LNTB Lateral nucleus of the trapezoid body solid form. LSO Lateral superior olrvary nucleus MGB Medial geniculate body Survival times varied from 4 to 96 hours, MNTB Medial nucleus of the trapezoid body but in most experiments the survival time was MSO Medial superior olivary nucleus 24 hours. The animals were then deeply an- PVCN Posteroventral cochlear nucleus esthetized with nembutal (4 mg/100 gm body SOC Superior olivary complex VMPO Ventromedial penolivary nucleus weight) and perfused through the heart with VNLL Ventral nucleus of the lateral lemniscus phosphate-buffered heparinized saline (pH 7.2) VNTB Ventral nucleus of the trapezoid body followed by the fixative solution. In most an- VOP Ventral periolivary nucleus imals the fixative consisted of 2% paraformal- dehyde + 1%'glutaraldehyde in 0.05 M phos- phate buffer (pH 7.2).After 20-30 minutes of RESULTS perfusion the brains were removed from the The presentation of the results is divided skull, covered with a thin layer of egg yolk, into two parts. Part one gives a brief descrip- and postfixed for 4-6 hours in the cold. The tion of the cytoarchitecture of the auditory cen- tissue was then washed overnight in two ters with the exception of the medial genicu- changes ofcold 0.1 M phosphate buffer (pH 7.2) late body and the auditory cortex. A detailed containing 20% sucrose. The following day the description of subdivisions of different audi- brains were cut on a freezing microtome into tory nuclei and the neurons composing these AUDITORY PATHWAYS OF BATS 27

Fig. 1. Frontal sections of the medulla oblongata and The rectangles in the right half of each section indicate the the overlying cerebellum (Ceb) (left) and of the midbrain location of the sections in figures 2 to 5. Ahhrev.: NLL- (right) showing the position of the lower auditory centers. nuclei of the lateral lemniscus. nuclei will be part of further studies. In part the lateral brain surface (Fig. 2). In the ventral two, the results of the HRP experiments are part a superficial ependymal cell layer is fol- presented. lowed medially by a molecular cell layer, a “fusiform” cell layer, and a polymorphic cell region. However, the fusiform cell layer can be Part I: Cytourchitecture of the lower auditory distinguished from the polymorphic cell region centers only by a more orderly arrangement of fusi- Figure 1 shows the position of the lower au- form cells, which are often oriented with their ditory centers in two frontal sections of the long axis running from lateral to medial, and brainstem. The description of these centers by a slightly higher density of granular cells. starts from caudal to rostra1 with the cochlear In contrast to the ventral DNC, the dorsocau- nucleus complex followed by the superior oli- dal region shows a disorderly cell arrange- vary complex, the nuclei of the lateral lemnis- ment. But the main difference is that a mo- cus, and the inferior colliculus. lecular layer is not developed in this part of The cochlear nucleus complex. As in other the DCN. mammals the cochlear nucleus complex (CN) of Rhinolophus ferrumequinum can be divided The Posterouentrul Cochlear Nucleus. The on the basis of cytoarchitectural features (cell PVCN is situated ventral to the DCN and just size, cell shape, packing density of cells) into caudal to the acoustic branch of the eighth three subnuclei (Fig. 2):dorsal (DCN), postero- nerve root. It will be divided in this study only ventral (PVCN), and anteroventral (AVCN) into three subdivisions-a central, a lateral, cochlear nucleus. The boundary between the and a ventral (Fig. 2). DCN, th PVCN, and the AVCN is formed by In the ventral subdivision the neurons are a dense layer of small, darkly stained granular generally smaller than in the other two regions cells. The PVCN and the AVCN are clearly and they are more densely packed. In the ros- separated by the incoming fibers of the eighth tral third of the PVCN the ventral subdivision nerve root. All three subnuclei can be divided is separated from the lateral subdivision by a into different subdivisions composed of differ- region composed of densely packed, small, ent cell types. round cells. The lateral subdivision is mainly composed The dorsal cochlear nucleus. The dorsal of large, spindle-shaped neurons which are ar- cochlear nucleus is the smallest of the three ranged in an orderly manner with their long subnuclei of the cochlear nucleus complex. In axes parallel to the outgoing fibers which con- cell-stained material, the DCN of Rhinolophus tribute to the trapezoid body. The lateral and can be divided into a dorsocaudal and a ventral the ventral subdivision together have a con- part. The boundary between the two DCN voluted “S” shape which gives the PVCN of parts is made apparent by a shallow sulcus on Rhinolophus its typical form. 28 H. SCHWEIZER

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Fig. 2. Frontal sections of the cochlear nucleus complex. Section 1 is most caudal, section 12 most rostral. The sections are spaced at 80 pm. Abbrev.: d-dorsocaudal region of the DCN; g-granular cell region (dotted area in each section); IAS-VIII-fibers of the intermediate acoustic stria and of the vestibular nerve (horizontal hatching); c-central, v-ventral, 1-lateral subdivision of the PVCN; VIII-acoustic nerve root (vertical hatching). AUDITORY PATHWAYS OF BATS 29

The central subdivision is separated from the cochlear nucleus, lateral to the rostral pole the lateral subdivision by a region of very low of the motor nucleus of the facial nerve, just cell density and many incoming cochlear fibers medioventral to the facial nerve root (Fig. 3). and it stands out by reason of its numerous More rostrally, in the middle parts of the SOC, large, faintly stained, ovoid and multipolar the nucleus has a convoluted shape with a dor- cells. The central subdivision may correspond sal and ventral hilus where the trapezoid body in position to the “octopus cell area” of other fibers enter the nucleus. The outline ofthe LSO mammals. is more rounded in its rostral parts and a sharp delineation against the surrounding perioli- The Anteroventral Cochlear Nucleus. Of vary nuclei becomes difficult. The nucleus the three subnuclei of the CN the anteroven- mainly consists of medium-sized, spindle- tral is by far the biggest. It is separated from shaped neurons arranged with their long axes the DCN by a thin granular cell layer which parallel to the convolutions of the nucleus. also covers the caudal two-thirds of the sub- Some large multipolar cells, more numerous nucleus laterally and dorsally (Fig. 2). The in lateral parts, are scattered throughout the ventral border of the AVCN is formed by the nucleus. incoming fibers of the acoustic nerve root. In cell-stained material the AVCN shows a more The Medial Superior Olivary Nucleus. Out uniform structure than do the PVCN and the of all components of the SOC the medial nu- DCN, because one main cell type (medium- cleus shows the strongest divergence from the sized, round to ovoid) is common throughout cytoarchitectural scheme of other mammals. the nucleus. Although the cell composition and It forms a broad slightly curved oval cell band especially the cell density vary considerably with a ventromedial to dorsolateral orienta- in different parts of the subnucleus, in this tion (Fig. 3). Its ventral pole is situated dor- study the AVCN will not be further subdi- solaterally to the MNTB. Most neurons in the vided. MSO are spindle-shaped or oval, but many tri- angular and multipolar cells are intermixed. The Superior Olivary Complex. The supe- The neurons are arranged in an irregular way, rior olivary complex (SOC) of Rhinolophus fer- but most spindle-shaped neurons are directed rumequinum consists of three main nuclei sur- with their long axes running ventromedial to rounded by several smaller cell groups (Fig. 3). dorsolateral. Although the cytoarchitecture differs in some The Medial Nucleus of the Trapezoid respects from that in other mammals the no- body. The MNTB is situated just dorsolateral menclature used in other mammals is adopted to the pyramidal tract. The cells composing the here. Therefore the three main nuclei are nucleus are very homogeneous in size and called lateral superior olivary nucleus (LSO), shape. They are round, medium-sized, and medial superior olivary nucleus (MSO), and have a small, eccentric nucleus. The neurons medial nucleus of the trapezoid body (MNTB). are arranged in ten to 15 horizontal rows be- The justification for the use of this nomencla- tween the crossing fibers of the ventral tra- ture is based not only on cytoarchitectural fea- pezoid body. tures, but is also taken from the results of the HRP experiments, as will be shown later. The The Nuclei of the Lateral Lemniscus. smaller cell groups of the SOC are divided into Between the SOC and the inferior colliculus two main groups, the nuclei of the trapezoid there are three nuclei which project to the IC body and the periolivary nuclei: ventral nu- as the HRP experiments have shown. These cleus of the trapezoid body (VNTB), lateral nuclei are surrounded by the ascending and nucleus of the trapezoid body (LNTB), ventral descending fibers of the lateral lemniscus. periolivary nucleus (VPO), ventromedial peri- They are called the ventral (VNLL), inter- olivary nucleus (VMPO), dorsal periolivary mediate (INLL), and dorsal (DNLL) nuclei of nucleus (DPO), and anterolateral periolivary the lateral lemniscus (Fig. 4). nucleus (ALPO) (Fig. 3). However, in this re- port the description of the SOC nuclei is re- The Ventral Nucleus of the Lateral Lemnis- stricted to the three main nuclei. cus. The VNLL can be divided on the basis of cell-stained material into a lateral and a The Lateral Superior Olivary Nucleus. The medial part. The medial part appears very ho- LSO is by far the largest of all nuclei of the mogeneous and consists of densely packed, SOC. Its first caudal neurons appear medial to medium-sized, spherical cells. Caudally the 30 H. SCHWEIZER

1

Fig. 3. Frontal sections of the superior olivary complex. cell group; V spnucleusof the spinal tract ofthe trigeminal Section 1is most caudal, section 6 most rostral. The sections nerve; VII-motor nucleus of the facial nerve; VIII-acous- are spaced at 180 km, Ahbrev.: mPO-marginal periolivary tic nerve root. cells are arranged in vertical rows between the sally, and forms a lateral bulge in the surface lemniscal fibers. In the lateral part the cells of the brainstem. The nucleus mainly consists are much more loosely packed and many large of fusiform cells which are arranged with their multipolar, globular, and elongate cells are long axes running in a lateral to medial ori- found in addition to the spherical cells. The entation. Many fiber bundles of the lateral region between the two parts shows a very low lemniscus cross through the nucleus like the cell density. The dorsal border of the VNLL is bundle at the dorsal pole of the VNLL. defined by lemniscal fibers crossing from the lateral side at a right angle to the main path- way to the medial side andlor vice versa. The Dorsal Nucleus of the Lateral Lemnis- cus. The DNLL is located dorsally to the The Intermediate Nucleus of the Lateral INLL just ventral to the external nucleus of Lemniscus. The INLL has about the same the inferior colliculus. It consists of different size as the VNLL, is continuous with it dor- cell types which have long dendrites often ex- AUDITORY PATHWAYS OF BATS 31

3

Fig. 4. Frontal sections of the nuclei of the lateral lemniscus. Section 1 is caudal, section 3 rostral. The sections are spaced at 180 km. tending dorsally into the ventral parts of the only gradual transitions between adjacent re- IC . gions. Furthermore, most of the cell types can be found in all three regions, but the relative The Inferior Colliculus. As in most other percentage of neuron types is quite different. microchiroptera, the IC of Rhinolophus fer- The most impressive difference, however, is rumequinum can be seen directly at the brain the density of cells and the distribution of large surface after removal of the dorsal parts of the cells. skull. Only its most caudal parts are covered On the basis of cell-stained material, two by the cerebellar vermis. The IC is bordered main classes of neurons can be distinguished ventrally by the superior cerebellar peduncle in the ICC: (1)multipolar cells, which appear and the parapeduncular nucleus and more ros- round, oval, elongate, or polygonal, with a trally by the DNLL, the cuneiform nucleus, broad range of soma diameters (from about 15 and the incoming fibers of the lateral lemnicus. to 35 pm), and (2) fusiform cells, which appear The rostral border is formed by the deep layers elongate or ovoid. of the superior colliculus and the intercolli- cular area. Medially the IC is bordered by the central gray matter. The Dorsolateral Region of the ICC. The The IC of Rhinolophus can be divided into neurons in the dorsolateral region are much four parts (Fig. 5). These parts are named ac- more densely packed than in the ventromedial cording to their spatial relationships: central region. The fusiform neurons are arranged in nucleus (ICC), pericentral nucleus (ICP), pos- layers, turning from dorsomedial to ventrola- terior nucleus, and external nucleus (ICX). All teral, and they run parallel to the ascending these nuclei are cytoarchitecturally different lateral lemniscus fibers. Medium-sized and a and seem to have different connections. few large, multipolar cells are intermixed with the fusiform cells, especially dorsally and dor- The Central Nucleus of the ZC. The central solaterally at the border to the ICP. The dor- nucleus is the main nucleus of the IC. It oc- solateral region is only 500 pm thick medially, cupies by far the largest portion of the posterior but extends laterally to a depth of about 1,800 midbrain tectum. Within the ICC it is possible CLm. to distinguish a dorsolateral, a ventromedial, and a dorsomedial region. Although the cy- The Ventromedial Region of the ZCC. In toarchitecture is clearly different there exist contrast to the dorsolateral region, the ventro- 32 H. SCHWEIZER

Fig. 5. Frontal sections of the inferior colliculus. Section 1 is caudal, section 6 rostra]. The sections are spaced at 180 pm. Abbrev.: AC-cerebral aquaeduct; ICCd-dorsolateral, ICCm-dorsomedial, ICCv-ventromedial region of the ICC; P-posterior nucleus; IV-fourth ventricle. AUDITORY PATHWAYS OF BATS 33 medial region does not show any stratification loosely packed large cells of different shape. In in the cell arrangement and the neurons are the caudal parts of the ICX, at the border to not densely packed. More multipolar and other the ICC, very large cells occur. These are the large cells are intermixed with the fusiform largest cells in the whole IC (40- to 50-pm soma cells. diameter) and they seem to connect the ICC The large cells show a typical arrangement with the ICX. The neurons are directed from in the ICC. In the caudal third they are con- dorsolateral to ventromedial and their long gregated ventrolaterally and ventromedially. dendrites (up to 400 pm) cross laterally In the middle parts of the ICC only a few large through the ICX and lead medially deep into cells can be observed. In the rostral third the the ICC. number of small cells and the cell density de- crease and this part of the ICC characteristi- Fiber Architecture of the ZC. In fiber- cally has a large number of medium-sized and stained material five fiber bundles can be seen large neurons. The ringlike distribution of coursing into or out of the IC, and they show large cells around the ICC can be seen most a characteristic course and distribution within clearly in sagittal sections. They are accu- the IC. These fiber bundels are the lateral lem- mulated all around the ICC, except in its dor- niscus (LL), the commissure of the IC (CIC), socaudal part, where they are only sparsely the commissure of Probst, the brachium of the distributed. IC (BIC), and the tectopontine tract. The lateral lemniscus represents the main The Dorsomedial Region of the ZCC. The auditory input to the IC from lower auditory characteristic feature of the dorsomedial re- nuclei. It contains fibers from and to CN, SOC, gion is a high number of small multipolar cells; and the nuclei of the lateral lemniscus. Before these, in addition to small fusiform cells, give reaching the IC the fibers of the LL cross as a granular appearance to this region. compact bundles through the region of the All these data based on cell-stained material DNLL. The LL fibers enter the IC ventrally, clearly show that the ICC is not homogeneous but ventrolaterally and ventromedially they in its structure but can be divided into different are combined into two main bundles. The main parts which might receive different connec- direction of the LL fibers within the IC is from tions from the lower and higher auditory cen- ventral to dorsomedial. ters and might have different functions in au- A strong connection exists between the two ditory processing. IC. Fibers having their somata in the ICP and the ICC of both sides take a ventral to dorso- The Pericentral Nucleus of the ZC. The peri- medial pathway within the IC and cross in the central nucleus in not very well developed in commissure of the IC. The course of the com- Rhinolophus and it is sometimes difficult to missural fibers within the IC is nearly parallel separate it from the dorsomedial and dorso- to the ascending lemniscal fibers. lateral region of the ICC. It covers the surface The fibers of the commissure of Probst enter of the ICC as a thin shell (100to 200 p,m thick) the IC ventromedially intermixed with LL fi- caudally, medially, dorsally, and rostrally. It bers. As the HRP experiments will show, the can mainly be distinguished from the ICC by fibers of the commissure of F’robst are axons its lower cell density. Most cells are spindle- of the contralateral DNLL neurons crossing shaped and are directed with their long axes the midline at the bottom of the central gray parallel to the brain surface. matter, just dorsal and ventral to the medial The Posterior Nucleus. This nucleus, which longitudinal fascicle. Their course within the seems to have no homologue in other mam- IC seems to be parallel to the LL fibers. mals, is situated caudally in the lateral portion The fibers of the brachium of the IC and of of the IC (Fig. 5). It is separated from the ICC the tectopontine tract take a totally different by a region of very low cell density. The nu- path within the IC with respect to the LL fi- cleus is homogeneous in appearance and is bers. In general they run from medial to lat- mainly composed of small, round cells which eral, nearly at a right angle to the ascending are densely packed. lemniscal fibers. At the border to the ICX they turn ventrally and form the BIC at the lateral The External Nucleus of the ZC. The rostral edge of the IC and just ventral to it is the tec- continuation of the posterior nucleus forms the topontine tract. external nucleus containing the fibers coursing In summary, the fiber pattern of the IC is to the brachium of the IC. It is composed of formed by incoming LL fibers running from 34 H. SCHWEIZER ventral to dorsal and crossing at nearly a right In the experiment where a large HRP de- angle the medially to laterally directed out- posit was placed into the caudal portions of the going fibers of the BIC and of the tectopontine IC (RF17; Fig. 61, the entire caudal half of the tract. IC was filled with HRP reaction product in- cluding ICP, the posterior nucleus, and parts Part 11: The connections of the inferior of the ICX. In the rostral half the affected re- colliculus gion was restricted to an elongated band reach- The results on the connections of the IC are ing from dorsomedial to ventrolateral through based on six representative experiments in the ICC. - which HRP was injected into different parts of The region of labeled cells in the contralat- the IC. Because of the relatively large HRP era1 1C corresponded to the injected IC portion injections and the overrepresentation of the but was smaller than the dark brown injection ICC compared to ICP and ICX, this study can zone. Because of the high HRP concentration only provide information on the afferent pro- most labeled neurons in the ICC were com- jections to the entire IC and only a limited pletely stained as in a Golgi preparation (Fig. amount of information on the terminations of 7A). No labeled cells were found in the con- the projecting fibers in different subdivisions tralateral ICX and only a few in the ICP. of the IC. In the nuclei of the lateral lemniscus labeled After HRP injections into the IC, labeled cells were found contralaterally in the DNLL cells were found in the following nuclei: ipsi- and ipsilaterally in the DNLL, the INLL, and laterally in the IC, the DNLL, the VNLL, the the VNLL. In the DNLL and INLL the labeled INLL, the MSO, the LSO, the VNTB, the VPO, cells were scattered throughout the nuclei. the VMPO, the LNTB, the ALPO, the DPO, Labeling in the VNLL was concentrated in the the DCN, the auditory cortex, the suprape- lateral half of the nucleus. In the medial half duncular nucleus, the nucleus of the BIC, the only a few cells were labeled in the most ven- cuneate nucleus, the gracile nucleus, the nu- tromedial region. cleus of the spinal tract of the trigeminal In the SOC, labeled neurons were found con- nerve, and the raphe nucleus; contralaterally tralaterally in the LSO, VNTB, and VPO, and labeled neurons were found in the IC, the they were found ipsilaterally in the LSO, MSO, DNLL, the LSO, the VNTB, the VPO, the VNTB, VPO, VMPO, and a few in the LNTB, VMPO, the DCN, the PVCN, the AVCN, the ALPO, and DPO. The labeled neurons in the cuneate nucleus, the gracile nucleus, and the MSO were distributed throughout the nucleus. nucleus of the spinal tract of the trigeminal In the LSO they were found ispi- and contra- nerve. laterally in all parts of the nuclei, but the cells Anterograde transport of HRP and terminal located in the medial part of each nucleus were axon arborization were evident ipsilaterally in much more heavily labeled than those found the medial geniculate body (MGB), the lateral in other parts of the LSO. pontine nuclei, the deep layers of the superior All three contralateral nuclei of the cochlear colliculus, the central gray matter adjacent to nucleus complex showed heavy labeling. Ipsi- the IC, scattered in the pontine reticular for- laterally labeled cells were found only in the mation, and contralaterally in the IC. Axon dorsocaudal region of the DCN. In the contra- terminal arborization also was found ipsilat- lateral DCN labeled cells were found in the erally in the VNLL, the MSO, the LSO, the dorsocaudal region and in the fusiform region VNTB, and contralaterally in the LSO, but of the remaining nucleus. The labeled neurons these projections must be confirmed by other, in the contralateral PVCN were situated in the anterograde methods. ventral, the lateral, and the central subdivi- In the following the position of the injection sions (Fig. 9A). The AVCN contained labeled sites in the IC and the distribution of labeled cells throughout the nucleus, but the density cells will be discribed in three representative of labeling was higher in caudal parts (Fig. experiments where HRP was injected into the 9A). Only the most rostral parts of the AVCN whole IC (one animal, experiment RF17), the were completely unlabeled. deep parts of the ICC (experiment RF12), and In addition to cells labeled in the lower au- into the dorsolateral region of the ICC, includ- ditory centers, HRP was taken up by the large ing parts of the ICP (experiment RF18). The pyramidal cells of layer V of the auditory cor- results of the other experiments are very sim- tex (Fig. 7B). Furthermore, some labeled cells ilar and are included in a summarized pres- were located in the thalamus medial to the BIC entation when they differ from that of three and dorsolateral to the cerebral peduncle. At described cases. the level of the LSO some neurons were labeled AUDITORY PATHWAYS OF BATS 35

n Rf 17

2000pm .

Fig. 6. Frontal sections of the brainstem showing the The black area in the IC surrounded by a dotted line indi- distribution of HRP-labeled neurons in the lower auditory cates the position of the HRP deposit. Abbrev.: VI1-motor nuclei in the experiment RF17. Section 1 is most caudal, nucleus of the facial nerve. section 7 most rostral. The sections are spaced at 400 pm. 36 H. SCHWEIZER

Fig. 7. A. Golgi-like labeled neurons in the contralateral terograde labeling within the ipsilateral pontine nuclei. ICC. Note that most neurons are multipolar. Calibration Calibration bar 200 pm. D. Anterograde labeling in the bar 100 Fm, B: Labeled pyramidal cells in layer V of the ipsilateral MGB and labeled fibers in the cerebral peduncle ipsilateral auditory cortex, Calibration bar 200 Fm. C: An- (CP). Calibration bar 200 Fm.

in the raphe nucleus between the two MNTBs. geniculate body (Fig. 7D) and in the ipsilateral Labeled cells were also found ipsi- and contra- pontine nuclei (Fig. 7C). laterally in the cuneate nucleus, in the gracile Figure 8 shows the injection site in the IC nucleus, and in the nucleus of the spinal tract and the distribution of labeled cells in the of the trigeminal nerve. brainstem in an experiment (RF 12) where Anterograde labeled axons and axonal ar- HRP was injected into middle and deep parts borization occurred ispilaterally in the medial of the ICC. Only small parts of the ICP and no AUDITORY PATHWAYS OF BATS 37

Rf 12

Fig. 8. Frontal sections of the brainstem showing the of the HRP deposit. Note the topographical arrangment of distribution of labeled cells after a deep IC injection (RF12). labeled cells in different nuclei. Abbrev.: VII-motor nu- Section 1 is most caudal, section 7 most rostral. The sections cleus of the facial nerve. are spaced at 400 pm.The black area indicates the position 38 H. SCHWEIZER

Fig. 9. A Labeled neurons in the contralateral cochlear nucleus. In the PVCN, cells are labeled in the ventral (v) as well as in the lateral (1)subdivision. Calibration bar 200 pm. B: Labeling pattern in the ipsilateral nuclei of the lateral lemniscus. Calibration bar 200 pm. C: Labeled bipolar cells in the VNTB. Calibration bar 100 pm. D: Labeled neurons in the contralateral DNLL. Calibration bar 200 pm. parts of the posterior nucleus or of the ICX beled neurons. Labeled axons could be traced were affected by the injection. from the contralateral DNLL neurons up to the Labeled cells in the contralateral ICC were injected region in the IC (Fig. 9D). The labeled found in locations corresponding to the ipsi- cells in the ipsilateral INLL and VNLL were lateral injection site. concentrated in the lateral half of each nu- Heavy labeling occurred in all three nuclei cleus, where they form a dorsoventral band of the lateral lemniscus ipsilaterally, whereas (Fig. 9B). Whereas in the INLL many unla- contralaterally only the DNLL contained la- beled cells were intermixed with labeled neu- AUDITORY PATHWAYS OF BATS 39 rons, in the lateral half of the VNLL nearly eral pontine nuclei. Some labeled axons could every neuron contained HRP reaction product. be followed into the central gray matter of both In the superior olivary complex labeled neu- sides and into the deep layers of the superior rons were found ipsilaterally in tbe LSO, MSO, colliculus of the same side. VNTB, VPO, DPO, and ALPO and contralat- In a further experiment (RF 18), the injec- erally in the LSO, VNTB, and VMPO. In cau- tion site was situated dorsolaterally in the IC dal sections, the first labeled neurons in the (Fig. 11).It covered dorsolateral parts of the SOC appeared in the VNTB ventromedial to ICP and parts of the dorsolateral region of the the motor nucleus of the facial nerve. Most ICC: the ventromedial and the dorsomedial neurons were bipolar with long dendrites run- region of the ICC and the ICX were spared. ning parallel to the trapezoid body fibers (Fig. Many labeled cells were found in the ipsi- 9C). Similar neurons were found in the VPO. lateral dorsomedial region of the ICC. A few Many cells were labeled in the VMPO espe- labeled cells were present dorsally and dorso- cially in the rostral parts of the SOC where the laterally in the contralateral ICC. The labeled labeled neurons formed a cellular band passing cells appeared in about the same region and from ventromedial to dorsolateral between the at the same depth as the electrode tip was po- LSO and the MSO. A few multipolar cells con- sitioned in the ipsilateral IC during ionto- taining HRP reaction product were present phoretic injection. just dorsal to the space between the MSO and In the nuclei of the lateral lemniscus labeled the LSO in the DPO. Most labeled cells in the cells were found ipsilaterally in the DNLL, LSO of both sides were fusiform and situated INLL, and VNLL, and contralaterally only in only in the medial curvature of the nuclei (Fig. the DNLL. While in the DNLL of both sides 10A). Only a few labeled cells were observed labeled neurons were scattered throughout the in the more ventrolateral parts. Labeling in nuclei, in the INLL and especially in the VNLL the MSO occurred ipsilaterally in the ventro- a clear topographical arrangement of labeled medial half of the nucleus in caudal sections, neurons could be observed. In the INLL the whereas in more rostral sections labeled cells labeled cells were loosely arranged in dorsal were scattered throughout the nucleus (Fig. and dorsolateral parts of the nucleus. In the 10B). VNLL labeled cells were located medially from Heavy labeling was found contralaterally in the dorsal to the ventral border of the nucleus. all three subnuclei of the cochlear nucleus com- Labeling in the SOC was rather faint. Most plex. The labeled neurons in the DCN were labeled cells appeared ipsilaterally in the concentrated in the dorsocaudal region; only VMPO. In the LSO the labeled neurons were a few labeled cells were found in the fusiform located ipsi- and contralaterally in the most cell region throughout the nucleus and in the lateral parts of the nuclei. Only a couple of polymorphic cell region. In the PVCN labeled faintly labeled neurons were present dorsally cells were concentrated in the lateral subdi- in the ipsilateral MSO. In addition some la- vision. No labeled cells were situated in the beled cells were observed ipsilaterally in the ventral subdivision and only a few occurred in VNTB, VPO, LNTB, and ALPO. the central subdivision. In the caudal third of In contrast to the SOC the contralateral the AVCN the labeled neurons were concen- cochlear nucleus showed heavy labeling. Many trated laterally, distributed throughout the cells were labeled in the fusiform cell layer and nucleus in middle parts, and only poorly rep- quite a few in the polymorphous cell region of resented in the anterior third. The most rostral the DNC, whereas the dorsocaudal region of portion of the AVCN was not labeled. the DCN was free of labeling. A couple of cells In addition to labeling in brainstem auditory were also labeled in the fusiform cell layer of nuclei, some labeled neurons were found in the the ipsilateral DCN. In the PVCN labeled cells raphe nucleus between the two MNTBs and in were were located exclusively in the ventral the reticular formation at the level of the motor subdivision, extending throughout the caudo- nucleus of the facial nerve. In the diencephalon rostral extent. In the AVCN labeled cells were labeled cells were found just medial to the BIC found primarily in the lateral portions of the and in a distinct cell group just dorsolateral to rostral third part. the lateral parts of the cerebral peduncle. La- Some labeled pyramidal cells were found in beled axons could be detected in the cerebral layer V of the ipsilateral auditory cortex. Sim- peduncle and these probably arose from the ilar to all other cases, anterograde labeled ax- auditory cortex. Heavy anterograde labeling ons occurred in the MGB and the lateral pon- was found in the ipsilateral MGB and the lat- tine nuclei. Many labeled axons could be 40 H. SCHWEIZER

Fig. 10. A: Labeling pattern within the ipsi- (on the right ipsilateral superior olivary complex. (The same section as side) and contralateral superior olivary complex after a ven- in Fig. 10A, at higher magnification). Calibration bar 250 tral IC injection. Note that no cells are labeled in the con- pm. tralateral MSO. Calibration bar 500 km. B: Labeling in the AUDITORY PATHWAYS OF BATS 41

2000 prn,

Fig. 11. Frontal sections of the brainstem showing the 400 wm.The black area indicates the position of the HRP distribution of labeled neurons in the lower auditory nuclei deposit in the IC. Note the difference projection pattern in in the case of a dorsal IC injection (RF18). Section 1 is most comparison to RF12 (Fgi. 8). Abbrev.: VII-motor nucleus caudal, section 7 most rostral. The sections are spaced at of the facial nerve. 42 H. SCHWEIZER followed from the injection site to the central dorsal IC injections labeled cells were found gray of both sides, but their terminal fields more dorsally in the MSO and laterally in the could not be determined. LSO and with deep IC injections ventromedi- The results of all HRP experiments can be ally in the MSO and medially in the LSO. No summarized as follows. Relatively large HRP topographical arrangement of labeled cells deposits were placed in the IC. Two main cases could be found in the periolivary nuclei. can be distinguished: (1)The HRP deposit was The three subnuclei of the cochlear nucleus located in the dorsal parts of the IC including complex mainly projected contralaterally to the ICP, the dorsal parts of the ICC, and dorsal the IC. Only a few labeled cells were found in parts of the ICX; (2) the HRP deposit was the ipsilateral DCN. In the contralateral CN placed deep into the ICC and the injection site distinct topographical representation occurred included parts of the ICP, the dorsolateral, and in relation to the position of the HRP deposit. the ventromedial region of the ICC. In most In the case of dorsal IC injections labeled neu- experiments the dorsomedial region of the ICC rons were located ventrally in the PVCN and and the ICX were missed. Also spared were the the DCN and rostrally in the AVCN. In the most anterior parts of the ICC to avoid spread- DCN most labeled cells were located in the ing of HRP into the superior colliculus. fusiform cell layer. In the case of deep IC in- After the HRP reaction labeled neurons and1 jections, labeled cells were found predomi- or labeled axon terminals were found in dif- nantly in the lateral subdivision of the PVCN, ferent brainstem nuclei and in layer V of the in the dorsocaudal region of the DCN, and pos- auditory cortex. While the overall projection teriorly in the AVCN. At the diencephalic level pattern was similar in all cases, some auditory labeled cells were found just medial to the BIC nuclei showed different labeling patterns de- in the nucleus of the brachium of the IC and pending on the injection site in the IC. a second cell group was located ventromedially In all experiments labeled cells were found to the MGB in the suprapeduncular nucleus. in the IC contralateral to the injection site and No labeled cells were found in the MGB itself, axons could be tracked in the commissure of but a very dense network of labeled axons and the IC. Only a few labeled cells were found in axonal arborization was observed. the contralateral ICP and none in the posterior In all cases in which the forebrain was sec- or the external nucleus. Most cells in the con- tioned, labeled pyramidal cells were found in tralateral ICC seemed to be small and me- layer V of the temporal cortex. This area cor- dium-sized multipolar cells, as judged from responds to the auditory cortex. Axons arising Golgi-like labeling. from pyramidal cells could be followed through In all cases labeled cells could be found in the internal capsule and the cerebral peduncle all three nuclei of the lateral lemniscus. How- descending to the IC. ever, whereas the DNLL projected bilaterally, In addition labeled cells were found in nuclei the INLL and VNLL projected only ipsilat- not directly correlated to the classical auditory erally. No topographic representation of la- pathways. In cases where the ICX was covered beled cells could be observed in the DNLL. In by the HRP deposit, labeled cells were found the INLL, labeled cells were sometimes con- bilaterally but mainly contralaterally in the centrated in more medial parts and sometimes cuneate nucleus, in the gracile nucleus, and in in more lateral parts, but the regions where the caudal part of the nucleus of the spinal labeled cells were found were not consistently tract of the trigeminal nerve. Single labeled related to the position of the injection site. In cells were found scattered in the reticular for- contrast to the DNLL and the INLL the posi- mation, but they were concentrated in the tion of labeled cells in the VNLL was clearly raphe nucleus, between the two MNTB. related to the injection site. Labeled cells were In every case a heavy anterograde transport localized in medial parts in the case of dorsal of HRP occurred and labeled axon terminals IC injections and were concentrated in a lat- could be detected in the lateral pontine nuclei eral band in the case of deep IC injections. and the medial geniculate body. Some labeled In all experiments the projection pattern of axons seemed to terminate in the pontine re- SOC neurons was very similar. Labeled cells ticular formation, in the central gray matter, were detected ipsilaterally in the LSO, MSO, and in the deep layers of the superior collicu- VNTB, VPO, VMPO, DPRO, LNTB, and ALP0 lus. and contralaterally in the LSO, VNTB, and the DISCUSSION VPO. In no case were labeled cells found in the Since the work of Poljak ('26a,b) and other medial nucleus of the trapezoid body. With (for review see Henson, '70) there have been AUDITORY PATHWAYS OF BATS 43 only a few anatomical investigations on the roptera a nucleus which is homologous to the central auditory system of microchiroptera MSO in other mammals. In earlier studies the (Schweizer, '78; Zook, '79; Schweizer and SOC of the bat Myotis myotis was subdivided Radtke, '80; Zook and Casseday, '80). The cy- into a superior olivary nucleus, a paraolivary toarchitectonic results presented in this report nucleus, and a lateral and a medial nucleus of demonstrate that the anatomy of the central the trapezoid body (Schober, '59). The SOC of auditory pathways of Rhinolophus ferrume- Rhinolophus was subdivided into a superior quinum does not differ in general from those olivary nucleus, an accessory olivary nucleus in other mammals. However, most auditory consisting in its rostra1 parts of an external centers are very well developed in this bat; this and an internal nucleus, a nucleus, of the tra- is true for most brainstem centers as well as pezoid body, and an internal and an external for the higher auditory centers (the MGB and preolivary nucleus (Poljak, '26a). the auditory cortex) (Schweizer and Ratke, In a comparative study Irving and Harrison '80). ('67) pointed out that the MSO is very small Three major findings on the anatomical or- or absent in the hedgehog, bat, mouse, and ganization of the auditory system of the mole. Harrison and Feldman '70) and 0110 and greater horseshoe bat have to be emphasized Schwartz ('79) found a large superior paraoli- and will be discussed in more detail in this vary nucleus in the rat and the mouse respec- paper: (1)Medial to the LSO a cell group exists tively but only a poorly developed MSO. The which seems to be homologous to the MSO in criterion for the presence or absence of MSO other mammals, judging from the projection is that MSO must be composed of bipolar cells pattern to the IC; (2) there is a large nucleus with one dendrite directed laterally and one between the VNLL and the DNLL which pro- medially. According to this criterion the para- jects to the IC and which was called the inter- olivary nucleus of microchiroptera and some mediate nucleus of the lateral lemniscus other mammals cannot be homologous with the (INLL); and (3) the tonotopical arrangement MSO because it contains a large number of of the projections to the ICC shows the over- multipolar cells in addition to the bipolar cells. representation of the high-frequency regions It has been homologized with the retroolivary in the auditory system. nucleus of the cat which gives rise to the oli- vocochlear bundle. Using phylogenetic criteria The superior olivary complex however, Brown and Howlett ('72) emphasized Of all mammalian auditory centers, the SOC that in the rat the superior paraolivary nu- is by far the most complicated, and there is cleus is homologous to the MSO in the cat. much confusion about its organization and The presence or absence of the MSO was fur- function in different mammals. In the cat, nor- ther correlated with the development of the mally three main nuclei are distinguished in visual system, the head size (distance between the SOC-the lateral superior olivary nucleus the two ears), and the capacity for high-fre- (LSO),medial superior olivary nucleus (MSO), quency hearing (Harrison and Irving, '66; Irv- and the medial nucleus of the trapezoid body ing and Harrison, '67). The MSO is the first (MNTB). These main nuclei are surrounded by auditory center to receive fibers directly from several trapezoid body and periolivary nuclei both sides, whereas the LSO receives direct (e.g., Elverland, '78). Most neurophysiological fibers from the ipsilateral cochlear nucleus and anatomical work was performed on these only; the contralateral input comes from the main nuclei and only little is known about the cochlear nucleus via the MNTB. The LSO proj- projection pattern and the function of the tra- ects bilaterally and the MSO ipsilaterally to pezoid body and periolivary nuclei. The no- the ICC. Neurophysiological data have shown menclature of the SOC becomes even more con- that intensity differences between the two ears fusing when attempts are made to homologize are processed in the LSO whereas the MSO several nuclei in comparative studies of dif- decodes time differences (Tsuchitani and Boud- ferent mammalian species based only on cy- reau, '64; Moushegian et al., '72). The LSO is toarchitectural investigations. The SOC in very well developed in microchiroptera; the Rhinolophus was divided in this study into MNTB which is a direct index for the size of LSO, MSO, and MNTB, which are surrounded the LSO has the highest progression index of by the VNTB, VPO, LNTB, DPO, ALPO, and all auditory nuclei in Rhinolophus (Baron, '741, the VMPO. Besides some differences in the suggesting that processing of intensity differ- nomenclature of periolivary nuclei the main ences is a very important cue for echolocation. question is whether there exists in microchi- Most microchiroptera have a poorly developed 44 H. SCHWEIZER visual system and a small interaural distance, a MSO to detect the interaural time delay of and the frequencies of their echolocation calls the envelope of the calls. This assumption may and of best hearing are in the ultrasonic range. be true, but it is somewhat contradicted by the Therefore one would expect from the hypo- fact that Molossus, emitting a short constant thesis of Harrison and Irving ('66) that bats frequency, frequency-modulated echolocation have a well-developed LSO and a small or ab- call, seems to have a very well-developed MSO sent MSO, as the encountered interaural time (personal observation). Therefore no current differences are extremely small. But for two hypothesis can explain why one bat species has reasons the arguments of Brown and Howlett a MSO and others do not. A comparative study ('72), to homologize the nucleus medial to the in different species of bats considering the LSO with the MSO of other mammals, were anatomy of the auditory system in the context adopted even though the cytoarchitecture of of their behavior may perhaps throw some this region is quite different. First, Harrison light on these questions. A further reason why and Irving ('66) homologized the paraolivary the paraolivary nucleus of microchiroptera nucleus with the retroolivary nucleus, the might be homologous to the MSO comes from source of the olivocochlear bundle. If this holds, recent experiments of Harnischfeger ('801, who the neurons of this nucleus should be AChE- recorded from neurons in the SOC of Molossus positive, which is not true in Rhinolophus for that responded to interaural time differences the paraolivary nucleus (MSO) (personal ob- as low as 10 psec, indicating that bats are able servations). Neurons which are AChE-positive to decode the low interaural time differences were only located within the SOC in the VPO produced by their small head size. and VMPO, and the olivocochlear bundle is poorly developed. The AChE results support The nuclei of the lateral lernniscus the hypothesis of Brown and Howlett ('72) in In Rhinolophus and Pteronotus (Zook, '79) the rat. The second reason is the projection there are three nuclei in the projection path pattern to the ICC. The retroolivary nucleus of the lateral lemniscus to the IC; these are the in the cat projects bilaterally to the ICC, ventral (VNLL), intermediate (INLL), and dor- whereas the paraolivary nucleus (MSO) of sal (DNLL) nuclei of the lateral lemniscus. Rhinolophus projects ipsilaterally only. Al- Whereas VNLL and DNLL are common in though the cell composition and cell arrange- mammals, the INLL seems to be especially ment in the paraolivary nucleus are different well developed or unique to bats. from the MSO in the cat, one can conclude from The VNLL is a very prominent nucleus in the data presented that this nucleus in Rhin- Rhinolophus and has about the same size as olophus is homologous, on the basis of its pro- the LSO. Its high development suggests im- jection pattern, to the MSO in other mammals, portant function in echolocation. In other and it is therefore called MSO. One main proof mammals the VNLL receives fibers from the for the homology, however, is still lacking in CN and the SOC which terminate in the ven- Rhinolophus, i.e., evidence of a direct projec- tral and dorsal parts respectively (Fernandez tion from the cochlear nuclei of both sides. In and Karapas, '67; van Noort, '69; Warr, '69; Pteronotus parnellii parnellii a very well-de- Browner and Webster, '75). Neurophysiologi- veloped MSO was found on the basis of the cal data support the idea that the VNLL must projection pattern from the cochlear nuclei and be divided into two functionally different re- to the ICC (Zook, '79). The cellular arrange- gions, as can be expected from the connection ment is somewhat different in Pteronotus and pattern. Dorsal neurons respond to binaural more bipolar cells seem to be present (Zook, and ventral neurons to monaural stimulation personal communication). With the same (Aitkin et al., '70). methods Zook and Casseday ('78) could not find As pointed out above, the INLL seems to be a MSO in the bat Artibeus jarnaicensis, which unique to bats. However, it is possible that this has a better-developed visual system and a nucleus is homologous to the nucleus sagulum larger head size than Pteronotus and Rhino- of other mammals and is only especially well lophus. According to the hypothesis of Harri- developed in microchiroptera. This explana- son and Irving ('661, Artibeus should have a tion would be in agreement with the findings better-developed MSO than Pteronotus and of Adams ('79), who found a strong projection Rhinolophus, which is not the case. Zook ('79) from the sagulum to the ICC in the cat. The concluded from these data that bats which emit INLL apparently has an auditory function a long constant-frequency echolocation call since it projects to the ICC and receives pro- (like Rhinolophus and Pteronotus) might have jections from the CN and the SOC, as was dem- AUDITORY PATHWAYS OF BATS 45 onstrated in Pteronotus (Zook, '79). Like the exceptionally well detectable in Rhinolophus, other two nuclei of the lateral lemniscus, the where the narrow-frequency range from 80 to physiological functions of the INLL are un- 86 kHz plays an important role for echoloca- known in microchiroptera and its seems prom- tion and is vastly overrepresented in the ICC. ising to investigate the connections and the Fitting the neurophysiological data with the functions of the VNLL and the INLL since they anatomical parcellation of the ICC suggests are especially well developed or unique to bats. that in the dorsolateral region of the ICC of Rhinolophus the frequency range from low fre- Tonotopical arrangement of projection quencies up to 80 kHz is represented and that The aim of the HRP experiments presented the frequencies of the acoustical filter, i.e., the here was to obtain information on the complete frequency region in the hearing threshold afferent connections of the IC and to find out curve between 80 and 86 kHz (Neuweiler, '70) the main efferent pathways. Therefore, for the and higher frequencies, are represented in the tonotopical studies only two cases were distin- ventromedial region. Therefore one would ex- guished, i.e., HRP deposits into dorsal parts of pect to find labeled cells in low-frequency re- the ICC and deposits into ventral regions. gions of auditory centers after HRP deposits The central nucleus of the IC is the main in dorsal parts of the ICC, and in high-fre- target for ascending lateral lemniscus fibers quency regions after HRP deposits in the ven- which project to the ICC in a tonotopic order. tromedial region of the ICC. A tonotopic arrangement of neurons in the As the HRP experiments have shown, there ICC, with low best frequencies localized in dor- exist strong connections between the inferior sal parts and high best frequencies in ventral colliculi of both sides. The data for correspond- parts, is a common feature in bats and other ing regions on both sides suggest that there is mammals. This has been shown in Myotis lu- a point to point connection between the right cifugus and Plecotus townsendii (Grinnell, '63). and left ICC and that predominantly multi- Myotis lucifugus (Friend et al., '661, the cat polar cells give rise to the commissure of the (e.g., Rose et al., '63; Merzenich and Reid, '74; IC. There must be a strong influence between Aitkin and Moore, '75; Aitkin et al., '75; Roth the two inferior colliculi in the processing of et al., '781, the rabbit (Aitkin et al., '72), and auditory information, which has so far never the squirrel monkey (FitzPatrick, '75). been investigated with neurophysiological However, in the ICC of Rhinolophus a spe- methods. The data presented show further that cialization has been found regarding frequency only a few neurons of the ICP give rise to the representation: The best frequencies of single commissural projection and none of the ICX. units (in the range from 80 to 86 kHz) corre- sponding to the frequencies of the contant fre- Projections from the cochlear nucleus complex quency component of the echolocation sounds Neurons of all three subnuclei, DCN, PVCN, and echoes (Schnitzler, '68) are topically over- and AVCN, project directly to the ICC. represented compared to other frequencies DCN projection: The DCN projects bilater- (Schuller and Pollak, '79; Pollak and Schuller, ally to the ICC, but the projection from the '81). These results fit well with the findings contralateral side was consistently much that the frequency range from 83 to 86 kHz stronger. Most projecting neurons are situated occupies about the same length on the cochlear in the fusiform cell layer. This is in agreement basilar membrane as an octave at lower fre- with the findings in Pteronotus (Zook, '791, in quencies (Bruns, '76). The tonotopical organi- the cat (e.g., Fernandez and Karapas, '67; van zation in the ICC of Rhinolophus shows, be- Noort, '69; Osen, '72; Adams, '79), in the rat sides the dorsoventral, a mediolateral (Beyerl, '781, and in the Rhesus monkey arrangement: High-frequency neurons have (Strominger, '73). The DCN projects topo- been found in more medial parts of the ICC graphically to the ICC. After dorsal IC injec- from 500 to about 2,000 km deep and in lateral tions labeling was found more ventrally in the parts at a depth of about 1,800 pm (Pollak and DCN and after ventral IC injections cells in Schuller, '81). Similar results are available for the dorsal and especially in the dorsocaudal the bats Myotis lucifugus (Grinnell, '63) and unlayered region of the DCN were labeled. Molossus molossus and Molossus ater (Vater These facts suggest that the high frequencies et al., '79). The fact that the frequency range are represented dorsally and the lower fre- of low threshold in the audiogram is overre- quencies ventrally. This agrees with the find- presented in the ICC might be a common prin- ings that the basal turn of the cochlea projects ciple in mammals. This overrepresentation is to dorsal parts and the apical turn to ventral 46 H. SCHWEIZER parts of the DCN in the guinea pig (Noda and ’78). It can be concluded from the data pre- Pirsig, ’74) and in the kangaroo rat (Webster, sented that the high frequency range is also ’71). The DCN of microchiroptera has, in com- highly represented in the AVCN. No labeled parison to basal insectivores, the lowest pro- cells were found ipsilaterally in the AVCN in gression index of all auditory nuclei (Baron, agreement with the findings in Pteronotus ’74) suggesting that DCN does not play an im- (Zook, ’79) and the cat (Roth et al., ’78) but in portant role in echolocation. In this study, how- disagreement with findings of Adams (’79) in ever, a specialized unlayered region was de- the cat. The AVCN of microchiroptera shows, tected in the dorsocaudal part of the DCN, in comparison to basal insectivores, the high- which seems to have no homologue in other est progression index of the cochlear nuclei mammals. In this region labeled cells were (Baron, ’741, thus suggesting an important always found when HRP was injected into the function in echolocation. The “small spherical high-frequency region, i.e., the ventromedial cells” of the AVCN receive input from only two region of the ICC. to three auditory nerve fibers, which might PVCN projection: After dorsal ICC injections enable them to play an important role in fine labeled cells were found in the contralateral frequency discrimination, especially if the au- ventral subdivision of the PVCN and in the ditory nerve fibers stem from neighboring lateral subdivision after ventral ICC injec- parts of the basilar membrane. “Large spher- tions. Although the cellular arrangement ical cells” are not present in the AVCN of seems to be distinct for both subdivisions in Rhinolophus and other microchiroptera cell-stained material, it is questionable (Baron, ’74; Poljak, ’26b). whether they constitute functionally different subdivisions. They appear to differ only in Projections from the superior olivary complex their tonotopical arrangement with the lower The topographical projection pattern of the frequencies represented in the ventral subdi- vision and the higher frequencies in the lateral SOC to the ICC is similar to that in other mam- mals (e.g., Roth et al., ’78; Adams, ’79; Zook, subdivision. The ventral and the lateral sub- division together form an “S”-shaped convo- ’79). Whereas in the trapezoid body and the periolivary nuclei a topographical projection luted structure. Such convolutions often ap- pattern could not be detected, the projections pear in the brain when one part of a nucleus is exceptionally well developed. The overre- from the LSO and the MSO to the ICC are presentation of high frequencies in the audi- highly ordered. Labeled cells were found after tory system of Rhinolophus might have led to dorsal IC injections in the lateral parts of the a strong development of the lateral subdivision LSO, dorsally in the MSO, and for deep IC and therefore to the 9”-shaped structure. In injections in the medial limb of the LSO and all HRP-injected brains only a few labeled cells ventrally in the MSO. were found in the central subdivision where the “octopus cell region” might be located. This Projections from the nuclei of the lateral corresponds to the findings in Pteronotus (Zook, lemniscus ’791, the cat (Adams, ’791, and the rat (Beyerl, In Rhinolophus the VNLL can be divided ’78), and to the findings that “octopus cells” into at least two subdivisions, which were des- mainly project to the periolivary nuclei and the ignated lateral and medial according to their nuclei of the lateral lemniscus, but do not have location. Both subdivisions show different pro- direct connections to neurons of the ICC (Warr, jection patterns. Whereas only a few cells were ’69; van Noort, ‘69; Osen, ’72; Zook, ’79). labeled in the medial subdivision when HRP AVCN projection: The topographical ar- was injected into the ICC, nearly every cell rangement of labeled cells in the AVCN after was labeled in the lateral part. A possible ex- HRP injections into the IC was not as clear as planation would be that the lateral subdivision in the two other cochlear subnuclei. However, serves as a relay station for ascending CN fi- in HRP injections restricted to dorsal parts of bers and that all neurons in this part project the IC (e.g., RF 18) labeled cells were found to the ICC and that neurons in the medial sub- only in the anterior part of the AVCN; HRP division receive SOC fibers and project only injections into ventral parts of the IC led to partially to the ICC. heavy labeling in the entire caudal two-thirds In every experiment labeled cells were found of the AVCN. The arrangement of labeled cells in the INLL. But with the large HRP deposits is very similar to that found in Pteronotus used in this study a topographical projection (Zook, ’79) and the cat (Adams, ’79; Roth et al., pattern of labeled cells could not be detected. AUDITORY PATHWAYS OF BATS 47

The DNLL is poorly developed in Rhinolo- ton, '69; Carey and Webster, '71; Kawamura, phus compared to other auditory nuclei and its '75). The tectopontine tract links the auditory connection pattern to the ICC is identical with system, via the lateral pontine nuclei, with the that in other mammals. cerebellum (e.g., Kawamura, '75). Neurons of the lateral pontine nuclei and of the cerebel- Projections from the auditory cortex lum respond to acoustic stimulation (Boyd and In all experiments, labeled pyramidal cells Aitkin, '76; Aitkin and Boyd, '75; Jen and were found in layer V of the temporal cortex. Schlegel, '80). A direct projection from the IC The region with labeled cells occupied a large to the cerebellum, as found in Tadarida bras- portion of the temporal cortex, varying accord- iliensis (Henson et al., '68) and in the cat (Pow- ing to the injection site in the IC. The region ell and Hatton, '691, could not be found in Rhin- where labeled cells were found covers at least olophus with the anterograde HRP method. the auditory cortex, which is very well devel- Smaller projections were found to the deep oped in Rhinolophus (Ostwald, '80; Schweizer layers of the superior colliculus and the central and Radtke, '80). The efferent fibers arising gray matter. Both projections have to be con- from pyramidal cells in the auditory cortex ter- sidered very carefully because both regions are minate within the pericentral and the central very close to the injected IC. But, the superior nucleus of the IC. Whether or not other parts colliculus projection is known in other mam- of the cortex (e.g., the somatosensory cortex) mals (Moore and Goldberg, '66; Powell and contribute to this projection could not be de- Hatton, '69; Carey and Webster, '71; Moore et termined in this study. al., '77; Edwards et al., '79) and might be in- volved in auditory motor reflexes, such as the Other projections control of the pinna movements (Henkel and In addition to projections from auditory nu- Edwards, '78). The projection from the IC to clei to the IC, a few other projections must be the central gray matter, which exists in many mentioned here. The most consistently found if not all mammals (e.g., van Noort, '691, may "nonauditory" projections were from the dorsal link the auditory system with parts of the vo- column nuclei, and they were found in cases calization system. It has been possible in bats in which the ICX was affected by the HRP to elicit echolocation calls with electrical stim- injection. These results agree with projections ulation of the central gray (Suga and Scblegel, to the ICX in other mammals (Schroeder and '73; Suga et al., '73; Schuller, '79). A connection Jane, '76; RoBarts, '79). By projections from between auditory and vocalization systems is the dorsal column nuclei to the IC, somatosen- important for the echolocation system of sory information is apparently linked with the Rhinolophus and other bats which change auditory system at the midbrain level and it their echolocation calls according to the ori- seems that the ICX is not part of the direct entation situation. But, the poorly developed ascending auditory pathway. Its connections connection between the IC and the central gray to and from the ICC have not yet been inves- disclosed by the anterograde HRP technique tigated in Rhinolophus. suggests that there must be additional links Labeled cells were found scattered in the between the auditory and vocalization systems pontine reticular formation. Most of them were in mammals. concentrated in the raphe nucleus just between the two MNTB. It seems likely that connec- CONCLUSION tions between the reticular formation and the The data presented here have shown that ICC may link arousal effects between the re- the central auditory system of the greater ticular formation and the auditory system and horseshoe bat Rhinolophus ferrumequinum vice versa. does not differ in general from that in other In all experiments anterograde labeling of mammals. Most auditory centers (including axon terminals occurred. As expected, heavy the medial geniculate body and the auditory labeling was found in the medial geniculate cortex) are especially well developed and the body which links the IC with the auditory cor- regions where the behavioral relevant fre- tex. The second main efferent IC projection is quencies are represented are extended in all a strong projection is a strong projection from nuclei. In contrast to earlier findings in the the IC to the lateral pontine nuclei. This pro- SOC of Rhinolophus a MSO has been defined jection has also been described in other mam- according to its projection pattern to the IC. In mals as the tectopontine tract (e.g., Moore and the bats investigated so far an additional nu- Goldberg, '66; van Noort, '69; Powell and Hat- cleus of the lateral lemniscus exists. 48 H. SCHWEIZER

The IC of Rhinolophus presents a very com- Boyd, J., and L.M. Aitkin (1976) Responses of single units plex projection pattern. All auditory nuclei in the pontine nuclei of the cat to acoustic stimulation. with the exception of the medial geniculate Neurosci. Lett. 3:259-263. Brown, J.C., and B. Howlett (1972) The olivo-cochlear tract body and the medial nucleus of the trapezoid in the rat and its bearing on the homologies of some con- body project directly to the IC. Therefore the stituent cell groups of the mammalian superior olivary auditory information reaches the IC directly complex. A thiocholine study. Acta Anat. 83:505-526. from the cochlear nuclei and after processing Browner, R.H., and D.B. Webster (1975) Projections of the trapezoid body and the superior olivary complex of the in the SOC andlor the nuclei of the lateral lem- Kangaroo rat (dipodomys merriamri. Brain. Behav. Evol. niscus and higher centers. Although the target 11:322-354. regions of ascending fibers from different au- Bruns, V. (1976) Peripheral auditory tuning for fine fre- quency analysis in the CF-FM bat, Rhinolophus ferru- ditory nuclei do not completely overlap within mequinum. 11. Frequency mapping in the cochlea. J. the ICC, one could suggest a time delay system Camp. Physiol. 106:87-97. within the ascending auditory system so that Carey, Ch.L., and D.B. Webster (1971) Ascending and de- auditory information processed at different scending projections of the inferior colliculus in the Kan- garoo rat (Dzpodomys rnerriarnii. Brain Behav. Evol. levels reaches the IC successively. It would be 4t400-412. of great interest to learn whether the different Edwards, St.B., Ch.L. Ginsburgh, C.K. Henkel, and B.E. incoming informations are passed separately Stein (1979) Sources of subcortical projections to the su- on to higher auditory centers or whether they perior colliculus on the cat. J. Camp. Neural. 184:309-330. are integrated within the IC. Elverland, H.H. (1978) Ascending and intrinsic projections ofthe superior olivary complex in the cat. Exp. Brain Res. ACKNOWLEDGMENTS 32:117-134. Fernandez, C., and F. Karapas (1967) the course and ter- The author wishes to thank the members of mination of the striae of Monakow and Held in the cat. the Arbeitskreis Neuro- und Rezeptorphysiol- J. Camp. Neural. 131:371-386. ogie for helpful discussion; doctors N. Neu- FitzPatrick, K.A. (1975) Cellular architecture and topo- graphic organization of the inferior colliculus in the squir- weiler, O.W. Henson, Jr., G. Schuller, and M. rel monkey. J. Camp. Neural. 164:185-208. Vater for their critical reading of the manu- Friend, J.H., N. Suga, and R.A. Suthers (1966) Neural re- script; and N. Chayegan, H. Hahn, and C. sponses in the inferior colliculus of echolocating bats to Ruehle for technical assistance. Data in this artificial orientation sounds and echoes. J. Cell Physiol. 67:319-332. report were part of the Ph.D. thesis of the au- Graham, R.C., and M.J. Karnovsky (1966) The early stages thor ('78). of absorption of injected horseradish peroxidase in the This investigation was supported by grants proximal tubules of the mouse kidney: Ultrastructural of the Deutsche Forschungsgemeinschaft, cytochemistry by a new technique. J. Histochem. Cyto- chem. 14:291-302. Schn. 13616; Br. 59312; SFB45lB22. Grinnell, A.D. (1963) The neurophysiology of audition in bats: Intensity and frequency parameters. J. Physiol. LITERATURE CITED 167:38-66. Adams, J.C. (1977) Technical considerations on the use of Harnischfeger, G. (1980) Brainstem units of echolocating horseradish peroxidase as a neuronal marker. Neurosci- bats code binaural time differences in the microsecond ence 2:141-145. range. Naturwissenschaften 671314-315. Adams, J.C. (1979) Ascending projections to the inferior Harrison, J.M., and M.L. Feldman (1970) Anatomical as- colliculus. J. Camp. 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