Ascending Projections to the Inferior Colliculus JOE C. ADAMS Laboratory of Neuro-otolaryngology, National Institute of NeuroLogical and Communicative Disorders and Stroke, National Institutes of Health, Building 36, Room 5032, Bethesda, Maryland 20014

ABSTRACT

Cells t h a t send ascending projections to t h e inferior colliculus were identified following injections of horseradish peroxidase into t h e colliculus. Labelled cells were found in all subcollicular auditory nuclei. Virtually all cells of t h e ipsilateral ventral nucleus of t h e lateral lemniscus and medial superior olive appear to project to the colliculus. Very few cells in these nuclei were labelled on t h e contralateral side. Heavy labelling on the contralateral side was found in the dorsal nucleus of t h e lateral lemniscus and cochlear nucleus, with less labelling being found ipsilaterally in these nuclei. The lateral superior olive was approximately evenly labelled on t h e two sides, with about half the cells from each side projecting to each colliculus. Cells in all periolivary cell groups were labelled, with most being found adjacent to t h e medial superior olive. An effort was made to identify individual cell types t h a t were labelled and some 24 cell types were identified. In the cochlear nucleus there were marked differences between cell types in t h e extent of their labelling. Topographic projections matched previously described tonotopic organization of t h e colliculus and all major subcollicular nuclei except the ventral nucleus of t h e lateral lemniscus. A description of t h e cells in t h e nucleus is provided.

The inferior colliculus (IC) occupies a central position in t h e auditory system where numerous ascending and descending pathways converge. Its connections with t h e cochlear nucleus (CN), t h e auditory cerebral cortex, and many intermediate nuclei make knowledge of its organization a prerequisite for t h e understanding of t h e structure and function of virtually all levels of t h e central auditory neuraxis. In view of its key importance, there have been surprisingly few studies of anatomical connections of t h e IC. The present study is concerned with its ascending inputs. Previous knowledge about origins of ascending inputs to t h e IC in the cat was learned from lesion studies (Stotler, '53; Warr, '66, '69, '72; Fernandez and Karapas, '67; Van Noort, '69, Osen, "72). The retrograde cell marking technique using horseradish peroxidase (HRP) (Kristenson and Olsson, '71; LaVail and LaVail, '72) was used in the present study. Large injections were made to identify as many projections as possible and to facilitate the identification of cell types t h a t were labelled. Identification of labelled cell types is J. COMP. NEUR. (1979) 183: 519-538.

necessary to fully exploit the HRP method and to provide a basis for further anatomical and physiological studies of t h e structures involved. MATERIALS AND METHODS

Adult cats were anesthetised with urethane (750 mg/kg) and ketamine (30 mg/kg). The posterior aspect of t h e IC was exposed by aspiration of the cerebellum. The intended site of injection was cut with a number 11 scalpel blade and about 1 p1 of a n aqueous solution of HRP (approximately 40%) was injected. Backflow often occurred so t h a t the exact volume of HRP solution at the injection site was not known. The skin incision was closed and normal body temperature maintained with a heating pad. Animals were kept deeply anesthetised until perfused. Survival times were from 24 to 33 hours. Results a r e from 20 animals with one IC injected and one animal with both IC injected. An additional nine animals served as controls, In three of these t h e cerebellum was removed and approximately 1 p1 of HRP injected into t h e CSF in

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the fourth ventricle. In six animals HRP was injected into a parasagittal cut of the trapezoid body following a ventral exposure of the medulla. Animals were perfused intracardially with 0.1 M phosphate buffer (pH 7.3) containing 0.5%sodium nitrate, and then with a fixative. The fixative was 1%paraformaldehyde and 4% glutaraldehyde in phosphate buffer (pH 7.31, except when silver impregnations were to be done. In these cases the fixative was 4% paraformaldehyde in phosphate buffer. Following perfusion, the brain was immediately removed from the skull and infiltrated with 30% sucrose. Infiltrated brains were frozen with carbon dioxide and cut in 52-pm sections, except when silver impregnations were planned. In these cases alternate sections were cut a t 26 pm and 52 pm. In early experiments sections were processed aLcording to the method of Graham and Karnovsky ('66), except that phosphate buffer was used in all solutions. In later experiments the sections were soaked in a solution of 0.5%cobalt chloride before incubation in diaminobenzidine (DAB) (Adams, '771.' A thionin counterstain was used on many sections. When silver impregnations were done, 26-pm sections were mounted on slides following incubation for reaction product, dried in a 37°C oven overnight, and processed in Protargol according to the method of Bodian. Locations of HRP labelled cells in individual sections were plotted using a PDP-11 computer which acted as a memory for cell locations. The outlines of low magnification projections of sections were entered into the computer using a sonic digitizer (Graf-Pen) with the dorsal and ventral midline used as fiducial points. The sections were then examined with a Zeiss Universal microscope equipped with a digital stepping stage with a step size of 0.5 pm. The direction and rate of movement of the stage was controlled by the microscopist through the computer by movement of a joystick. The fiducial points of each section were entered to permit computation of scale factor and orientation. The stage was then moved to position HRP-labelled cells under the crosshairs of an ocular. The stage coordinates of each labelled cell were stored in t h e computer. In this way, cells were accurately mapped and counted, with repeated counts of the same cell being rejected. Finally, graphic representations of section outlines and the locations of labelled cells were produced with an electrostatic plotter.

RESULTS

Nature of labelling Following injections of HRP into cuts in the IC, labelled cells were found in all subcollicular auditory nuclei. Labelled cells were always found in clusters or limited portions of the nuclei after injections into dorsal portions of the IC and often following ventral injections. The latter cases are particularly noteworthy because cuts made across the IC preceding ventral injections surely severed ascending fibers that terminate in the dorsal IC. Despite the fact that these fibers must have been exposed to the injected peroxidase the distribution of labelled cells indicated that the effective injection site was often limited to the region of the injection. The extent of labelling of cells following injections of large amounts of peroxidase into cuts often far exceeded that commonly reported following injections of smaller amounts when no cut is made. In extreme cases, cells were completely filled with reaction product so that their somata and dendritic trees were visible. More commonly, labelled cells were partially filled with diffuse and granular reaction product. In some cases, however, labelling was in the form of a few granules of reaction product within somata. As a rule, when cells in a given nucleus were heavily labelled, more labelled cells were found within that nucleus. Consequently, in the present study procedures were employed to produce the heaviest possible labelling so that all cells that could be labelled would be seen. The locations of labelled cells within a given nucleus generally matched those in the homonymous contralateral nucleus even though the number of cells and the extent of their labelling was usually asymmetric. In those cases where few cells were labelled, their locations matched the place of heaviest labelling in the homonymous contralateral nucleus. The symmetry of the labelling indicates that fibers from bilateral pairs of subcollicular nuclei terminate in the same parts of the IC. The injections into the IC were made a t ' After this series of experiments was finished other methods of visualizing HRP were published. To compare the present results with those obtainable with a more sensitive method one cat was injected with HRP and some sections incubated with tetramethylbenzedine (TMB) according to the method of Mesulum ('78). Adjacent aections were incubated with DAB. Results obtained wing T L B were essentially the same as with DAB.

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various dorso-ventral levels, but all injections involved the central nucleus of the IC. Following every injection some cells in most subcollicular nuclei were labelled. Because large volumes of HRP solutions were injected into cuts in the IC, association of the locations of labelled cells with rostro-caudal or medio-latera1 placements of the injections was precluded. However, there was a consistent relation between the locations of labelled cells and the dorso-ventral locations of the IC injections. This relation matched that predicted from the reported tonotopic organizations of the IC and most of the subcollicular nuclei. Examples of dorsal and ventral IC injections will be given to demonstrate these patterns of labelling. Cochlear nucleus Labelled cells were always found in both CN. For a description of the distribution of the labelled cells the traditional division of the nucleus into three principal parts will be used. The dorsal cochlear nucleus (DCN) is a layered structure situated caudal and dorsal to the ventral cochlear nucleus. The latter is divided into anteroventral (AVCN) and posteroventral (PVCN) portions. The region immediately caudal to the eighth nerve root is included in the AVCN (Brawer et al., '74). Labelled cells were found in all three parts of the nucleus, with their locations dependent upon the site of the IC injection. Labelling within specific regions of the CN contralateral to the injection is shown in figure 1.This figure shows the locations of all labelled cells in a series of transverse sections after an injection into the dorsal part of the IC. There were no labelled cells in the posterior or anterior extremes of the DCN. In the middle region, where the DCN has its greatest dorsoventral expanse, labelled cells were located ventrally (fig. 1A-C). In the PVCN there were no labelled cells in the caudal pole (fig. lA,B). More rostrally labelled cells were located in a band across the ventral border of the nucleus (fig. 1C). Still more rostrally little labelling was present medially (fig. 1D). In the caudal AVCN labelled cells were located ventrolaterally (fig. 1F). More rostrally, labelling was largely in the ventral portion with some labelling along the dorsal margins (fig. 11-K). The locations of labelling after injections into the ventral IC contrasted with and roughly complemented those of dorsal injections as is shown in figure 2. In the DCN, cells in the

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extreme posterior part of the nucleus were labelled. More rostrally, labelling was heaviest in dorsal regions. In the PVCN there was little labelling in the caudal pole, with the most posterior group of labelled cells located in the medial half of the nucleus (fig. 2B). Rostra1 to this, labelled cells appeared as a band extending across the PVCN and caudal AVCN, but few were found dorsally. Dorsal to the nerve root, t h e AVCN contained labelled cells throughout its dorsoventral extent (fig. 2F), but there were columns where little or no labelling occurred. Evidence of alternate labelled and unlabelled columns were found in all cases of abundant labelling of posterodorsal AVCN (see also fig. 16). Alternate columns of labelling were not readily evident in cases where HRP labelling was sparse and a counterstain was employed because spatial patterns of labelled cells become obscured under these conditions. The alternating columns of labelling in the AVCN became apparent in almost all cases when the labelled cells' locations were displayed in the computer-generated plots. Whenever cells in the dorsal part of the caudal AVCN were labelled there was a concentration of them along the margins (fig. 2E-J). The rostra1 pole of the AVCN was always sparsely labelled, particularly following injections of the ventral IC (fig. 2K,L). The labelled nuclei shown in figures 1 and 2 were contralateral to the injected IC. Ipsilaterally, labelled cells were found in regions corresponding to those of heaviest labelling in the contralateral CN, but far fewer were found and labelling was always faint. Superior olivary complex

Labelled cells were found in all nuclei and cell groups of the SOC. Figure 3 shows the distribution of labelled cells in the SOC following a peroxidase injection into the dorsal IC. Labelling was limited to the dorsal part of the medial superior olive (MSO) and there were far more labelled cells in the ipsilateral MSO than in the contralateral MSO. Only the lateral portion of the lateral superior olive (LSO) was labelled, and here approximately to the same extent on both sides. Labelled periolivary cells were predominantly ipsilateral to the injection. These labelled cells were found in all periolivary regions except lateral to the LSO; most were medial to the MSO. Labelled periolivary cells were widely scattered, with no apparent concentration within any cell group.

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Fig. 1 Frontal sections of the left cochlear nucleus (A-L) showing the locations of all HRP labelled cells in these sections following injection into the dorsal part of the right IC. Lateral is to the left. Section A is most caudal; L is most rostral. IC shows the injection site. The black region depicts the cut and the crosshatching the diffusion spot. NR is the auditory nerve root. Calibration bar is 2 mm and applies only to A-L.

The distribution of labelled SOC cells following a ventral IC injection is shown in figure 4. The ventral tip of t h e MSO was heavily labelled ipsilaterally, but less so contralaterally. The extent to which individual cells were filled was much greater than in t h e

case shown in figure 3 and, in t h e region of labelling, a greater percentage of labelled cells was found. Labelling in the LSO was limited to t h e medial part and was approximately the same on t h e two sides. Labelling was seen in all periolivary cell groups, with a high concen-

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Fig. 2 Frontal sections of the left cochlear nucleus (A-L) showing the locations of all HRP labelled cells in these sections following injection into the ventral part of the right IC. The organization of the figure is the same as in figure 1. G N is the granular cell layer. Calibration bar is 2 mm and applies only to A-L.

tration found medial to t h e ipsilateral MSO. Labelled cells found in posterior regions included some in t h e region of t h e nucleus reticularis paragiguntocellularis lateralis, small

cells located ventral to t h e facial nucleus (figs. 4 B,C), and small cells located lateral to the inferior olive immediately adjacent to t h e postolivary sulcus (fig. 4A). Labelled cells

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Fig. 3 Frontal sections of the SOC showing the locations of all HRP labelled cells in these sections following injection into the dorsal part of the right IC. Section A is most caudal. I 0 is inferior olive; VII is facial nucleus. Arrow heads in A and B point to post-olivary sulcus. Periolivary cells are those lying outside the boundaries of the MSO and LSO. Sections are evenly spaced a t = 900 fim. Calibration bar is 3 mm and applies only to the SOC.

were concentrated around the ventral portion of the MSO ipsilateral to the injection (fig. 4D-F), and were scattered from the dorsomedial periolivary group t o the ventral nucleus of the trapezoid body. In general, more labelled cells were found adjacent to the MSO than to the LSO. Although it is not evident in figures 3 and 4 there was a consistent difference in the proportion of cells that was labelled in the MSO and LSO. Even in cases of most intense labelling, there were never more than half the cells in LSO that were labelled, and the proportion was approximately the same on the two sides.

In the MSO when labelling was heavy in the side ipsilateral to the injection nearly all cells in the labelled region were labelled. There were always far fewer cells labelled in the contralateral MSO. The distribution of labelled cells in the SOC for the case in which both IC were injected with KRP distinguished that case from all others. When both IC were injected, in the regions where labelled cells were present, virtually all cells in both the MSO and LSO were labelled on both sides.

Nuclei of the lateral lemniscus Labelled cells were located in all portions of

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Fig. 4 Frontal sections of the SOC showing the locations of all HRP labelled cells in these sections following injections of the ventral part of the right IC. Section A is most caudal. Sections are evenly spaced a t 900 /un. Calibration bar is 3 mm and applies only to the SOC. Arrow heads in A point t o post-olivary sulcus.

the dorsal (DNLL) and ventral (VNLL) nuclei HI. The dorso-ventral distribution of labelled of the lateral lemniscus. Labelled cells in cells in the VNLL shown in the two cases is these nuclei following a n HRP injection into similar. The principal difference between the the dorsal IC are shown in the left column of two cases is that after the ventral injection figure 5. In VNLL, labelling ipsilaterally was more labelled cells were found. Unlike the CN, found scattered throughout the nucleus, but the MSO, and the LSO, the VNLL showed no in the DNLL it was restricted to the dorsal region bilaterally (fig. 5 C ) . Labelling in the lem- regional concentrations of labelled cells that niscal nuclei following a ventral injection of could be associated with the dorso-ventral the IC is shown in the right column of figure 5 . placement of the HRP injected site. In all In the DNLL contralateral to the injection la- cases labelling in the contralateral VNLL was belled cells were scattered through the nu- sparse, but more was found accompanying procleus, with fewer in the dorsal portion (fig. 5F- fuse ipsilateral labelling.

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Comparison of labelling between nuclei Table 1 gives t h e numbers of labelled cells found in t h e principal auditory nuclei in one ventral injection case. These counts were made in a sample of one-twelfth of t h e total amount of tissue from this animal. The table shows that there is a wide range over which subcollicular nuclei vary in the relative numbers of their inputs to t h e IC. Because t h e limitations of the HRP technique are not fully known these counts cannot be taken as estimates of the total number of cells projecting to the IC. However, t h e numbers in t h e table are representative of t h e rank order of labelling between nuclei. The contralateral ventral CN always had the most labelled cells and was always followed closely by t h e ipsilateral VNLL. The contralateral VNLL always had the fewest labelled cells; there were always very few in t h e contralateral MSO and few in the ipsilateral CN. There was considerable variability between animals in the rank orders of t h e remaining nuclei, but their relative numbers, compared to the most heavily labelled nuclei, were similar to those given in the table. Identification of labelled cells One of the advantages of the peroxidase labelling method is t h a t the individual cells that give rise to a projection can be visualized. In some cases the identification of labelled cells belonging to a specific cell class is relatively easy because of such features as cell size and characteristic location, for example t h e pyramidal cells of t h e DCN. In especially favorable cases labelled cells can be identified readily by their appearance because their somata and dendrites are filled with t h e reaction product. In other cases t h e amount of reaction product can be scant enough SO t h a t when sections are stained for Nissl substance, cells can be identified by their Nissl pattern and also be seen to contain the HRP reaction product. Another useful "counterstain" is the Protargol method of Bodian. All of these methods were used in the present study to identify labelled cells. Cell types in t h e DCN a r e distinct from those in t h e ventral CN. In the DCN labelled cells included a few small cells located in the superficial (plexiform) layer, pyramidal cells (sometimes called fusiform cells) located in t h e granular layer, and a n assortment of cell types located in t h e deep layer. Figure 6 shows

examples of labelled pyramidal cells in t h e posterior DCN contralateral to the injection. Among t h e pyramidal cell dendrites are several labelled cells of t h e plexiform layer (upper half of fig. 6). In t h e deep layer of t h e DCN labelled cell types included highly elongate fusiform cells located in t h e medial margin of t h e DCN with their dendrites oriented parallel to the margin (fig. 7 ) ,large cells with many long, spine-covered dendrites (fig. 71, and large cells with only a few thin dendrites (fig. 8 ) . Small cells in t h e deep DCN were not labelled. Labelled DCN cells ipsilateral to t h e injections included pyramidal cells and large cells of t h e deep DCN. Because of t h e sparsity of labelling i t was not possible to be sure how many large cell types labelled ipsilaterally. In t h e PVCN both small and large cells were labelled. The small cells were elongate and were most often located along the margins of t h e nucleus (fig. 9). In the posterior third of t h e PVCN labelled large cells were mostly confined to the medial border of the nucleus. Anterior to this, large and medium-sized labelled cells spanned t h e entire width of t h e nucleus. The dendrites of these cells were oriented across t h e PVCN inclined at a n angle of 30-40"from horizontal (fig. 10). A large part of t h e posterior PVCN is occupied by octopus cells, which are characterized by large somata containing finely dispersed Nissl substance (Osen, '69a) and a covering of houtons on their somata and proximal dendrites (Osen, '69a; Van Noort, '69; Kane, '73). Figure 11 shows a bouton on a large, labelled cell located in t h e PVCN. The terminal suggests that this cell is a n octopus cell. Positive identification of labelled octopus cells was never certain and many octopus cells always remained unlabelled. In several cases there was labelling of only t h e medial border of the posterior PVCN. Protargol impregnations showed t h a t virtually no cells in t h e region of labelling were covered with boutons terminaux. Adjacent TABLE 1

The number of cells labelled following a n HRP injection o f t h e ventral IC Ipsilateral

Contralateral

24 29 546 126 1,160 59

1,333 142 9 101 3 267

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Ventral cochlear nucleus Dorsal cochlear nucleus Medial superior olive Lateral superior olive Ventral lateral lemniscus Dorsal lateral lemniscus

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Fig. 5 Frontal sections of the nuclei of the lateral lemniscus for two cases of HRP injection of the IC. The left column is a dorsal injection case, the right column a more ventral injection. Sections A and E are most caudal. VLL = VNLL, DLL = DNLL. Calibration bar is 8 mm.

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sections with no counterstain showed no labelled cells in t h e zone immediately lateral to the labelled zone. This more lateral zone contains almost exclusively octopus cells and most cells there had boutons terminaux. Thus, labelling in the posterior PVCN was limited t o a cytologically distinct part of t h e nucleus containing few, if any, octopus cells. In t h e anterior PVCN numerous cells were labelled. These cells lacked boutons on their somata but were readily identified as t h e multipolar cells described by Osen (’69a) by their size and their large clumps of Nissl substance (fig. 12). In the region of t h e eighth nerve root many multipolar cells and some small cells were labelled. Granule cells in t h e layer forming t h e lateral surface of this part of t h e nucleus remained unlabelled (figs. 2E, 13). This region contains a high concentration of globular cells which have a finely dispersed Nissl substance. In all cases many globular cells remained unlabelled by peroxidase, and only a few small labelled cells were found t h a t had a dispersed Nissl pattern (fig. 14). Globular cells have been identified as “bushy cells,” a term used to describe their dendrites as seen in Golgi impregnations (Tolbert and Morest, ’77). In no case was a peroxidase labelled cell in the region of the eighth nerve root identified as a bushy cell by HRP filling of its dendritic tree. As was shown in figures 1 and 2 t h e number of labelled cells in the AVCN rostral to t h e eighth nerve root is relatively small. Osen (’69a) described t h e prominent cell type of t h e anterior AVCN as a spherical cell (which could be large or small) t h a t had a characteristic cap of Nissl substance close to t h e nucleus as well as clumps of Nissl substance farther from the nucleus. Few, if any, large ( > 20 pm) spherical cells were labelled with HRP but smaller cells with t h e spherical cell Nissl pattern were labelled. Measurements of cells in t h e AVCN t h a t were impregnated by the rapid Golgi method (Brawer and Morest, ’75) suggest t h a t bushy cells seen in Golgi impregnations correspond to spherical cells seen in Nissl-stained material. In t h e present experiments, when labelled cells were completely filled with reaction product, only one bushy cell was identified. This was in t h e posterior dorsal part of t h e AVCN. On t h e other hand, many labelled stellate cells were found (fig. 15). There were also many small cells labelled in the AVCN, particularly along t h e margin of t h e nucleus (figs. 13, 16). Figure 16 shows a striking case of a heavily labelled

margin with a n adjacent zone of unlabelled cells. The perikarya of a great many labelled cells were too filled with reaction product to permit visualization of a Nissl pattern while their dendrites were inadequately filled to permit their identification as a bushy cell or stellate cell. Because of this some sections were “counterstained” using t h e Protargol impregnation. This impregnation is useful for identifying cells in t h e CN because i t reveals some types of terminals on cells. In Protargol material several hundred HRP labelled cells in t h e AVCN were located t h a t contained sufficiently little reaction product to make it possible to see impregnated terminals. Out of these less t h a n 10 cells had a bifurcated terminal on them and none of these terminals were as large or elaborate as those commonly seen on t h e large spherical cells in t h e rostral AVCN (Adams, ’77: fig. 4). Labelled cells in t h e SOC were located in all nuclei. I n t h e LSO large multipolar and smaller bipolar cells were labelled (fig. 17). In t h e MSO bipolar cells located centrally and multipolar cells along t h e margins were labelled (fig. 18).Most labelled periolivary cells were located medial and ventral to the MSO, including slender (’5 p m across), elongate cells, large ( > 2 5 pm) multipolar cells with long dendrites, and cells of intermediate size (fig. 19). Within t h e medial nucleus of t h e trapezoid body few cells were labelled and these appeared to correspond to t h e stellate cells described by Morest (’68).Anterior to t h e LSO large multipolar and large (=25 pm) spherical cells t h a t stain very lightly for Nissl s u b s t a n c e were labelled. Because of t h e paucity of labelled cells and t h e multiplicity Fig. 6 Labelled cells in and superficial to the granular layer of the caudal DCN following an HRP injection of the contralateral IC. Pyramidal cells are in a row across the bottom one third of the picture. Note the labelled cells among their apical dendrites. Calibration 50 pm. Fig. 7 Labelled cells in the deep DCN following an HRP injection of the contralateral IC. The large cell at the top has many dendrites, some of which are covered with spines. Calibration 100 pm. Fig. 8 A labelled cell in the deep DCN following an HRP injection of the contralateral IC. In contrast to that in figure 7 this cell has a few long, thin, smooth dendrites. It is situated in the acoustic striae at the medial border of nucleus, with the dendrites extending toward the external surface. Calibration 70 pm. Fig. 9 Labelled cells along the medial border of the PCVN with dendrites extending laterally across the nucleus. Note the large cells with thick dendrites and the smaller, elongate cells. Calibration 70 pm.

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of cell types in periolivary cell groups, i t is not possible to state with assurance how many types were labelled or if all types were present on both sides. Extending caudally from the posterior periolivary cell group is the nucleus reticularis paragigantocellularis lateralis, which contains small, medium and large cells (Taber, '61). In all injected animals cells of all three sizes were labelled and were found scattered throughout the length of this nucleus, with cells of about 19 pm average diameter being most plentiful. No consistent spatial distributions of labelled cells were observed in this region with dorso-ventral changes in the site of the collicular injection, but more labelled cells were found following ventral injections. Small cells located immediately dorsomedial to t h e postolivary sulcus (figs. 3A,B, 4A) were regularly found, with more located on the side ipsilateral to the injected IC. Also, small cells adjacent to t h e facial nucleus were consistently found with most located ventrally. I t could not be determined how many distinct cell types were represented by the labelled small cells located caudal to t h e SOC. Only a brief description of t h e cytology of lateral lemniscal nuclei appears in t h e modern literature (Taber, '61), so a more detailed one will be provided here to permit reporting of labelled cell types. The nuclei lie within t h e lateral lemniscus as a continuous band of cells from just ventral to t h e IC to t h e region immediately rostra1 to t h e LSO. A distinction between a dorsal nucleus (DNLL) and a ventral nucleus (VNLL) has long been recognized. The DNLL is characterized by large (19-27 p m diameter) cells with darkly staining Nissl substance. Interspersed among the large cells a r e elongate cells (10-13 p m across, 35-40 pm in length) and multipolar cells (17-24 pm in diameter). All of these cells have finely dispersed Nissl substance and they frequently lack a distinct cap of Nissl substance around their nuclei. The VNLL can be divided into zones: the dorsal, middle, and ventral zones, each of which contain cytologically distinct cell types. The boundaries between zones are not distinct and regions of overlap commonly occur. Cells of the dorsal zone resemble those of the DNLL in t h a t they have finely dispersed Nissl substance and frequently lack a cap of Nissl substance around their nuclei. These cells are multipolar in shape and range from approximately 12-25 pm in diameter. A predominant cell type of t h e middle zone is t h e

multipolar cell. I t is medium-sized (12-20 p m diameter) and contains large, densely staining clumps of Nissl substance. The other principal cell type of t h e middle zone is the horizontal cell. This cell is 9-11 p m across and has faint Nissl substance t h a t extends into the proximal dendrites. The dendrites radiate in t h e horizontal plane so t h a t the cells appear with a long axis running across the ascending lemniscal fibers when they are viewed in frontal and in parasagittal sections. Frequently these horizontal cells occur in groups t h a t interrupt t h e continuity of cells in t h e vertical axis of t h e VNLL. In t h e middle zone there are also small cells (6-12 pm diameter) containing dark Nissl substance. Most cells in t h e middle zone, but especially t h e multipolar cells, have a distinct cap of Nissl substance around their nuclei. The ventral zone of the VNLL contains several cell types. The predominant type is a n oval shaped cell ranging from 10-20 pm in diameter. This cell has finely dispersed, densely staining Nissl substance. The Nissl substance seldom extends into the basal dendrites so t h a t the cells have a smooth profile resembling a n oval pebble. These cells bear a close resemblance to the principal cells of the medial nucleus of t h e trapezoid body because of their size and appearance in Nissl as well as Protargol preparations. In Protargol material these cells a r e covered with large end bulbs, or calyces of Held. A high concentration of oval cells is invariably found in t h e ventro-lateral limit of the VNLL. The full extent of the space occupied by oval cells varies greatly from cat to cat. In some cases the multipolar cells of the middle zone extend ventrally so as to lie medial to some oval cells of t h e ventral zone. Oval cells are found scattered as far medial as the MSO in t h e region anterior to t h e LSO. This region is more appropriately considered a n anterior periolivary region, however, because the predominant cell types here, large multipolar cells (20-35 pm) and lightly staining round cells (25 pm diameter), are also found caudally, ventral and medial to the MSO. The ventral zone of the VNLL also contains elongate cells (8-12 pm across, 20-35 pm long) t h a t have darkly staining clumped Nissl substance. The other cells found in this zone a r e small cells (10-15 Fm diameter) with sparse Nissl substance. Following HRP injections of the IC all cell types of t h e ipsilateral DNLL and VNLL were labelled. Virtually every cell type was also labelled in the contralateral lemniscal nuclei

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53 1

Fig. 10 Labelled cells in the anterior PVCN. Note the orientation of the dendrites from upper left toward lower right. This direction is from dorsolateral toward ventromedial. Calibration 60 pm. Fig. 11 A large cell in the PVCN was labelled with HRP and also treated with Protargol. Note the bouton terminal (arrow). Calibration 14 pm, Fig. 12 A labelled cell in the anterior PVCN t h a t was counterstained with thionin. The large dark clumps are Nissl substance that are characteristic of multipolar cells. Calibration 10 pm. Fig. 13 Labelled cells in t h e posterior AVCN. There is a sharp contrast in the distribution of labelled cells between the granular cell layer (upper left) and the layer containing many labelled multipolar cells just below it. The clear region in the lower right is an auditory nerve fascicle. Thionin counterstain. Calibration 100 pm.

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Fig. 14 Labelled cells (center) of the interstitial region of the CN t h a t were labelled sufficiently lightly to permit visualization of their dispersed Nissl pattern. Thionin counterstain. Calibration 20 pm. Fig. 15 A labelled bushy cell (arrow) and numerous labelled stellate cells located in the posterior dorsal part of the AVCN. Thionin counterstain. Calibration 150 pm. Fig. 16 Heavy labelling of cells along the margms a t the anterior AVCN, especially along the dorsomedial pole (upper right). Many of the dark figures within the nucleus are erythrocytes. Calibration 300 pm. Fig. 17 Labelled cells in the LSO. Note t h e abundance of multipolar cells and the one clear case of an elongate cell (upper right). Dark field micrograph. Calibration 50 pm.

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Fig. 18 Labelled cells in t h e ventral MSO and adjacent regions. The dark line running across the upper right of the picture is a blood vessel that lies along the main axis of the MSO. Note the elongate and multipolar cells within the MSO. The labelled cells in the lower left quadrant lie ventral and medial to the MSO. Darkfield micrograph. Calibration 150 p. Fig. 19 Labelled cells in the posterior periolivary group ipsilateral to an HRP injection of the IC. Note the range of sizes of labelled cells. Calibration 100 Fm. Fig. 20 Labelled cells of t h e VNLL ipsilateral to t h e injected IC. Calibration 45 Fm. Fig. 21 Labelled cells in the DNLL contralateral to t h e injected IC. Calibration 45 pm,

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t h a t has been described for t h e IC and most subcollicular nuclei. Rose et al. ('63) and Merzenich and Reid ('74) described t h e tonotopic organization of t h e central nucleus of IC as having low characteristic frequencies located dorsally and high characteristic frequencies ventrally. Correspondingly, in t h e present study when t h e dorsal IC was injected with HRP, cells in the CN, MSO, LSO and DNLL t h a t were labelled were located in t h e regions of those nuclei where maximal responsiveness to low frequency tones has been found in physiological studies (Rose et al., '59; Kiang et al., '75; Bourk, '76; Tsuchitani and Boudreau, '66; Guinan et al., '72; Aitkin e t al., '70). In t h e case of periolivary cell groups there were not enough labelled cells to determine if topographic projection from these areas matched their tonotopic organization (Tsuchitani, '77) and t h a t of t h e injection site in t h e IC. In t h e case of t h e VNLL, however, the topographic projections found in the present study do not match t h e tonotopic organization described for t h a t nucleus (Aitkin e t al., '70). Aitkin et al. described a regular progression of change in frequency responsiveness from dorsal to ventral VNLL, whereas in the present study there were labelled cells scattered throughout t h e dorso-ventral extent of t h e VNLL regardless of t h e site of t h e H R P injection into t h e IC. The reason of this apparent conflict of results may lie in t h e preliminary nature of t h e physiological results, which were from only four electrode penetrations. Others (Guinan et al., '721, recording in the VNLL, were unable to confirm the findings of Aitkin et al. Furthermore, projections to the VNLL from t h e CN are non-symmetric (Warr, '66, '69, '721, in contrast to CN projections to the MSO. This difference in the topographic organization of ascending inputs to the VNLL and MSO appears to be accompanied by a difference in t h e topographic organization of their outputs, as evidenced in the present study. Reconciling differences in topographic connections of t h e VNLL with its reported tonotopic organization can best be achieved following further physiological studies. The amount of HRP injected in the present series is greater than that commonly used. The injection of more than 1 p1 resulted in good labelling but also in a large diffusion spot. The possibility t h a t the effective injecDISCUSSION tion site may have been larger than desirable Topographic projections found in the pres- must be examined. In particular, the possient study match t h e tonotopic organization bility t h a t results could be confounded by

with t h e exception of t h e oval cells of t h e ventral zone of t h e VNLL. Of the types t h a t were labelled, not all types on t h e contralateral side were labelled in every case however. Most cells in ipsilateral lemniscal nuclei labelled heavi 1y . Lateral to t h e lateral leminscus is t h e sagulum. This nucleus extends from t h e brachium conjunctivum to t h e lateral zone of t h e IC. There are two distinct cell types in t h e sagulum. The predominant type is t h e elongate cell which is usually 5-6 pm across with its long axis parallel to t h e lemniscus. Elongate cells stain very lightly for Nissl substance. Scattered among t h e elongate cells are larger (8-13 pm diameter) cells with somewhat circular profiles. The cells have large nuclei and a few dark-staining clumps of Nissl substance. Similar cells are more abundant in the adjacent middle zone of t h e VNLL. Following HRP injections into t h e IC, both cell types of the sagulum were labelled on both sides. Although some were near t h e injection site, no cells in t h e sagulum were ever labelled heavily. Controls Animals with the cerebellum removed and HRP injected into the CSF of t h e fourth ventricle showed no labelled cells in auditory nuclei. These animals and animals with HRP injected into t h e IC had labelled cells in nuclei known to project to the cerebellum, including the pons, the inferior olive, and widely scattered cells of t h e reticular formation. Injections of HRP were made into t h e trapezoid body to determine if t h e cell types of the ventral CN t h a t seldom labelled following injections of t h e IC were refractory to labelling with HRP. Large spherical cells of t h e rostra1 AVCN and t h e globular cells of t h e interstitial region of t h e CN were readily labelled following HRP injections into t h e trapezoid body. The distribution of labelled cells in t h e case that was incubated with TMB was t h e same as was found in adjacent sections incubated with DAB even though more labelled cells were found with TMB. The more sensitive method showed no new cell types labelled and t h e relative numbers of labelled cells of each type was approximately t h e same in TMB and DAB material.

Y

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spread of H R P to the DNLL must be considered. That this was not t h e case is indicated by the pattern of labelling seen in t h e lower nuclei as t h e injection site was moved from the dorsal limit of the IC toward t h e ventral limit. With dorsal injections the diffusion of injected HRP was limited to t h e dorsal portions of the IC. With more ventral injections the location of the most intensely labelled cells within most nuclei changed systematically but no new class of cells became labelled as t h e injection sites were moved very close to the ventral extent of IC. This finding suggests t h a t either t h e cells t h a t project to t h e DNLL were not labelled following ventral IC injections or t h a t t h e same cells project to both the IC and t h e DNLL. In either case t h e interpretation of t h e present results is not changed. The consistent labelling of some cell types more heavily than others indicates a difference in t h e rates at which different somata accumulate peroxidase. I t may be t h a t differences in rates of HRP accumulation are due to differences in diameters of t h e axons carrying t h e HRP. This interpretation is supported by the present findings. Labelling of cells in t h e ipsilateral CN was always less complete than in the contralateral CN. Warr ('72) has described fibers t h a t ascend to the IC from the ipsilateral CN as small fibers t h a t travel in t h e "lateral trapezoid tract." Their small diameter probably accounts for their having been frequently undetected by silver degeneration techniques (Fernandez and Karapas, '67; Van Noort, '69; Warr, '69; Osen, '72). The diameter of these fibers contrasts with the larger size of those projecting to the contralateral IC which are readily seen by silver degeneration methods. The findings t h a t cells of some classes consistently labelled faintly and t h a t only a few of these cells ever labelled raise questions about t h e uniformity of projections from these cell classes. If the percentages of labelled cells of various classes can be taken at face value, then t h e present results indicate t h a t small proportions of cells of some classes send projections to t h e IC. These cell classes include spherical cells, globular cells, and octopus cells of the contralateral CN, those cell classes of the ipsilateral CN and contralateral VNLL t h a t project to the IC, as well as several cell types found in periolivary cell groups. The faint labelling of t h e CN cells is not due to a refractoriness of these cells to labelling with

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HRP. This is evidenced by their clear labelling following HRP injections into the trapezoid body in the present study or by injections into t h e acoustic striae (Adams and Warr, '76). It may be that all cells of those classes t h a t labelled faintly project to the IC but were not seen because the methods employed were not sensitive enough to demonstrate their projections. Extra large amounts of HRP were injected in a n effort to visualize all projections possible. In addition, some sections of one case were incubated with TMB. This procedure is more sensitive than t h a t employing DAB and is very likely as sensitive as any method available for demonstrating t h e presence of HRP. Nevertheless, the same patterns of labelled cells were seen in the TMB-treated tissue as in t h a t treated with DAB. This supports the suggestion t h a t only small percentages of t h e cells in classes t h a t label faintly project to t h e IC. Alternatively, if all cells of these classes project to t h e IC, other methods will have to be employed to demonstrate this. Cells caudal to the SOC were found to project to t h e IC i n t h e present study. Cells in this region have also been shown to project to t h e CN (Adams and Warr, '76) and this region receives projections from t h e CN (Van Noort, '69; Warr, '72). In view of the connections of cells in this region with auditory nuclei, and because they a r e continuous with t h e loosely organized posterior periolivary cell groups, i t seems reasonable to regard cells in this region t h a t have auditory connections as periolivary cells. An organization of ascending projections from the principal nuclei of the SOC was evident from t h e pattern of labelled cells. The pattern of labelling in t h e LSO contrasted markedly with t h a t in t h e MSO. The number and degree of labelling of cells in the LSO was always approximately t h e same on both sides. Furthermore, no matter how dense t h e labelling, when one IC was injected, only approximately half the cells in each LSO were labelled. This finding agrees with retrograde degeneration results (Stotler, '53) and suggests t h a t half t h e cells in each LSO project to each IC. This suggestion was supported by the finding t h a t , following HRP injections of both IC, all LSO cells in t h e region of labelling were labelled. This pattern of projections contrasts with t h a t seen in t h e MSO where the degree of labelling was asymmetric and all cells appear to project to the ipsilateral IC, and perhaps they project to both IC. Projections to t h e IC

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from the contralateral MSO were not reported by Brunso-Bechtold and Thompson ('76) or Elverland ('78). These negative findings are likely due to lack of sensitivity of t h e HRP methods employed as is suggested by the lack of complete filling of any cells with reaction product in either study. Elverland ('78) also failed to find projections to t h e contralateral IC following tritrated leucine injections of t h e MSO. Negative findings from anterograde studies using amino acids are much stronger when more than one tracer is employed. The pattern of labelling in t h e CN, taken with results of previous studies, suggests a n organization of its ascending and recurrent projections. There is evidence t h a t cell types t h a t were labelled lightly, infrequently, or not a t all in the present study (spherical cells, globular cells, and octopus cells) have promin e n t projections t o subcollicular nuclei (spherical cells to the MSO and LSO, Warr, '66; Osen, '69b; globular cells to t h e MNTB, Warr, '72; Tolberg and Morest, '77; octopus cells to t h e VNLL, Van Noort, '69; Warr, '69). On the other hand, cells t h a t labelled heavily in the present study (large cells of the DCN and multipolar cells of the PVCN) may innervate periolivary cells by way of collaterals (Fernandez and Karapas, '67; Warr, '69; Van Noort, '69). Periolivary cells are t h e sources of recurrent innervation of t h e cochlea and CN (Rasmussen, '46, '60, '64; Van Noort, '69; Luk et al., '74; Warr, '75; Adams and Warr, '76; Adams, '76; Kane, '76; Elverland, '77) and their inputs appear to be segregated according to cell classes within t h e CN. In t h e present study only two classes of cells in the cochlear nucleus were never found labelled after IC injections. These were granule cells and small cells of t h e deep DCN. Previously, only pyramidal cells and a few giant cells of the DCN had been identified as projecting to the IC (Osen, '72). It was evident from anterograde degeneration studies t h a t other CN cells also project to t h e IC, but they could not be identified using retrograde degeneration techniques. The finding t h a t small cells located in t h e dorsal and ventral CN project to t h e IC was somewhat surprising in view of previous assertions t h a t these small cells are interneurons and do not send axons out of t h e CN (Lorente d e NO, '76). It may be that these small cells a r e a subclass of t h e multipolar cells t h a t labelled so readily in the rest of the ventral CN. In the present study three classes of large

cells of the ventral CN were seldom labelled: large spherical cells, globular cells, a n d octopus cells. The large spherical cells of t h e rostra1 AVCN are most likely bushy cells described in Golgi material (Brawer and Morest, '75). Only one bushy cell was ever found completely filled with reaction product (fig. 15) and i t was located in t h e postero-dorsal part of t h e AVCN. Globular cells found in t h e region of t h e eighth nerve root were seldom labelled. These cells are also bushy cells (Tolbert and Morest, '77) and no cells in the region of t h e eighth nerve root were identified as bushy cells by t h e reaction product filling their dendritic trees. Octopus cells were rarely, if ever, labelled. The lack of labelling of octopus cells was accented by t h e fact t h a t when labelling occurred in t h e posterior PVCN, where octopus cells a r e most abundant, labelled cells were largely or solely confined to a band lying along t h e medial border of t h e nucleus where there a r e few, if any, octopus cells. In t h e PVCN single unit recordings have shown t h a t units with high characteristic frequencies are located dorsally and low characteristic frequencies ventrally (Rose e t al., '59; Kiang e t al., '75) so t h a t the band of labelling in the posterior PVCN presumably cut across isofrequency contours. Octopus cells lying lateral to t h e band of labelled cells remained unlabelled. The fact t h a t unlabelled octopus cells were located lateral to labelled cells indicates t h a t t h e injection sites in the IC were sufficiently ventral to label cells in the part of t h e PVCN where many octopus cells are located. Despite t h e paucity of labelling of t h e three above mentioned cell types, the ventral CN contralateral to the injected IC consistently contained more labelled cells than other subcollicular nuclei (table 1).The large majority of labelled cells in t h e ventral CN were multipolar cells and small cells. It is interesting t h a t Osen ('69a) was unable to specify differences (except for size) between multipolar cells and small cells seen in Nissl material. Further, Brawer et al. ('74) noted t h a t small cells resembled stellate cells when seen in Golgi impregnation. It seems likely t h a t t h e stellate cells of Brawer et al. correspond to multipolar cells of Osen. The structural similarity of these cells are accompanied by similarities in their projections, as evidenced by present results. Given the relative numbers and extent of labelling of these cells, i t seems likely t h a t their output may be responsible for t h e primacy of contralateral stimulation in

ASCENDING PROJECTIONS TO THE INFERIOR COLLICULUS

producing evoked responses in the IC (Ades and Brookhart, '50). Physiological effects in the IC due to direct projections of other cell classes of the ventral CN remain to be established. In this regard findings regarding antidromic stimulation of cells in the CN are noteworthy. Bourk ('76) was unable to antidromically activate large spherical cells of the AVCN by stimulation of the IC, although other nearby cells, believed to be stellate cells, were readily activated. Physiological studies of antidromic activation of globular cells and octopus cells would certainly be of interest in light of present findings. Results of the present experiments show that there are some 24 classes of cells from each half of the brainstem that give rise to ascending projections to the IC. Exact numbers cannot be given with confidence because of uncertainties in defining the classes of small cells in the CN and SOC. The large number of inputs of the IC emphasizes the enormity of the task of describing the organization of the IC. Furthermore, this gives a perspective regarding the difficulties of interpreting physiological studies of single unit activity in the IC. There are indications that the central nucleus of the IC is not equipotential regarding termination of ascending inputs (Goldberg and Moore, '67; Van Noort, '69; Osen, '72; Jones, '76) and physiological characteristics (Aitkin et al., '76). The present findings of the number of ascending inputs demonstrate the need for an intensified effort in order t o obtain a rudimentary knowledge of collicular organization. ACKNOWLEDGMENTS

The author is grateful to friends who commented on various versions of the manuscript. L. E. Lipkin provided valuable advice concerning the use of the computer to plot results. W. D. Livingston programmed and interfaced the computer. Barbara Judy, Gabrielle Nieses, and Marianne Parakkal provided technical assistance. LITERATURE CITED Adams, J. C. 1976 Central projections to t h e cochlear nucleus. Neuroscience Abstracts, 11: 12. 1977 Technical considerations on t h e use of horseradish peroxidase as a neuronal marker. Neurosci., 2: 141-145. Adams, J. C., and W. B. Warr 1976 Origins of axons in t h e cat's acoustic striae determined by injection of horseradish peroxidase into severed tracts. J. Comp. Neur., 270: 107-122. Ades, H. W., and J. M. Brookhart 1950 The central auditory pathway. J. Neurophysiol., 13: 189-205. Aitkin, L. M., D. J. Anderson and J. F. Brugge 1970

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Tonotopic organization and discharge characteristics of single neurons in nuclei of t h e lateral lemniscus of the cat. J. Neurophysiol., 33: 421-440. Aitkin, L. M., L. Roth and M. M. Merzenich 1976 Differential projections of ascending inputs to the central nucleus of the inferior colliculus. Neuroscience Abstracts, II: 1. Bourk, T. R. 1976 Electrical Responses of Neural Units i n t h e Anteroventral Cochlear Nucleus of the Cat. Ph.D. Thesis, Massachusetts Institute of Technology. Brawer, J. F., and D. K. Morest 1975 Relations between auditory nerve endings and cell types in t h e cat's anteroventral cochlear nucleus seen with the Golgi method and Nomarski optics. J. Comp. Neur., 260: 491-506. Brawer, J. R., D. K. Morest and E. C. Kane 1974 The neuronal architecture of t h e cochlear nucleus of the cat. J. Comp. Neur., 155: 251-300. Brunso-Bechtold, J . K., and G. C. Thompson 1976 Auditory hind brain projections to the inferior colliculus as demonstrated by horseradish peroxidase in the cat. Anat. Rec., 284: 365. Elverland, H. H. 1977 Descending connections between t h e superior olivary and cochlear nuclear complexes in t h e cat studies by autoradiographic and horseradish peroxidase methods. Exp. Brain Res., 27: 397-412. 1978 Ascending and intrinsic projections of the superior olivary complex in t h e cat. Exp. Brain Res., 32: 117-134. Fernandez, C., and F. Karapas 1967 The course and termination of t h e stria of Monakow and Held in t h e cat. J. Comp. Neur., 232: 371-386. Goldberg, J . M., and R. Y. Moore 1967 Ascending projections of t h e lateral lemniscus in the cat and monkey. J. Comp. Neur., 229: 143-156. Graham, R. C. Jr., and M. J. Karnovsky 1966 The early stages of absorption of injected horseradish peroxidase in t h e proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., 14: 291-302. Guinan, J. J., B. E. Norris and S. S. Guinan 1972 Single auditory units in t h e superior olivary complex. 11. Locations of unit categories and tonotopic organization. Internat. J. Neurosci., 4: 147-166. Jones, D. R. 1976 Ascending auditory pathways to the inferior colliculus in t h e tree shrew. Neuroscience Abstracts, ZZ: 20. Kane, E. C. 1973 Octopus cells in t h e cochlear nucleus of t h e cat: Heterotypic synapses upon homotypic neurons. Internat. J. Neurosci., 5: 251-279. Kane, E. S. 1976 Descending inputs to caudal cochlear nucleus in cats: A horseradish peroxidase (HRP) study. Am. J. Anat., 246: 433-441. Kiang, N. Y. S., D. A. Godfrey, B. E. Norris and S.Moxon 1975 A block model of t h e cat cochlear nucleus. J. Comp. Neur., 262: 221-246. Kristensson, K., and Y. Olsson 1971 Uptake and retrograde axonal transport of peroxidase in hypoglossal neurones. Acta Neuropath. (Berlin), 29: 1-9. LaVail, J. H., and M. W. LaVail 1972 Retrograde axonal transport i n t h e central nervous system. Science, 276: 1416-1417. Lorente de NO. R. 1976 Some unresolved problems concerning t h e cochlear nerve. Ann. Otol., Rhinol., and Laryngol. 85 (Suppl.), 34: 1-28. Luk, G. D., D. K. Morest and N. M. McKenna 1974 Origins of t h e crossed olivocochlear bundle shown by a n acid phosphatase method i n t h e cat. Ann. Otol., Rhinol. and Laryngol., 83: 382-394. Merzenich, M. M., and M. D. Reid 1974 Representation of t h e cochlea within the inferior colliculus of t h e cat. Brain Res., 77: 397-415.

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Mesulam, M. 1978 Tetramethyl henzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents. J. Histochem. Cytochem., 26: 106-117. Morest, D. K. 1968 The collateral system of the medial nucleus of the trapezoid body of the cat, its neuronal architecture and relation to t h e olivo-cochlear bundle. Brain Res., 9: 288-311. Osen, K. K. 1969a Cytoarchitecture of the cochlear nuclei of the cat. J. Comp. Neur., 136: 453-484. 1969h The intrinsic organization of the cochlear nuclei in the cat. Acta oto-laryng. (Stockholm), 67: 352-359. 1972 Projection of the cochlear nuclei on the inferior colliculus in t h e cat. J. Comp. Neur., 144: 355-372. Rasmussen, G . L. 1946 The olivary peduncle and other fiber projections of the superior olivary complex. J. Comp. Neur., 84: 141-219. 1960 Efferent fibers of the cochlear nerve and cochlear nucleus. In: Neural Mechanisms of the Auditory and Vestibular Systems. G. L. Rasmussen and W. F. Windle, eds. Thomas, Springfield, pp. 105-115. 1964 Anatomic relationships of the ascending and descending auditory systems. I n : Neurological Aspects of Auditory and Vestibular Disorders. W. S. Field and B. R. Alford, eds. Thomas, Springfield, pp. 5-19. Rose, J. E., R. Galambos and J. R. Hughes 1959 Microelectrode studies of the cochlear nuclei of the cat. Bull. Johns Hopkins Hosp., 204: 211-251. Rose, J. E., D. D. Greenwood, J. M. Goldherg and J. E. Hind 1963 Some discharge characteristics of single neurons in the inferior colliculus of t h e cat. I: Tonotopical organization, relation of spike counts to tone intensity, and firing ~

patterns of single elements. J. Neurophysiol., 26: 294-320. Stotler, W. A. 1953 An experimental study of the cells and connections of the superior olivary complex of the cat. J. Comp. Neur., 98: 401-432. Taber, E. 1961 The cytoarchitecture of the brain stem of the cat. I. Brain stem nuclei of the cat. J. Comp. Neur., 116: 27-69. Tolbert, L. P., and D. K. Morest 1977 Cambined Golgi, horseradish peroxidase (HRP), and electron microscopic study of bushy cells in the cochlear nucleus. SOC.Neurosci. Ahstr. Ill: 12. Tsuchitani, C. 1977 Functional organization of lateral cell groups of cat superior olivary complex. J. Neurophysiol., 40: 296-318. Tsuchitani, C., and J. C. Boudreau 1966 Single unit analysis of cat superior olive S sequent with tonal stimuli. J. Neurophysiol., 29: 684-697. Van Noort, J. 1969 The Structure and Connections of the Inferior Colliculus. An Investigation of the Lower Auditory System. Van Gorcum, Leiden. Warr, W. B. 1966 Fiber degeneration following lesions in the anterior ventral cochlear nucleus of the cat. Exp. Neurol., 24: 453-474. 1969 Fiber degeneration followinglesions in the posteroventral cochlear nucleus of the cat. Exp. Neurol., 23: 140-155. 1972 Fiber degeneration following lesions in the multipolar and globular cell areas in the ventral cochlear nucleus of the cat. Brain Res., 40: 247-270. 1975 Olivocochlear and vestibular efferent neurons of the feline brain stem: Their location, morphology and number determined by retrograde axonal transport and acetylcholinesterase histochemistry. J. Comp. Neur., 161: 159-182.

Note added in proof: Since this paper was completed a report has appeared (Beyerl, B. D., 1978, Brain Res., 145: 209-223) on HRP injection in the r a t IC. Reported results are in agreement with those of the present study. However, no mention was made of

projections from the ipsilateral DNLL, either VNLL, contralateral MSO, or several periolivary cell groups. To check on the possibility of species differences of IC projections, two rats were injected with HRP. Results were in complete agreement with those described above in the cat. Beyerl’s lack of finding the projections mentioned above may be due to the use of a less sensitive HRP technique, perhaps due to shall amounts of injected HRP and a prolonged period of fixation and infiltration of the tissue with sucrose.