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Afferent projections to the central nucleus of the inferior colliculus in the rat
Brain Research, 145 (1978) 209-223 © Elsevier/North-HollandBiomedicalPress
209
Research Reports
AFFERENT PROJECTIONS TO THE CENTRAL NUCLEUS OF THE INFERIOR COLLICULUS IN THE RAT BRIAN D. BEYERL*
Department of Neurophysiology and Waisman Center on Mental Retardation and Human Development, University of Wisconsin Medical School, Madison, Wisc. 53706 (U.S.A.) (Accepted August 12th, 1977)
SUMMARY
The afferent projections to the inferior colliculus of the rat were studied using the method of retrograde transport of horseradish peroxidase (HRP). Following large injections of HRP into the central nucleus, cells within the cochlear nuclei, superior olivary complex and auditory cortex were stained. Within the contralateral dorsal cochlear nucleus, fusiform cells were heavily labeled. Giant cells were also labeled in deeper layers. In the contralateral ventral cochlear nucleus, virtually all major cell types were labeled, with some types being labeled in greater numbers than others. Octopus cells of posteroventral division of ventral cochlear nucleus (PVCN) were never labeled. HRP-positive cells were found in ipsilateral and contralateral lateral superior olivary nucleus (LSO), ipsilateral medial superior olivary nucleus (MSO), ipsilateral and contralateral lateral nucleus of the trapezoid body (LTB), ipsilateral ventral nucleus of the trapezoid body (VTB), and ipsilateral superior paraolivary nucleus (SPN). Pyramidal cells of layer V of auditory cortex were heavily labeled. Small injections of HRP into the central nucleus resulted in labeled cells within restricted regions of the cochlear nuclei, superior olivary complex and auditory cortex. Injections into dorsal regions of the central nucleus resulted in cells labeled in ventral regions of the dorsal and ventral cochlear nuclei, and in lateral regions of LSO. These regions contain neurons which are considered to have low best frequencies. Injections placed in more ventral regions of the central nucleus led to labeling of cells in more dorsal regions of the cochlear nuclei and more medial regions of LSO in agreement with the tonotopical progressions within these structures.
* Address reprint requests to Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Md. 21205, U.S.A.
210 INTRODUCTION The inferior colliculus receives ascending afferent input from the cochlea," nuclei and superior olivary complex ~t,s,92a,2G,2s as well as a corticofugal projection from auditory cortex 7,21. The use of modern anatomical tracer methods in recent experiments in cat 1-a,'~, monkey 11, guinea pig 1, and tree shrew s,ls have allowed more precise identification of the sources of some of these inputs. However, little is known of the specific cell types which contribute to these pathways. The rat is well suited for study of this question, since celt types within the cochlear nuclei have been described 12,~a and the cytoarchitecture of the auditory nuclei is known 14,1¢;. In addition, the tonotopic organization of the inferior colliculus has been characterized". In the experiments reported here the sources of afferent fibers reaching the inferior colliculus were studied using the method of retrograde transport of horseradish peroxidase (HRP). It has been possible using this method to identify several of the cell types within the brain stern and cortex which contribute to this afferent supply. In addition, it has been possible to describe the topography of the projection: pattern and to relate it to the tonotopical organization of the brain stem auditory pathway. MATERIALS AND METHODS Experiments were conducted on 22 male rats of the Holtzman and Charles River strains, weighing between 200 and 500 g. Each rat was anesthetized with sodium pentobarbital (20 mg/kg body weight) and placed in a stereotaxic head holder. The dorsal surface of the skull was exposed and a small rectangular bone flap over the occipital cortex was carefully removed with the help of a high speed dental drill. The dura mater was removed and the cortex was aspirated to expose the dorsal surface of the inferior colliculus. A small calibrated glass microplpette with a tip diameter less than 20/~m was filled with a 40 % solution (0.4 mg HRP/1.0/A H20) of horseradish peroxidase (Type VI, Sigma Chemical Co.). The micropipette was advanced under visual control into the inferior colliculus by means of a stereotaxic micromanipulator. The H R P solution was injected under pressure (2-5 lbs/sq.in.) through the pipette. Larger volumes (1.0-2.0/~l) of HRP were injected by making multiple small injections along the dorsoventral axis of the inferior colliculus. The pipette remained stationary for at least 5 rain after each of these smaller injections. The large volumes required approximately 35-45 min for the entire series of injections. Injections of smaller volume (0.4 .ul) were made in the same general manner, at a single predetermined depth within the central nucleus. In two of the experiments the H R P solution was in)ected through a 5.0/~1 Hamilton microsyringe which was also advanced into the colliculus by means of a stereotaxic micromanipulator.
Histologicalprocedures After a survival period of 24-48 h, the rats were anesthetized with sodium pentobarbital and perfused through the heart with saline followed immediately with approximately 500 ml of a 4% buffered paraformaldehyde solution (pH 7,2). Both
211 perfusates were cooled to 4 °C before use. After perfusion, the brain was removed from the skull and placed in buffered paraformaldehyde for 12 h at 4 °C. It was then placed in a 5 o/sucrose solution, buffered at pH 7.4 with 0.2 M sodium phosphate for 8 h followed by a solution of 3 0 ~ buffered sucrose for 16 h. Alternate frozen sections were cut at thicknesses of 90 # m and 30/~m and placed in Trisma buffer (pH 7.6). The 90 #m sections were used for the HRP-diaminobenzidine reaction. The alternate 30 #m sections were stained with thionin. The substrate used for the peroxidase in the 90 #m sections was 3,3'-diaminobenzidine tetrahydrochloride (I00 mg diaminobenzidine in 200 ml distilled water at 22-25 °C). One drop of 30% HzOz was added to this solution immediately before processing the tissue. The sections were exposed to substrate for 25 rain, carried through three baths of Trisma buffer (pH 7.6), and mounted onto gelatinized slides. The sections were then dried in a 37 °C incubator before coverslips were applied. Alternate 30 #m sections stained with thionin were used to study the cytoarchitecture. Sections were studied under both bright- and dark-field illumination. Thick sections were traced at a magnification of 30 x with the aid of a Bausch and Lomb projector, or were photographed; dots were placed on the drawings or negatives to show the locations of HRP-positive cells observed in the original sections. Cytoarchitectural parcellation was readily derived from adjacent thionin sections after adjustments were made for tissue shrinkage. RESULTS Three major cell groups are recognized in Nissl-stained sections of the inferior colliculus of the rat. The central nucleus is composed of densely packed small neurons, and constitutes the core of the inferior colliculus. The central nucleus is bounded laterally by the external nucleus, which is composed of large, loosely packed neurons. The pericentral nucleus comprises a sheet of small neurons which envelops the dorsal and caudal surfaces of the central nucleus, and the lateral surface of the external nucleus. Its neurons are similar in appearance to those of the central nucleus but are more densely packed than those of either the central or external nuclei. A population of larger cells is seen in the dorsomedial part of the central nucleus, as is the case in the cat 2~ and monkey 10. Injections of HRP were placed in the central nucleus of the inferior colliculus in all of the animals used in these experiments. The evidence indicates that the uptake of HRP was confined to the central nucleus in the region around the pipette tip, although there was in all cases widespread diffusion of the tracer.
(a) D&tribution of labeled cells within the cochlear nuclear complex The cochlear nuclear complex of the rat can be parcellated into dorsal and ventral nuclei based on its appearance in Nissl-stained material (Fig. 1). The dorsal cochlear nucleus (DCN) is composed of three distinct neuronal laminae that are recognized in most mammalian species. Within the ventral cochlear nucleus (VCN), anteroventral (AVCN) and posteroventral (PVCN) divisions are recognizable. Locations
Fig. 1. Normal cytoarchitecture of the cochlear nuclei and superior olivary complex ol the rat. A: coronal section through rostral anteroventral cochlear nucleus (AVCN). B: coronal section through dorsal cochlear nucleus (DCN) and po~teroventral cochlear nucleus (PVCN). RB, restiform body: 5ST, spinal trigeminal tract. C: coronal section through the right superior otivary complex. LSO, lateral superior olivary nucleus; MSO, medial superior olivary nucleus; LTB, lateral nucleus Of the trapezoid body; VTB, ventral nucleus of the trapezoid body; MTB, medial nucleus of the trapezoid body: SPN, superior paraolivary nucleus. Nissl stain. ,: 60.
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Fig. 2. A : tracing of a coronal section through the inferior colliculus of rat 76-21M. A volume of 2.0 FI 4 0 ~ w/v HRP was injected into the central nucleus; the total injection was composed of ten 0.2/~1 injections (black dots). The dashed ouline within the inferior colliculus represents the limits of the central nucleus; 41-h survival. B-K: a series of camera lucida drawings of coronal sections through the contralateral (with respect to the injection site) cochlear nucleus showing the distribution or labeled cells. Section B is most rostral, section K is most caudal. Dots within sections represent labeled cells. Arrowheads in sections I and J point to the octopus cell region of the PVCN. L N: series of coronal sections showing the distribution of labeled cells through the superior olivary complex of both sides in rat 76-21M. Section L is most rostral, section N is most caudal. See Fig. l for abbreviations.
214
Fig. 3. Photomicrographs (× 1050) of HRP-labeled (right) and corresponding normal Nissl-stained cochlear nucleus neurons. A and B: D C N fusiform cell. C and D: D C N giant (deep) cell, E and F: PVCN type j (small pale) cell. G and H: PVCN type 1 (medium-sized pale multipolar) cell, 1 and J: PVCN fusiform cell (region V). K and L: AVCN type i (round) cell. M and N: AVCN type d (fuslform) cell. O and P: AVCN type f (large round) cell.
215 and identification of labeled cells are based on the cytoarchitectonic studies of the cochlear complex of the rat by Harrison and irving 12,13. In the present experiments, virtually all major cell types within the cochlear nuclear complex have been labeled with HRP. However, some cell types have been labeled in greater numbers than others. The general distribution of labeled cells in the cochlear nuclei following a large injection of H RP into the central nucleus is illustrated in Fig. 2. In the AVCN, type i (round) cells in region I were labeled in greatest numbers (Fig. 3K and L). Region ! is located in the dorsal two-thirds of the AVCN. Compared with numbers of labeled cells observed in region I, fewer labeled ceils were seen in region II, located in the ventral one-third of the AVCN and PVCN. Labeled cells observed in greatest numbers within region II included type f (large round) and type d (fusiform) cells (Fig. 3M-P). Type c (plump round) cells of region Ill, located in the rostral pole of the AVCN, and type g (globular) cells of region I I were rarely labeled. Within the PVCN, cells of region V, located in intermediate and certain dorsal regions of the PVCN, were labeled in greatest numbers. Labeled cells in region V included type j (small pale) cells and type 1 (medium-sized pale multipolar) cells (Fig. 3E-H). In addition, region V contained many labeled fusiform cells (Fig. 31 and J). Numbers of labeled cells were significantly fewer within region II of the PVCN; they included cell types which were labeled in the corresponding region I1 of the AVCN. No labeled cells were ever observed in region IV (octopus cell region) of the PVCN. The distribution of HRP-positive cells within the contralateral dorsal cochlear nucleus is illustrated in Fig. 2. Fusiform cells within DCN are heavily labeled in all of the present experiments (Fig. 3A and B). In addition, a sizeable number of neurons within the deeper layers of DCN are labeled (Fig. 3C and D). With the exception of an occasional displaced fusiform cell, labeled cells have not been observed in the molecular layer of DCN in any of the brains in this series. In those brains where there was heavy labeling in the contralateral cochlear complex, only a few scattered HRPpositive cells were observed in the ipsilateral cochlear nuclei. Single small injections of HRP into the central nucleus resulted in labeled cells within restricted regions of the contralateral cochlear nuclear complex. A small injection in the ventral region of the central nucleus of rat 76-4M resulted in labeling of cells confined to dorsal regions of the contralateral DCN and AVCN (Fig. 4B-E). No labeled cells were seen in PVCN of this brain. This observation is consistent with results from other experiments in which dorsally situated octopus cells in PVCN were not labeled (Fig. 2). A small injection into the central region of the central nucleus resulted in labeled cells confined to intermediate levels of the contralateral AVCN, PVCN and DCN (Fig. 5B-E). Again, cells within the octopus cell region were not labeled. An injection into dorsal areas of the central nucleus labeled cells only in more ventral areas of each of the three major subdivisions of the cochlear complex (Fig. 6B-E).
(b) Distribution of labeled cells within the superior olivary complex The superior olivary complex of the rat is comprised of 6 cell groups which
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Fig. 4. A: tracing of coronal section through the inferior colliculus of rat 76-4M showing the focus of the single HRP injection in black (0.4 M 4 0 ~ w/v HRP). The dashed outline within the inferior colliculus represents the limits of the central nucleus: 46-h survival. B-E: a series of coronal sections through the contralateral (with respect to the site of injection) cochlear nucleus. Section B is most rostral, section E is most caudal. The arrowhead in section D points to the octopus cell region in the PVCN. F and G: coronal sections through the superior olivary complex of both sides: section F is rostral to section (3. See legend of Fig. 1 for abbreviations. are easily recognized in Nissl-stained sections 14 (Fig. 1). The lateral superior olivary nucleus (LSO) is a p r o m i n e n t cell mass which is S-shaped i n coronal sections. Medial to the LSO is the medial superior olivary nucleus (MSO) composed o f fusiform cells with their long axes oriented horizontally. The celt groups lying in or near the trape-
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Fig. 5. A: tracing of coronal section through the inferior colliculus of rat 76-3M showing the focus of the single HRP injection in black (0.4/~l 4 0 ~ w/v HRP). The dashed outline within the inferior colliculus represents the limits of the central nucleus; 45-h survival. B-E: a series of coronal sections through the contralateral (with respect to the injection site) cochlear nucleus. Section B is most rostral, section E is most caudal. F and G : coronal sections through the superior olivary complex of both sides; section F is rostral to section G. See legend of Fig. 1 for abbreviations. zoid b o d y have been collectively labeled as the nuclei o f the t r a p e z o i d body. Three distinct nuclei can be seen: the medial (MTB), lateral (LTB), and ventral (VTB) nuclei o f the t r a p e z o i d body. The LTB is c o m p o s e d o f round, fusiform, and m u l t i p o l a r cells; it is located i m m e d i a t e l y ventral to the LSO. The cells o f the M T B are g r o u p e d near the midline o f the b r a i n stem; they are r o u n d or o v a l - s h a p e d with eccentric
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Fig. 6. A: tracing of coronal section through the inferior colliculus of rat 76-I09M showing the focus of the single HRP injection in black (0.4 pl 4 0 ~ w/v HRP). The dashed outline within the inferior colliculus represents the limits of the central nucleus; 42-h survival. B-E: a series of coronal sections through the contralateral (with respect to the site of injection) cochlear nucleus. Section B is most rostral, section E is most caudal. F and G : coronal sections through the superior olivary complex of both sides; section F is rostral to section 13. See legend of Fig. 1 for abbreviations. nuclei. The VTB is located v e n t r o m e d i a l to the LTB, a n d contains small, lightly staining cells. The superior paraolivary nucleus (SPN) is situated medial to the LSO a n d is composed of large a n d small m u l t i p o l a r cells.
219 The retrograde HRP reaction within cells in the superior olivary complex after injections of a large volume of HRP into the central nucleus is shown in Fig. 2L-N. Heavy labeling is seen in neurons throughout the rostrocaudal extent of the following cell groups: ipsilateral and contralateral LSO, ipsilateral and contralateral LTB, ipsilateral MSO, ipsilateral VTB, and ipsilateral SPN. Labeled cells are seen throughout the dorsoventral extent of the ipsilateral MSO. HRP-positive cells have not been observed in either the ipsilateral or contralateral MTB. In the present experiments, labeled cells are not uniformly distributed in most cell groups within the superior olivary complex. I n general, the lateral limbs of both the contralateral and ipsilateral LSO are only weakly labeled as compared to the remainder of this nucleus. The lateral regions of SPN are more heavily labeled than more medial regions of the nucleus. Labeled cells seem to cluster in intermediate regions of VTB. After a small injection into the most ventral areas of the central nucleus, labeled cells are observed in the most dorsal portion of the medial limb of the contralateral and ipsilateral LSO (Fig. 4). Fig. 5 illustrates the distribution of positive cells in the LSO after an HRP injection into a more intermediate region of the central nucleus. In this brain, positive cells are found in the ventral portions of the medial limb of LSO bilaterally (sections F and G). A small injection into the dorsal region of the central nucleus results in the labeling of cells confined to the dorsal portion of the lateral limb of LSO bilaterally (Fig. 6). The topographic projection pattern of the other cell groups in the superior olivary complex could not be determined due to tile relatively small number of cells labeled by small injections of HRP.
(c) Distribution of labeled cells within auditory areas of the cerebral cortex The cytoarchitecture of the auditory cortex in the rat has been described by Krieg15,16. Layers II and Ill are fairly thin and are composed of pyramidal and granular cells of varying sizes. Layer IV is composed of a large number of granule cells and is difficult to distinguish from supragranular layers. Layer V is composed of pyramidal cells of varying sizes and is easily distinguished from the layers above and below it. A variety of cell types comprise layer VI, including fusiform and spherical cells. After injections of HRP into various parts of the central nucleus, labeled cells are seen in the ipsilateral cerebral cortex. The distribution of labeled cells after a large injection roughly corresponds to auditory area 41 of Krieg's topographic map of rat cortex (Fig. 7). Virtually all of the labeled cells appear to be medium-to-large pyramidal cells and are confined to layer V (Fig. 8). No labeled cells are seen in the contralateral cortex or in the thalamus of either side. Small injections of HRP within the central nucleus resulted in relatively small patches of labeled cells in layer V. (d) Distribution of other labeled eel& Occasionally, a few scattered HRP-positive cells were observed in the contralateral inferior colliculus as a result of large injections of HRP into the central nucleus of the inferior colliculus. On the other hand, many HRP-positive cells were observed in the contralateral dorsal nucleus of the lateral lemniscus as a result of both large and small injections of HRP into the central nucleus. One brain, which
220 A
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Fig. 7. A series of coronal sections through the ipsilateral (with respect to the injection site) cortex of rat 76-21M. The stippled area on the inset drawing indicates the area m which labeled cells were observed in the cortex. Dashed lines on the inset indicate the planes and locations of sections shown in the coronal sections. Dots within the section represent labeled cells. The location of the injection within the inferior colliculus is seen in Fig. 2. received no injection o f H R P , was processed in the same way as the e x p e r i m e n t a l brains to search for e n d o g e n o u s H R P within a u d i t o r y n e u r o n s o f the b r a i n stem '7. N o labeled n e u r o n s were f o u n d in any o f the a u d i t o r y structures in this brain. DISCUSSION The inferior colliculus in the rat receives strong, t o p o g r a p h i c a l l y organized p r o j e c t i o n s from the three m a j o r subdivisions o f the c o n t r a l a t e r a l cochlear nuclear complex, f r o m 5 cell g r o u p s o f the s u p e r i o r olivary complex, a n d f r o m p y r a m i d a l cells o f layer V o f a u d i t o r y cortex. W h i l e any o r all o f the cell g r o u p s which m a k e u p the inferior colliculus m a y receive afferent fibers f r o m these sources, the results presented here indicate t h a t the central nucleus is the prime t a r g e t o f n e u r o n s l a b e l e d i n these e x p e r i m ents. The foci o f all injections were well within the c e n t r a l nucleus, a l t h o u g h a b r o w n reaction p r o d u c t was seen to s p r e a d widely t h r o u g h o u t the inferior collicutus. Small injections o f H R P resulted in labeling o f cells within restricted regions in all o f the lower
221
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Fig. 8. Photomicrograph of a coronal section through the ipsilateral cortex of rat 76-21M. This section corresponds to section B in Fig. 7. Dots indicate locations of labeled cells seen in the adjacent section. Cortical laminae are indicated along the right side of the photomicrograph. Nissl stain..! 56.
nuclei. Furthermore, the distribution of labeled cells was systematically related to the site of the injection. This observation would suggest that the most active uptake of H R P occurred in a restricted region of the central nucleus immediately surrounding the tip of the micropipette, regardless of the more widespread diffusion of H R P indicated by the brown background reaction product. Results obtained from brains that received small injections of H R P indicate that projections from the contralateral cochlear nuclear complex to the central nucleus are arranged topographically. Cells located in intermediate regions of contralateral AVCN, PVCN, and D C N project to intermediate regions of the central nucleus, cells located in dorsal areas of DCN and AVCN project to ventral areas of the central nucleus and cells in ventral regions of all 3 cochlear nuclei project to dorsal regions of the central nucleus. Similar results have been described in the cat TM. in general, cells with high best frequencies are located ventrally and cells with low best frequencies are located dorsally within the central nucleus in the rat 6. Thus, the pattern of cochlear nucleus afferents to the central nucleus suggests that within each of the 3 subdivisions of the rat cochlear nucleus, cells with high best frequencies are located dorsally and cells with low best frequencies are located ventrally, as indeed they are in the cat 22. As is the case in the contralateral cochlear nuclei, the pattern of ascending projections from the ipsilateral and contralateral LSO is an orderly one and may be correlated with the tonotopical organization of the LSO 24. Results of others concerning projections from the cochlear nucleus to the ipsi-
222 lateral inferior colliculus are not consistent; some studies25, 26,2s have demonstrated a significant projection, while others4,9,19 have not. In the present experiments, a strong ipsilateral projection has not been observed. However. these negauve results do not eliminate the possibility of such an ipsilateral pathway in the rat, considering the limited value of negative results obtained with the H R P technique. Results from all brains examined in this series indicate a strong projection from the ipsilateral superior paraotivary nucleus to the central nucleus. The SPN in the rat is located in the same relative position with respect to other cell groups of the supermr olivary complex as is the dorsomedial periolivary group m the cat 17. suggesting a possible functional homology between these two nuclei. However. the dorsomedial periolivary group in the cat appears to project to the inferior colliculus bilaterally 2°. whereas the present experiments suggest that the SPN in the rat projects only to the ipsilateral inferior colliculus. Furthermore. the cytoarchitecture of these two nuclear groups is quite different in the two species. The dorsomedial periolivary group in the cat is composed primarily of small 'elongate' neurons tT. whereas the SPN in the rat is composed primarily of larger multipolar cells ~'~. The termination of cortico-collicular fibers in cat 7,zl and tree shrew ~s appear confined to the cell groups which surround the central nucleus or to the large-cell dorsomedial sector of the central nucleus. In the monkey H. on the other hand. the laminated portion of the central nucleus is a target of fibers originating in area AI. The present experiments indicate that the central nucleus in the rat also receives substantial input from the cerebral cortex. However. the organization of auditory cortex in the rat is not known and. thus. it is not possible to determine whether this projection originates in the primary auditory area or in one or more of the surrounding auditory fields. Regardless of the cortical fields within which they are located, cortico-collicular neurons are, for the most part, pyramidal cells of layer V. Recently, Zook and Casseday 29 demonstrated a similar distribution of cortico-fugal cells projecting to the inferior colliculus in the bat. ACKNOWLEDGEMENTS The author wishes to express his gratitude to Dr. John F. Brugge tbr unfailing advice during the course of these experiments, as well as for valuable help with the manuscript. Thanks are also due to Sigrid Borgner for the typing of the manuscript, and Shirley Hunsaker for assistance with photography. This research was supported by N I H Program Project Grant NS05326. the K n a p p Honors Research Scholarship (University of Wisconsin Honors Program), and by the B. H. Hibbard Award (Phi Kappa Phi H o n o r Society).
REFERENCES 1 Adams, J. C., Ascending and descending projections to the inferior colliculus, Neurosci. A b s t r . . 1 (1975) 37. 2 Adams, J. C., Types of cells in the cochlear nucleus that pro lect to the inferior colliculus in the cat. Anat. Rec., 184 (1976) 340.
223 3 Aitkin, L. M., Roth, L. and Merzenich, M. M., Differential projections of ascending inputs to the central nucleus of the inferior colliculus, Neurosci. Abstr., 2 (1976) 3. 4 Barnes, W. T., Magoun, H. W. and Ranson~ S. W., The ascending auditory pathway in the brainstem of the monkey, J. romp. Neurol., 79 (1943) 129 152. 5 Brunso-Bechtold, J. K. and Thompson, G. C., Auditory hindbrain projections to the inferior colliculus as demonstrated by horseradish peroxidase in the cat, Anat. Rec., 184 (1976) 365. 6 Clopton, B. M. and Winfield, J. A., Tonotopic organization in the inferior colliculus of the rat, Brain Research, 56 (1973) 355-358. 7 Diamond, l. T., Jones, E. G. and Powell, T. P. S., The projection of the auditory cortex upon the diencephalon and brain stem in the cat, Brain Research, 15 (1969) 305 340. 8 Jones, D. R., Ascending auditory pathways to inferior colliculus in the tree shrew, Neurosci. Abstr., 2 (1976) 20. 9 Fernandez, C. and Karapas, F., The course and termination of the striae of Monakow and Held in the cat, J. romp. Neurol., 131 (1967) 371-386. 10 Fitzpatrick, K. A., Cellular architecture and topographic organization of the inferior colliculus of the squirrel monkey, J. comp. Neurol., 164 (1975) 185 208. I I Fitzpatrick, K. A. and Imig, T. J., Projections of the auditory cortex in the Owl monkey, Am~t. Rec., 184 (1976) 403. 12 Harrison, J. M. and Irving, R., The anterior ventral cochlear nucleus, J. eomp. Nem'oL, 124 (1965) 15 -~12.
13 Harrison, J. M. and Irving, R., The organization of the posterior ventral cochlear nucleus in the rat, J. romp. Neurol., 126 (1966) 391 402. 14 Harrison, J. M. and Feldman, M. L., Anatomical aspects of the cochlear nucleus and superior olivary complex. In W. D. Neff (Ed.), Contributions to Sensory Physiology, Vol. 4, Academic Press, New York, 1970, pp. 95 142. 15 Krieg, W. J. S., Connections of the cerebral cortex. I. The albino rat. (A) Topography of the cortical areas, J. romp. Neurol., 84 (1946) 221 276. 16 Krieg, W. J. S., Connections of the cerebral cortex. 1. The albino rat. (B) Structure of the cortical areas, J. romp. Neurol., 84 (1946) 277-323. 17 Morest, D. K., The collateral system of the medial nucleus of the trapezoid body of the cat, its neuronal architecture and relation to the olivocochlear bundle, Brain Research, 9 (1968) 288-31 I. 18 Oliver, D.L., Midbrain projections to the medial geniculate body and their relationship to corticofugal projections of the auditory cortex, Neurosci. Abstr., 2 (1976) 8. 19 Osen, K. K., Projection of the cochlear nuclei on the inferior colliculus in the cat, J. romp. Neurol., 144 (1972) 355-372. 20 Rasmussen, G. L., Concerning certain projection fibers of the superior olivary complex, Anat. Rec., 88 (1944) 453. 21 Rockel, A. J. and Jones, E. G., The neuronal organization of the inferior colliculus of the adult cat. I. The central nucleus, J. romp. Neurol., 147 (1973) 11-60. 22 Rose, J. E., Galambos, R. and Hughes, J. R., Microelectrode studies of the cochlear nuclei of the cat, Bull. Johns Hopk. Hosp., 104 (1959) 211-251. 23 Stotler, W. A., An experimental study of the cells and connections of the superior olivary complex of the cat, J. romp. Neurol., 98 (1953) 401-432. 24 Tsuchitani, C. and Boudreau, J. C., Single unit analysis of cat superior olive S segment with tonal stimuli, J. Neurophysiol., 29 (1966) 684-697. 25 Warr, W. B., Fiber degeneration following lesions in the anterior ventral cochlear nucleus of the cat, Exp. Neurol., 14 (1966) 453-474. 26 Wart, W. B., Fiber degeneration following lesions in the posteroventral cochlear nucleus of tl~e cat, Exp. Neurol., 23 (1969) 140-155. 27 Wong-Riley, M. T. T., Endogenous peroxidatic activity in brain stem neurons as demonstrated by their staining with diaminobenzidine in normal squirrel monkeys, Brain Research, 108 (1976) 257 278. 28 Woollard, H. H. and Harpman, J. A., The connexions of the inferior colliculus and of the dorsal nucleus of the lateral lemniscus, J. Anat. (Lond.), 74 (1940) 441-458. 29 Zook, J. M. and Casseday, J. H., Projections to the inferior colliculus in the echolocating bat, Pteronotus parnellii p~rnellii, Neurosci. Abstr., 2 (1976) 29.
209
Research Reports
AFFERENT PROJECTIONS TO THE CENTRAL NUCLEUS OF THE INFERIOR COLLICULUS IN THE RAT BRIAN D. BEYERL*
Department of Neurophysiology and Waisman Center on Mental Retardation and Human Development, University of Wisconsin Medical School, Madison, Wisc. 53706 (U.S.A.) (Accepted August 12th, 1977)
SUMMARY
The afferent projections to the inferior colliculus of the rat were studied using the method of retrograde transport of horseradish peroxidase (HRP). Following large injections of HRP into the central nucleus, cells within the cochlear nuclei, superior olivary complex and auditory cortex were stained. Within the contralateral dorsal cochlear nucleus, fusiform cells were heavily labeled. Giant cells were also labeled in deeper layers. In the contralateral ventral cochlear nucleus, virtually all major cell types were labeled, with some types being labeled in greater numbers than others. Octopus cells of posteroventral division of ventral cochlear nucleus (PVCN) were never labeled. HRP-positive cells were found in ipsilateral and contralateral lateral superior olivary nucleus (LSO), ipsilateral medial superior olivary nucleus (MSO), ipsilateral and contralateral lateral nucleus of the trapezoid body (LTB), ipsilateral ventral nucleus of the trapezoid body (VTB), and ipsilateral superior paraolivary nucleus (SPN). Pyramidal cells of layer V of auditory cortex were heavily labeled. Small injections of HRP into the central nucleus resulted in labeled cells within restricted regions of the cochlear nuclei, superior olivary complex and auditory cortex. Injections into dorsal regions of the central nucleus resulted in cells labeled in ventral regions of the dorsal and ventral cochlear nuclei, and in lateral regions of LSO. These regions contain neurons which are considered to have low best frequencies. Injections placed in more ventral regions of the central nucleus led to labeling of cells in more dorsal regions of the cochlear nuclei and more medial regions of LSO in agreement with the tonotopical progressions within these structures.
* Address reprint requests to Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Md. 21205, U.S.A.
210 INTRODUCTION The inferior colliculus receives ascending afferent input from the cochlea," nuclei and superior olivary complex ~t,s,92a,2G,2s as well as a corticofugal projection from auditory cortex 7,21. The use of modern anatomical tracer methods in recent experiments in cat 1-a,'~, monkey 11, guinea pig 1, and tree shrew s,ls have allowed more precise identification of the sources of some of these inputs. However, little is known of the specific cell types which contribute to these pathways. The rat is well suited for study of this question, since celt types within the cochlear nuclei have been described 12,~a and the cytoarchitecture of the auditory nuclei is known 14,1¢;. In addition, the tonotopic organization of the inferior colliculus has been characterized". In the experiments reported here the sources of afferent fibers reaching the inferior colliculus were studied using the method of retrograde transport of horseradish peroxidase (HRP). It has been possible using this method to identify several of the cell types within the brain stern and cortex which contribute to this afferent supply. In addition, it has been possible to describe the topography of the projection: pattern and to relate it to the tonotopical organization of the brain stem auditory pathway. MATERIALS AND METHODS Experiments were conducted on 22 male rats of the Holtzman and Charles River strains, weighing between 200 and 500 g. Each rat was anesthetized with sodium pentobarbital (20 mg/kg body weight) and placed in a stereotaxic head holder. The dorsal surface of the skull was exposed and a small rectangular bone flap over the occipital cortex was carefully removed with the help of a high speed dental drill. The dura mater was removed and the cortex was aspirated to expose the dorsal surface of the inferior colliculus. A small calibrated glass microplpette with a tip diameter less than 20/~m was filled with a 40 % solution (0.4 mg HRP/1.0/A H20) of horseradish peroxidase (Type VI, Sigma Chemical Co.). The micropipette was advanced under visual control into the inferior colliculus by means of a stereotaxic micromanipulator. The H R P solution was injected under pressure (2-5 lbs/sq.in.) through the pipette. Larger volumes (1.0-2.0/~l) of HRP were injected by making multiple small injections along the dorsoventral axis of the inferior colliculus. The pipette remained stationary for at least 5 rain after each of these smaller injections. The large volumes required approximately 35-45 min for the entire series of injections. Injections of smaller volume (0.4 .ul) were made in the same general manner, at a single predetermined depth within the central nucleus. In two of the experiments the H R P solution was in)ected through a 5.0/~1 Hamilton microsyringe which was also advanced into the colliculus by means of a stereotaxic micromanipulator.
Histologicalprocedures After a survival period of 24-48 h, the rats were anesthetized with sodium pentobarbital and perfused through the heart with saline followed immediately with approximately 500 ml of a 4% buffered paraformaldehyde solution (pH 7,2). Both
211 perfusates were cooled to 4 °C before use. After perfusion, the brain was removed from the skull and placed in buffered paraformaldehyde for 12 h at 4 °C. It was then placed in a 5 o/sucrose solution, buffered at pH 7.4 with 0.2 M sodium phosphate for 8 h followed by a solution of 3 0 ~ buffered sucrose for 16 h. Alternate frozen sections were cut at thicknesses of 90 # m and 30/~m and placed in Trisma buffer (pH 7.6). The 90 #m sections were used for the HRP-diaminobenzidine reaction. The alternate 30 #m sections were stained with thionin. The substrate used for the peroxidase in the 90 #m sections was 3,3'-diaminobenzidine tetrahydrochloride (I00 mg diaminobenzidine in 200 ml distilled water at 22-25 °C). One drop of 30% HzOz was added to this solution immediately before processing the tissue. The sections were exposed to substrate for 25 rain, carried through three baths of Trisma buffer (pH 7.6), and mounted onto gelatinized slides. The sections were then dried in a 37 °C incubator before coverslips were applied. Alternate 30 #m sections stained with thionin were used to study the cytoarchitecture. Sections were studied under both bright- and dark-field illumination. Thick sections were traced at a magnification of 30 x with the aid of a Bausch and Lomb projector, or were photographed; dots were placed on the drawings or negatives to show the locations of HRP-positive cells observed in the original sections. Cytoarchitectural parcellation was readily derived from adjacent thionin sections after adjustments were made for tissue shrinkage. RESULTS Three major cell groups are recognized in Nissl-stained sections of the inferior colliculus of the rat. The central nucleus is composed of densely packed small neurons, and constitutes the core of the inferior colliculus. The central nucleus is bounded laterally by the external nucleus, which is composed of large, loosely packed neurons. The pericentral nucleus comprises a sheet of small neurons which envelops the dorsal and caudal surfaces of the central nucleus, and the lateral surface of the external nucleus. Its neurons are similar in appearance to those of the central nucleus but are more densely packed than those of either the central or external nuclei. A population of larger cells is seen in the dorsomedial part of the central nucleus, as is the case in the cat 2~ and monkey 10. Injections of HRP were placed in the central nucleus of the inferior colliculus in all of the animals used in these experiments. The evidence indicates that the uptake of HRP was confined to the central nucleus in the region around the pipette tip, although there was in all cases widespread diffusion of the tracer.
(a) D&tribution of labeled cells within the cochlear nuclear complex The cochlear nuclear complex of the rat can be parcellated into dorsal and ventral nuclei based on its appearance in Nissl-stained material (Fig. 1). The dorsal cochlear nucleus (DCN) is composed of three distinct neuronal laminae that are recognized in most mammalian species. Within the ventral cochlear nucleus (VCN), anteroventral (AVCN) and posteroventral (PVCN) divisions are recognizable. Locations
Fig. 1. Normal cytoarchitecture of the cochlear nuclei and superior olivary complex ol the rat. A: coronal section through rostral anteroventral cochlear nucleus (AVCN). B: coronal section through dorsal cochlear nucleus (DCN) and po~teroventral cochlear nucleus (PVCN). RB, restiform body: 5ST, spinal trigeminal tract. C: coronal section through the right superior otivary complex. LSO, lateral superior olivary nucleus; MSO, medial superior olivary nucleus; LTB, lateral nucleus Of the trapezoid body; VTB, ventral nucleus of the trapezoid body; MTB, medial nucleus of the trapezoid body: SPN, superior paraolivary nucleus. Nissl stain. ,: 60.
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Fig. 2. A : tracing of a coronal section through the inferior colliculus of rat 76-21M. A volume of 2.0 FI 4 0 ~ w/v HRP was injected into the central nucleus; the total injection was composed of ten 0.2/~1 injections (black dots). The dashed ouline within the inferior colliculus represents the limits of the central nucleus; 41-h survival. B-K: a series of camera lucida drawings of coronal sections through the contralateral (with respect to the injection site) cochlear nucleus showing the distribution or labeled cells. Section B is most rostral, section K is most caudal. Dots within sections represent labeled cells. Arrowheads in sections I and J point to the octopus cell region of the PVCN. L N: series of coronal sections showing the distribution of labeled cells through the superior olivary complex of both sides in rat 76-21M. Section L is most rostral, section N is most caudal. See Fig. l for abbreviations.
214
Fig. 3. Photomicrographs (× 1050) of HRP-labeled (right) and corresponding normal Nissl-stained cochlear nucleus neurons. A and B: D C N fusiform cell. C and D: D C N giant (deep) cell, E and F: PVCN type j (small pale) cell. G and H: PVCN type 1 (medium-sized pale multipolar) cell, 1 and J: PVCN fusiform cell (region V). K and L: AVCN type i (round) cell. M and N: AVCN type d (fuslform) cell. O and P: AVCN type f (large round) cell.
215 and identification of labeled cells are based on the cytoarchitectonic studies of the cochlear complex of the rat by Harrison and irving 12,13. In the present experiments, virtually all major cell types within the cochlear nuclear complex have been labeled with HRP. However, some cell types have been labeled in greater numbers than others. The general distribution of labeled cells in the cochlear nuclei following a large injection of H RP into the central nucleus is illustrated in Fig. 2. In the AVCN, type i (round) cells in region I were labeled in greatest numbers (Fig. 3K and L). Region ! is located in the dorsal two-thirds of the AVCN. Compared with numbers of labeled cells observed in region I, fewer labeled ceils were seen in region II, located in the ventral one-third of the AVCN and PVCN. Labeled cells observed in greatest numbers within region II included type f (large round) and type d (fusiform) cells (Fig. 3M-P). Type c (plump round) cells of region Ill, located in the rostral pole of the AVCN, and type g (globular) cells of region I I were rarely labeled. Within the PVCN, cells of region V, located in intermediate and certain dorsal regions of the PVCN, were labeled in greatest numbers. Labeled cells in region V included type j (small pale) cells and type 1 (medium-sized pale multipolar) cells (Fig. 3E-H). In addition, region V contained many labeled fusiform cells (Fig. 31 and J). Numbers of labeled cells were significantly fewer within region II of the PVCN; they included cell types which were labeled in the corresponding region I1 of the AVCN. No labeled cells were ever observed in region IV (octopus cell region) of the PVCN. The distribution of HRP-positive cells within the contralateral dorsal cochlear nucleus is illustrated in Fig. 2. Fusiform cells within DCN are heavily labeled in all of the present experiments (Fig. 3A and B). In addition, a sizeable number of neurons within the deeper layers of DCN are labeled (Fig. 3C and D). With the exception of an occasional displaced fusiform cell, labeled cells have not been observed in the molecular layer of DCN in any of the brains in this series. In those brains where there was heavy labeling in the contralateral cochlear complex, only a few scattered HRPpositive cells were observed in the ipsilateral cochlear nuclei. Single small injections of HRP into the central nucleus resulted in labeled cells within restricted regions of the contralateral cochlear nuclear complex. A small injection in the ventral region of the central nucleus of rat 76-4M resulted in labeling of cells confined to dorsal regions of the contralateral DCN and AVCN (Fig. 4B-E). No labeled cells were seen in PVCN of this brain. This observation is consistent with results from other experiments in which dorsally situated octopus cells in PVCN were not labeled (Fig. 2). A small injection into the central region of the central nucleus resulted in labeled cells confined to intermediate levels of the contralateral AVCN, PVCN and DCN (Fig. 5B-E). Again, cells within the octopus cell region were not labeled. An injection into dorsal areas of the central nucleus labeled cells only in more ventral areas of each of the three major subdivisions of the cochlear complex (Fig. 6B-E).
(b) Distribution of labeled cells within the superior olivary complex The superior olivary complex of the rat is comprised of 6 cell groups which
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Fig. 4. A: tracing of coronal section through the inferior colliculus of rat 76-4M showing the focus of the single HRP injection in black (0.4 M 4 0 ~ w/v HRP). The dashed outline within the inferior colliculus represents the limits of the central nucleus: 46-h survival. B-E: a series of coronal sections through the contralateral (with respect to the site of injection) cochlear nucleus. Section B is most rostral, section E is most caudal. The arrowhead in section D points to the octopus cell region in the PVCN. F and G: coronal sections through the superior olivary complex of both sides: section F is rostral to section (3. See legend of Fig. 1 for abbreviations. are easily recognized in Nissl-stained sections 14 (Fig. 1). The lateral superior olivary nucleus (LSO) is a p r o m i n e n t cell mass which is S-shaped i n coronal sections. Medial to the LSO is the medial superior olivary nucleus (MSO) composed o f fusiform cells with their long axes oriented horizontally. The celt groups lying in or near the trape-
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Fig. 5. A: tracing of coronal section through the inferior colliculus of rat 76-3M showing the focus of the single HRP injection in black (0.4/~l 4 0 ~ w/v HRP). The dashed outline within the inferior colliculus represents the limits of the central nucleus; 45-h survival. B-E: a series of coronal sections through the contralateral (with respect to the injection site) cochlear nucleus. Section B is most rostral, section E is most caudal. F and G : coronal sections through the superior olivary complex of both sides; section F is rostral to section G. See legend of Fig. 1 for abbreviations. zoid b o d y have been collectively labeled as the nuclei o f the t r a p e z o i d body. Three distinct nuclei can be seen: the medial (MTB), lateral (LTB), and ventral (VTB) nuclei o f the t r a p e z o i d body. The LTB is c o m p o s e d o f round, fusiform, and m u l t i p o l a r cells; it is located i m m e d i a t e l y ventral to the LSO. The cells o f the M T B are g r o u p e d near the midline o f the b r a i n stem; they are r o u n d or o v a l - s h a p e d with eccentric
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Fig. 6. A: tracing of coronal section through the inferior colliculus of rat 76-I09M showing the focus of the single HRP injection in black (0.4 pl 4 0 ~ w/v HRP). The dashed outline within the inferior colliculus represents the limits of the central nucleus; 42-h survival. B-E: a series of coronal sections through the contralateral (with respect to the site of injection) cochlear nucleus. Section B is most rostral, section E is most caudal. F and G : coronal sections through the superior olivary complex of both sides; section F is rostral to section 13. See legend of Fig. 1 for abbreviations. nuclei. The VTB is located v e n t r o m e d i a l to the LTB, a n d contains small, lightly staining cells. The superior paraolivary nucleus (SPN) is situated medial to the LSO a n d is composed of large a n d small m u l t i p o l a r cells.
219 The retrograde HRP reaction within cells in the superior olivary complex after injections of a large volume of HRP into the central nucleus is shown in Fig. 2L-N. Heavy labeling is seen in neurons throughout the rostrocaudal extent of the following cell groups: ipsilateral and contralateral LSO, ipsilateral and contralateral LTB, ipsilateral MSO, ipsilateral VTB, and ipsilateral SPN. Labeled cells are seen throughout the dorsoventral extent of the ipsilateral MSO. HRP-positive cells have not been observed in either the ipsilateral or contralateral MTB. In the present experiments, labeled cells are not uniformly distributed in most cell groups within the superior olivary complex. I n general, the lateral limbs of both the contralateral and ipsilateral LSO are only weakly labeled as compared to the remainder of this nucleus. The lateral regions of SPN are more heavily labeled than more medial regions of the nucleus. Labeled cells seem to cluster in intermediate regions of VTB. After a small injection into the most ventral areas of the central nucleus, labeled cells are observed in the most dorsal portion of the medial limb of the contralateral and ipsilateral LSO (Fig. 4). Fig. 5 illustrates the distribution of positive cells in the LSO after an HRP injection into a more intermediate region of the central nucleus. In this brain, positive cells are found in the ventral portions of the medial limb of LSO bilaterally (sections F and G). A small injection into the dorsal region of the central nucleus results in the labeling of cells confined to the dorsal portion of the lateral limb of LSO bilaterally (Fig. 6). The topographic projection pattern of the other cell groups in the superior olivary complex could not be determined due to tile relatively small number of cells labeled by small injections of HRP.
(c) Distribution of labeled cells within auditory areas of the cerebral cortex The cytoarchitecture of the auditory cortex in the rat has been described by Krieg15,16. Layers II and Ill are fairly thin and are composed of pyramidal and granular cells of varying sizes. Layer IV is composed of a large number of granule cells and is difficult to distinguish from supragranular layers. Layer V is composed of pyramidal cells of varying sizes and is easily distinguished from the layers above and below it. A variety of cell types comprise layer VI, including fusiform and spherical cells. After injections of HRP into various parts of the central nucleus, labeled cells are seen in the ipsilateral cerebral cortex. The distribution of labeled cells after a large injection roughly corresponds to auditory area 41 of Krieg's topographic map of rat cortex (Fig. 7). Virtually all of the labeled cells appear to be medium-to-large pyramidal cells and are confined to layer V (Fig. 8). No labeled cells are seen in the contralateral cortex or in the thalamus of either side. Small injections of HRP within the central nucleus resulted in relatively small patches of labeled cells in layer V. (d) Distribution of other labeled eel& Occasionally, a few scattered HRP-positive cells were observed in the contralateral inferior colliculus as a result of large injections of HRP into the central nucleus of the inferior colliculus. On the other hand, many HRP-positive cells were observed in the contralateral dorsal nucleus of the lateral lemniscus as a result of both large and small injections of HRP into the central nucleus. One brain, which
220 A
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Fig. 7. A series of coronal sections through the ipsilateral (with respect to the injection site) cortex of rat 76-21M. The stippled area on the inset drawing indicates the area m which labeled cells were observed in the cortex. Dashed lines on the inset indicate the planes and locations of sections shown in the coronal sections. Dots within the section represent labeled cells. The location of the injection within the inferior colliculus is seen in Fig. 2. received no injection o f H R P , was processed in the same way as the e x p e r i m e n t a l brains to search for e n d o g e n o u s H R P within a u d i t o r y n e u r o n s o f the b r a i n stem '7. N o labeled n e u r o n s were f o u n d in any o f the a u d i t o r y structures in this brain. DISCUSSION The inferior colliculus in the rat receives strong, t o p o g r a p h i c a l l y organized p r o j e c t i o n s from the three m a j o r subdivisions o f the c o n t r a l a t e r a l cochlear nuclear complex, f r o m 5 cell g r o u p s o f the s u p e r i o r olivary complex, a n d f r o m p y r a m i d a l cells o f layer V o f a u d i t o r y cortex. W h i l e any o r all o f the cell g r o u p s which m a k e u p the inferior colliculus m a y receive afferent fibers f r o m these sources, the results presented here indicate t h a t the central nucleus is the prime t a r g e t o f n e u r o n s l a b e l e d i n these e x p e r i m ents. The foci o f all injections were well within the c e n t r a l nucleus, a l t h o u g h a b r o w n reaction p r o d u c t was seen to s p r e a d widely t h r o u g h o u t the inferior collicutus. Small injections o f H R P resulted in labeling o f cells within restricted regions in all o f the lower
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Fig. 8. Photomicrograph of a coronal section through the ipsilateral cortex of rat 76-21M. This section corresponds to section B in Fig. 7. Dots indicate locations of labeled cells seen in the adjacent section. Cortical laminae are indicated along the right side of the photomicrograph. Nissl stain..! 56.
nuclei. Furthermore, the distribution of labeled cells was systematically related to the site of the injection. This observation would suggest that the most active uptake of H R P occurred in a restricted region of the central nucleus immediately surrounding the tip of the micropipette, regardless of the more widespread diffusion of H R P indicated by the brown background reaction product. Results obtained from brains that received small injections of H R P indicate that projections from the contralateral cochlear nuclear complex to the central nucleus are arranged topographically. Cells located in intermediate regions of contralateral AVCN, PVCN, and D C N project to intermediate regions of the central nucleus, cells located in dorsal areas of DCN and AVCN project to ventral areas of the central nucleus and cells in ventral regions of all 3 cochlear nuclei project to dorsal regions of the central nucleus. Similar results have been described in the cat TM. in general, cells with high best frequencies are located ventrally and cells with low best frequencies are located dorsally within the central nucleus in the rat 6. Thus, the pattern of cochlear nucleus afferents to the central nucleus suggests that within each of the 3 subdivisions of the rat cochlear nucleus, cells with high best frequencies are located dorsally and cells with low best frequencies are located ventrally, as indeed they are in the cat 22. As is the case in the contralateral cochlear nuclei, the pattern of ascending projections from the ipsilateral and contralateral LSO is an orderly one and may be correlated with the tonotopical organization of the LSO 24. Results of others concerning projections from the cochlear nucleus to the ipsi-
222 lateral inferior colliculus are not consistent; some studies25, 26,2s have demonstrated a significant projection, while others4,9,19 have not. In the present experiments, a strong ipsilateral projection has not been observed. However. these negauve results do not eliminate the possibility of such an ipsilateral pathway in the rat, considering the limited value of negative results obtained with the H R P technique. Results from all brains examined in this series indicate a strong projection from the ipsilateral superior paraotivary nucleus to the central nucleus. The SPN in the rat is located in the same relative position with respect to other cell groups of the supermr olivary complex as is the dorsomedial periolivary group m the cat 17. suggesting a possible functional homology between these two nuclei. However. the dorsomedial periolivary group in the cat appears to project to the inferior colliculus bilaterally 2°. whereas the present experiments suggest that the SPN in the rat projects only to the ipsilateral inferior colliculus. Furthermore. the cytoarchitecture of these two nuclear groups is quite different in the two species. The dorsomedial periolivary group in the cat is composed primarily of small 'elongate' neurons tT. whereas the SPN in the rat is composed primarily of larger multipolar cells ~'~. The termination of cortico-collicular fibers in cat 7,zl and tree shrew ~s appear confined to the cell groups which surround the central nucleus or to the large-cell dorsomedial sector of the central nucleus. In the monkey H. on the other hand. the laminated portion of the central nucleus is a target of fibers originating in area AI. The present experiments indicate that the central nucleus in the rat also receives substantial input from the cerebral cortex. However. the organization of auditory cortex in the rat is not known and. thus. it is not possible to determine whether this projection originates in the primary auditory area or in one or more of the surrounding auditory fields. Regardless of the cortical fields within which they are located, cortico-collicular neurons are, for the most part, pyramidal cells of layer V. Recently, Zook and Casseday 29 demonstrated a similar distribution of cortico-fugal cells projecting to the inferior colliculus in the bat. ACKNOWLEDGEMENTS The author wishes to express his gratitude to Dr. John F. Brugge tbr unfailing advice during the course of these experiments, as well as for valuable help with the manuscript. Thanks are also due to Sigrid Borgner for the typing of the manuscript, and Shirley Hunsaker for assistance with photography. This research was supported by N I H Program Project Grant NS05326. the K n a p p Honors Research Scholarship (University of Wisconsin Honors Program), and by the B. H. Hibbard Award (Phi Kappa Phi H o n o r Society).
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