The morphology of the cochlear nucleus in the normal, adult chinchilla, as demonstrated by. Nissl staining, was examined. The cytoarchitecture was determined ...
Acta Otolaryngol (Stockh) 1991; Suppl 489: 12-22
Cytoarchitecture of Cochlear Nucleus in the Chinchilla C. E. FLECKEISEN,’ R. V. HARRISON’,Z.3and R. J. MOUNT3 From the Departments of ‘Physiology and ’Otolaryngology, University of Toronto, Toronto, Ontario, Canada M5S IA8, and 3ResearchInstitute, Hospital for Sick Children, Toronto, Ontario, Canada, M5G 1x8
Fleckeisen CE, Harrison RV, Mount RJ. Cytoarchitecture of cochlear nucleus in the chinchilla. Acta Otolaryngol (Stockh) 1991; Suppl489: 12-22. The morphology of the cochlear nucleus in the normal, adult chinchilla, as demonstrated by Nissl staining, was examined. The cytoarchitecturewas determined from sections viewed at the light microscope level. The chinchilla cochlear nucleus was found to possess most of the features reported in other mammalian cochlear nuclei. It could easily be divided into dorsal and ventral components due to an intervening layer of granule cells, and most cell types previously reported in mammals were also found in the chinchilla cochlear nucleus. A distinct distribution pattern of cell types exists within each part. Key words: auditory pathway, brainstem, hearing.
INTRODUCTION The cochlear nucleus is the first auditory area of the mammalian brainstem and is the first region in which complex sound processing occurs. As such, it has been the focus of a great number and variety of studies. Extensive data has been collected on the cochlear nucleus morphology in a wide range of species, determined at both light microscopic (Harrison & Irving, 1965; Osen, 1969; Bacsik & Strominger, 1973; Disterhoff et al., 1980; Webster & Trune, 1982; Heiman-Patterson & Strominger, 1985; Hackney & Pick, 1986) and electron microscopic levels (Cant & Morest, 1979; Parks, 1981; Wouterlood et al., 1984). These reports have shown that there is a general similarity in the structure of the mammalian cochlear nucleus. Brain sections stained for Nissl substance most clearly show the overall cytoarchitecture and reveal that there are two major subdivisions, designated the ventral cochlear nucleus (VCN) and the dorsal cochlear nucleus (DCN). The cell types of the cochlear nucleus have been characterized by various techniques, including Nissl staining and Golgi impregnation. The different appearance of cells determined by these two processes has resulted in parallel nomenclatures. For example, bushy cells are named after their dendritic pattern as revealed by the Golgi technique and correspond to spherical cells named for their most prominent feature in Nissl staining, cell body shape. This duality of nomenclature exists for most cell types in the cochlear nucleus. The particular distribution of each cell type within the cochlear nucleus combined with electrophysiological characterization of the cells facilitates comparison of cell types studied by different methods. Intensive work on the eIectrophysiology of the cochlear nucleus (Evans & Nelson, 1973; Perry & Webster, 1981; Oertel, 1983; Rhode, Oertel & Smith, 1983; Rhode, Smith & Oertel, 1983; Rhode & Smith, 1986a, b) has shown a variety of cell responses, from primary-like to complex. Few electrical studies have looked at the chinchilla cochlear nucleus and no descriptions of its morphology have been published to date. This is surprising since the chinchilla is a popular animal for auditory research, especially in North America, and has been used in a wide range of studies including morphological, electrophysiological and behavioral work (Mast, 1970; Miller, 1970; McGee et al., 1976; Hunter-Duvar, 1977; Hunter-Duvar et al., 1982; Kaltenbach & Saunders, 1987; Shirane & Harrison, 1987). The cochlea of this animal is very accessible, particularly for evaluation of haircell damage in various animal models of
Cytoarchitecture of cochlear nucleus sensorineural hearing loss. We have therefore made a study of the cytoarchitecture of the cochlear nucleus of the normal animal. We describe here the organization of the nucleus, and the distribution of cell types as determined by Nissl staining. We compare the findings to similar studies of the cochlear nuclei in other species.
METHODS Seven normal, adult chinchillas (approximate weight = 500 g) were evaluated. The animals were given an overdose of sodium pentobarbitol before sacrifice. The ascending aorta was perfused with saline followed by universal fixative (phosphate buffered 4% paraformaldehyde, 1 Yo glutaraldehyde; pH 7.4)at room temperature. The head was removed and placed in refrigerated fixative overnight. The following day, the brain was removed from the skull and stored in fresh fixative. After 7-10 days fixation the brain was bisected and the right half prepared for Nissl staining. The material was dehydrated in ethanol, cleared in xylene, infiltrated with paraffin, embedded and sectioned parasagittally at 20 pm on a rotary microtome. This plane was chosen because subdivisions of the cochlear nucleus are best distinguished in this aspect. The left half of the brain was used in other studies. Every fourth section was stained with cresyl fast violet, and differentiated in 95 percent ethanol. Standard bright field illumination was used when viewing the slides. Cell details were determined at 600 x magnification and general cell distribution examined at 300 X magnification. For this assessment the nomenclature developed by Osen (1969) and used extensively in work done by others on the cochlear nucleus at a light microscope level was adopted and adhered to as closely as possible.
RESULTS
General cochlear nucleus structure The chinchilla cochlear nucleus lies superficially on the brainstem and is covered dorsally by the cerebellum. Anatomically and functionally the nucleus is conveniently separated into two
Table 1. Legend of abbreviations used infigures AVCN CBL CR DCN FL GC LSA M-GA ML OA-
PVCN SSA VCN
'2 -91 841 7
Anteroventral cochlear nucleus Cerebellum Central region Dorsal cochlear nucleus Fusiform cell layer Granule cell layer Large-dark spherical cell area Multipolar-globular cell area Molecular cell layer Octopus cell area Posteroventral cochlear nucleus Small-pale spherical cell area Ventral cochlear nucleus
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14
A ) @
MULTIPOLAR CELL
0
LARGE-DARK SPHERICAL CELL
OCTOPUSCELL
@
SMALL-PALESPHERICALCELL
FUSIFORM CELL
6
CARTWHEEL CELL
0
a
SMALL CELL GLOBULAR CELL
Fig. 1. Legend to cell types in Fig. 2 ti4 b.
2b Fig. 2. Photomicrograph ( a )and diagram (b)of a parasagittal section of chinchilla dorsal cochlear nucleus taken from medial location in the cochlear nucleus. DCN subdivisions and cell types illustrated in diagram (refer to Table I and Fig. I).
Cvtoarchitecture of cochlear nucleus
Fig. 3. Photomicrograph (a)and diagram (b)of parasagittal cochlear nucleus section lateral to Fig. 2. All dorsal and ventral subdivisions shown. Cell types and distributions illustrated in diagram (refer to Table I and Fig. I).
major regions, a dorsal cochlear nucleus (DCN) and a ventral area (VCN) often subdivided into anterior and posterior regions (AVCN, PVCN). Table I and Fig. I provide legends for the schematics and abbreviations used in the following description of the chinchilla cochlear nucleus. The micrographs in Fig. 2-4 are of parasagittal sections proceeding in a medial to lateral
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A+
4b
Fig. 4. Photomicrograph (a) and diagram (b)of lateralmost, parasagittal ventral cochlear nucleus section. Cell types and distribution illustrated in diagram (refer to Table I and Fig. 1).
order through the cochlear nucleus showing its gross structure. The DCN is the most medial part of the nucleus (Fig. 2). The VCN is lateral and rostroventral to the DCN (Fig. 3). The two divisions are partially separated by a granule cell layer. In the most laterai parasagittal section of the cochlear nucleus (Fig. 4) there is no DCN present.
Cell types There are a number of cell types found in the chinchilla cochlear nucleus which can be distinguished in Nissl stained material by size, shape and affinity for the dye (Osen, 1969); the distribution of the various cell types also is specific. A description of the different cell types in the nucleus follows. A. Granule cells. Granule cells are the smallest cells found in the cochlear nucleus (Fig. 5). They appear mainly as a nucleus and cell membrane containing no visible cytosol. There are no Nissl granules present. The distribution of granule cells is shown in the diagrams of Figs.
Cytoarchitecture of cochlear nucleus 2-4. The cells are located mainly in the fusiform cell layer of the DCN and discrete granule cell layer of the VCN (Fig. 4 b). B. Small cells. Fig. 6 shows small cells. Other than granule cells these are the smallest found in the nucleus. They exhibit a variety of shapes in sections-from round to bipolar-and have very little cytosol. Their Nissl substance can be diffuse or clumped; there is no consistent pattern. This classification encompasses a wide range of cells found throughout both VCN and DCN. C. Small-pale spherical cells. Small-pale spherical cells are ovoid with eccentrically placed nucleus and nucleolus (Fig. 7). They have less cytosol and Nissl material than the large-dark spherical cell type and thus are paler staining. The nucleus is totally or partially surrounded by a band of discontinuous, medium sized Nissl granules. Aggregates of Nissl substance often form a cap on the nuclear membrane. Small-pale spherical cells are found caudal to the area occupied by large-dark spherical cells, as indicated in Fig. 3 b. D. Large-dark spherical cells. The large-dark spherical cells range in shape from spherical to ovoid and have medium sized Nissl granules arranged in concentric rings around a centrally placed nucleus. Some examples are shown in Fig. 8. The nucleolus is eccentrically placed within the nucleus. These cells can also have a Nissl cap associated with the nucleus, similar to that of the small-pale spherical cells. The large-dark spherical cells are very prominent in VCN, forming a rostra1 cap of darkly staining cells, as indicated in the distribution diagram of Fig. 3 . E. Multipolar cells. Multipolar cells are round to elongate and may have two or three dendritic projections which stain only in the region closest to the soma. This can give them a bipolar to triangular appearance. Some examples are shown in Fig. 9. Multipolar cells vary greatly in size, ranging from the largest sized cells in the cochlear nucleus to a medium size comparable to that of the large-dark spherical cells. Multipolar cells have abundant cytosol which contains medium to coarse Nissl substance arranged evenly throughout. The nuclei are situated centrally. Multipolar cells are found primarily in the central region of the VCN (Fig. 3 b). F. Globular cells. Globular cells have an ellipsoid shape and possess fine Nissl material evenly distributed throughout abundant cytosol (Fig. 10). The nuclei are eccentric giving some cells the appearance of “trailing” their cytosol behind. Globular cells can be pale or dark staining and on occasion exhibit a heavy, continuous crescent of Nissl material at the cell periphery. Globular cells occupy the same central VCN area as the multipolar cells. G. Octopus cells. Octopus cells are the second largest cells in the VCN having plentiful cytosol containing fine Nissl substance evenly dispersed, as shown in Fig. 11. They generally have a spherical shape, but can appear steerhorn or octopus-like when the roots of some dorsal dendrites have been stained. Occasionally an octopus cell has a few coarse granules gathered at the periphery of the cytosol. The nucleus is placed centrally and has an eccentric nucleolus. Octopus cells are located at the dorso-medial pole of caudal VCN, as indicated in Fig. 3 b. H. Fusiform cells. Fusiform cells (Fig. 12) are the largest cells found within the fusiform cell layer and are oriented perpendicularly to the surface of the DCN (see Figs. 2 b-3 6). They are elongated bipolar cells that contain medium sized Nissl granules which run parallel to the long axis of the cell. This configuration is most apparent in a parasagittal section. There are pale and dark fusiform cells but whether there is a normal gradation in staining or two distinct types was not determined. Fusiform cells form clusters creating a heterogeneous distribution within the layer. I. Cartwheel cells. Cartwheel cells are round in shape with a dense distribution of fine Nissl substance in the cytosol giving these cells a darkly stained appearance. Examples are shown in Fig. 13. The nucleus is eccentrically placed. This cell type is found in the molecular layer of the DCN and varies in distribution, being scattered dorsally and concentrated more ventrally,
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Fig. 5. Photomicrograph of granule cells (arrows) found in the chinchilla cochlear nucleus.
Fig. 6. Photomicrograph of small cells (orrows) representative of their type found in the chinchilla cochlear nucleus.
Cytoarchitecture of cochkar nucleus
Fig. 1 1 . Photomicrograph of octopus cells (arrows) representative of their type found in the chinchilla ventral cochlear nucleus. Fig. 12. Photomicrograph of fusiform cells (arrows) representative of their type found in the chinchilla dorsal cochlear nucleus. Fig. 13. Photomicrograph of cartwheel cells (arrows) representative of their type surrounding a fusiform cell (star) found in the chinchilla dorsal cochlear nucleus.
along the edge of the fusiform cell layer. Cartwheel cells can also be seen in the central region of the DCN.
Ceif areas Some cell types have a particular distribution in the chinchilla cochlear nucleus. As a result, cochlear nucleus areas can be defined according to the predominant cell type. The DCN is stratified. The fusiform cell layer forms a prominent arch of fusiform and granule cells, separating the dorsal lying molecular layer from the central region of the DCN Fig. 7. Photomicrograph of small-pale spherical cells (arrows) representative of their type found in the chinchilla ventral cochlear nucleus. Fig. 8. Photomicrograph of large-dark spherical cells (arrows) representative of their type found in the chinchilla ventral cochlear nucleus.
Fig. 9. Photomicrograph of multipolar cell (arrow) representative of its type found in the chinchilla ventral cochlear nucleus. Fig. 10. Photomicrograph of globular cells (arrows) representative of their type found in the chinchilla cochlear nucleus.
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(Fig. 2 b). This central region is more polymorphic having a variety of cells including multipolar cells, globular cells, and small cells. The occasional cartwheel or fusiform cells are situated at the boundary of the fusiform cell layer. Subdivision of the VCN is not as easily made as in the DCN since cell area borders are not readily defined. The most conspicuous cell type area in VCN is the large-dark spherical cell area which consists of a homogeneous population at the anterior-most pole of the VCN. As shown in Fig. 3 b, this is bordered posteriorly by an area containing small-pale spherical cells as well as other cell types. The octopus cell area lies at the posterodorsal pole of the VCN and the multipolar-globular cell area lies in the central region of the VCN, as indicated in Fig. 3 b. These cell type areas do not have as homogeneous a cell population as the large-dark spherical cell area. DISCUSSION The chinchilla cochlear nucleus can be easily divided into dorsal and ventral parts by an intervening layer of granule cells, a division reported in investigations of other mammals (Osen, 1969; Perry & Webster, 1981; Webster & Trune, 1982; Moore, 1988). The chinchilla DCN is stratified when visualized by Nissl staining. This feature is common to most mammals, with the exception of ferrets and humans (Osen, 1969; Moore & Osen, 1979; Perry & Webster, 1981; Webster & Trune, 1982; Heiman-Patterson & Strominger, 1985; Moore, 1988). Heiman-Patterson & Strominger (1985) also noted that the granule cell population in human DCN was relatively small compared to other species. These cells are known to form synapses on the apical dendrites of fusiform cells and a decrease in this population in more advanced species may indicate reduced intranuclear integration and a shift of information processing to higher levels (Moore & Osen, 1979; Heiman-Patterson & Strominger, 1985). Moore (1988) did not comment on the size of the granule cell population in ferrets, but since this species possesses a highly convoluted neocortex it has been speculated that the ferret brain has evolved to the point where its sound processing is also shifting to higher levels. The fusiform cells of the chinchilla are oriented perpendicularly to the dorsal surface of the DCN, a pattern also observed in the cat, rabbit and mouse (Osen, 1969; Perry & Webster, 1981; Webster & Trune, 1982). In the human DCN, fusiform cells have no particular orientation (Heiman-Patterson & Strominger, 1985). This has been attributed to the lack of granule cells and the resulting decrease in input to the apical dendrites of fusiform cells (Moore & Osen, 1979; Heiman-Patterson & Strominger, 1985). Chinchilla fusiform cells are distributed in clusters, a characteristic which has been noted in cats (Osen, 1969). In the present study we have found a gradient in the staining density of fusiform cells, but it was not possible to determine a difference in distribution. Larsen (1984) described dark and light staining fusiform cells which occupied different locations in the fusiform layer of the cat cochlear nucleus. He compared this finding to the observations made by Webster & Trune (1982) on Golgi prepared material from CBA/J mice. The mouse fusiform cell layer included two distinct medium sized cells: fusiform cells and Purkinje-like cells. It will require a study of Golgi material to determine whether the chinchilla DCN also possesses two distinct populations of medium-sized cells. No giant cells were noted in the central region of the chinchilla DCN. Osen (1969), in Nissl stained material from cats and Perry & Webster (1981), in Golgi prepared material from rabbits, described giant cells in the central region of the DCN. The rare “giant” multipolar cell was seen in the chinchilla VCN, but was included in the multipolar category. In most anatomical studies of the mammalian cochlear nucleus, the VCN is divided into the anteroventral cochlear nucleus and posteroventral cochlear nucleus. This division is useful for
Cytoarchitecture of cochlear nucleus descriptive purposes but was not done in the present study since, in Nissl stained material, the delineation can only be made with confidence in the region of the entry of the nerve root. The chinchilla VCN has a distinct population of darkly staining, spherical cells (the largedark spherical cells) which correspond to the large spherical cells of Osen’s (1969) classification in cats. Immediately caudal to the large-dark spherical cells are smaller, paler ovoid cells (small-pale spherical cells) which fit the criterion for Osen’s small spherical cells. Hackney & Pick (1986) examined the spherical cell population of guinea pigs and found that there was a single population of spherical cells with a normal range of sizes. In her review article (1988), Osen agreed that there was a single population of spherical cells, but that a distinct distribution existed, with the larger spherical cells being located most rostrally. On the other hand, Webster & Trune (1982) found a significant difference in size between the large and small spherical cell population. The spherical cells in this study were treated as two separate populations on the basis of a distinct distribution according to size and staining density. Multipolar and globular cells occupy the midregion of the chinchilla VCN, but cannot be subdivided into distinct regions as is possible in the cat cochlear nucleus (Osen, 1969). Studies of human and rabbit cochlear nuclei have also found no distinct regions of multipolar or globular cells (Moore & Osen, 1979; Perry & Webster, 1981). The small cells of the chinchilla cochlear nucleus are scattered throughout the VCN and are not concentrated in any particular region. This is in contrast to Osen’s (1969) finding in cats, where small cells form a “cap” along the ventral surface of the granule cell layer. Despite these differences, the chinchilla cochlear nucleus shares a remarkably similar structure with other mammals. It is composed of a dorsal and ventral part separated by a layer of granule cells and each subdivision can in turn be partitioned into different areas according to the cell types present. All major cell types described from basic stained cochlear nucleus material in other species, are also found in the chinchilla cochlear nucleus. REFERENCES Bacsik, R. D. & Strominger, N. L. (1973). The cytoarchitecture of the human anteroventral cochlear nucleus. J Comp Neurol, 147, 281-289. Cant, N. B. & Morest, D. K. (1 979). The bushy cells in the anteroventral cochlear nucleus of the cat. A study with the electron microscope. Neurosci, 4, 1925-1945. Disterhoft, J. F., Perkins, R. E. & Evans, S. (1980). Neuronal morphology of the rabbit cochlear nucleus. J Comp Neurol, 192, 687-702. Evans, E. F. & Nelson, P. G. (1973). The responses of single neurones in the cochlear nucleus of the cat as a function of their location and the anaesthetic state. Exp Brain Res, 17,402-427. Hackney, C. M. & Pick, G. F. (1986). The distribution of spherical cells in the anterior ventral cochlear nucleus of the guinea pig. Br J Audiol, 20, 21 5-220. Harrison, J. M. & Irving, R. (1965). The anterior ventral cochlear nucleus. J Comp Neurol, 124, 15-42. Heiman-Patterson, T. D. & Strominger, N. L. (1985). Morphological changes in the cochlear nucleus complex in primate phylogeny and development. J Morphol, 186, 289-306. Hunter-Duvar, I. M. (1977). Morphology of the normal and the acoustically damaged cochlea. S.E.M./1977. 11, 421-428. Hunter-Duvar, I. M., Suzuki, M. & Mount, R. J. (1982). Anatomical changes in the organ of Corti after acoustic stimulation. In: Hamernik, R. P., Henderson D. and Salvi R. (Eds.). New perspectives on noise induced hearing loss. New York: Raven Press, 3-22. Kaltenbach, J. A. & Saunders, J. C . (1987). Spectral and temporal response patterns of single units in the chinchilla dorsal cochlear nucleus. Exp Neurol, 96, 406-41 9. Larsen, S. A. (1 984). Postnatal maturation of the cat cochlear nucleus complex. Acta Otolaryngol (Stockh), Suppl, 417, 1-43. Mast, T. E. (1970). Study of single units of the cochlear nucleus of the chinchilla. J Acoust SOCAm, 48, 505-5 12. McGee, T., Ryan, A. & Dallos, P. (1976). Psychophysical tuning curves of chinchillas. J Acoust SOCAm, 60, 1146-1150. Miller, J. D. (1970). Audibility curves of the chinchilla. J Acoust SOCAm, 48, 513-523.
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Moore, J. K. (1988). Auditory brainstem of the ferret: sources of projections to the inferior colliculus. J Comp Neurol, 269, 342-352. Moore, J. K. & Osen, K. K. (1979). The cochlear nuclei in man. Am J Anat, 154, 393-418. Oertel, D. (1983). Synaptic responses and electrical properties of cells in brain slices of the mouse anterior ventral cochlear nucleus. J Neurosci, 3, 2043-2053. Osen, K. K. (1969). Cytoarchitecture of the cochlear nuclei in the cat. J Comp Neurol, 136, 453-484. Osen, K. K. ( 1 988). Anatomy of the mammalian cochlear nuclei: a review. In: Syka, J. and Masterton, R. B. (Eds.). Auditory pathway: structure and function. New York: Plenum Press, 65-75. Parks, T. N. (1981). Morphology of axosomatic endings in an avian cochlear nucleus: nucleus magnocellulark of the chicken. J Comp Neurol, 2 0 3 , 4 2 5 4 4 0 , Perry, D. R. & Webster, W. R. (1981). Neuronal organization of the rabbit cochlear nucleus: some anatomical and electrophysiological observations. J Comp Neurol, 197, 623-638. Rhode, W. S., Oertel, D. & Smith, P. H. (1983). Physiological response properties of cells labelled intracellularly with horseradish peroxidase in cat ventral cochlear nucleus. J Comp Neurol, 2 13, 448-463. Rhode, W. S. Smith, P. H. & Oertel, D. (1983). Physiological response properties of cells labelled intracellularly with horseradish peroxidase in cat dorsal cochlear nucleus. J Comp Neurol, 21 3, 426-447. Rhode, W. S. &Smith, P. H. (1986~).Encoding timing and intensity in the ventral cochlear nucleus ofthe cat. J Neurophysiol, 56, 261-286. Rhode, W. S. & Smith, P. H. (1986 b). Physiological studies on neurons in the dorsal cochlear nucleus of the cat. J Neurophysiol, 56, 287-307. Shirane, M. & Harrison, R. V. (1987). The effects of deferoxamine mesylate and hypoxia on the cochlea. Acta Otolaryngol (Stockh), 104, 99-107. Webster, D. B. & Trune, D. R. (1982). Cochlear nucleus complex of mice. Am J Anat, 163, 103-130. Wouterlood, F. G., Mugnaini, E., Osen, K. K. & Dahl, A. L. (1984). Stellate neurons in rat dorsal cochlear nucleus studied with combined Golgi impregnation and electron microscopy: synaptic connections and mutual coupling by gap junctions. J Neurocytol, 13,639-664. Address for correspondence: R. V. Harrison, Department of Otolaryngology, Hospital for Sick Children, 5 5 5 University Ave., Toronto, Ontario, Canada M5G 1 x 8
