- Email: [email protected]
The bushy cells in the anteroventral cochlear nucleus of the cat. A study with the electron microscope
0306.4522>79/1201-1925502.00/O
Neuros
THE BUSHY CELLS IN THE ANTEROVENTRAL COCHLEAR NUCLEUS OF THE CAT. A STUDY WITH THE ELECTRON MICROSCOPE NELL B. CANT’ and D. K. MOREST Department of Anatomy, Harvard Medical School, Boston, MA 02115, U.S.A., and Department of Anatomy, The University of Connecticut Health Center, Farmington. CT 06032, U.S.A. bushy cells in the anterior division of the anteroventral cochlear nucleus of the cat were studied with the electron microscope. In the anterior part of the anterior division, profiles of bushy cells and their processes are easily identified, since few cells of other types are found in this region. In the posterior and posterodorsal parts of the anterior division, the bushy cells are intermingled with stellate and small cells but can be identified on the basis of light-microscopic descriptions and comparisons with the results from the anterior part. Bushy cells are large, spherical cells with a centrally located nucleus enveloped by sheets of rough endoplasmic reticulum. Thin proximal dendrites jut abruptly from the cell body and contain a relatively pale cytoplasm. The distal dendrites contain few organelles other than numerous, very large mitochondria. The cell soma and proximal dendrites, as well as the axon hillock, receive numerous synaptic terminals, but the distal dendritic processes are contacted by relatively few endings. At least four types of terminals form synaptic contacts with the bushy cells. Very large terminals, containing large, spherical synaptic vesicles and forming multiple asymmetrical contacts, correspond to the end-bulbs of Held from the cochlea. These terminals disappear after cochlear ablations, but the other three types remain. The most numerous of these is a large terminal that contains flattened synaptic vesicles and forms long, nearly symmetrical contacts with the soma and dendrites of bushy cells. The second type of non-cochlear terminal is smaller and contains small, pleiomorphic synaptic vesicles that are not flattened. The third type occurs mainly on bushy cell dendrites, contains small. spherical synaptic vesicles, and forms moderately asymmetrical contacts. The bushy cells probably correspond to the primarylike units described in electrophysiological studies of the anterior division. Primarylike units respond to activity in auditory nerve fibers in a one-to-one manner, a finding compatible with the observation that much of the surface of the soma and dendrites of the bushy cells is contacted by auditory nerve terminals (end-bulbs of Held). Neither the origins nor the functions of the several types of non-cochlear inputs to the bushy cells are known. Further analysis of these inputs and of the other neuronal types in the anterior division, when correlated with physiological and biochemical data from the same cell types, could clarify the functional significance of the observed patterns of synaptic organization. Abstract-The
THE ANTEROVENTRAL cochlear nucleus (AVCN) of the cat comprises a number of subdivisions, each with a characteristic population of neurons (OSEN, 1969; BRAWER, MOREST & KANE, 1974; CANT & MOREST, 1979). Light-microscopic studies have shown that the terminal patterns formed by the cochlear nerve vary among the subdivisions (RAM~N Y CAJAL, 1909; LORENTE DE N& 1933, 1976; BRAWER & MOREST, 1975). Moreover, inputs from non-cochlear source; are differentially distributed within the AVCN (CANT & MOREST, 1978). Electron-microscopic identification of the neuronal types and their inputs is necessary to
define principles of synaptic organization in the nucleus. Studies of the fine structure of neurons in the ’ Present address: Department of Anatomy, Duke University Medical Center, Durham, NC 27710, U.S.A. Abbreviations: AA, AP, APD, anterior, posterior and posterodorsal parts, respectively, of the anterior division of the AVCN; AVCN, anteroventral cochlear nucleus; LV-S, SV, SV-F, SV-P, SV-S, large spherical, small, small flattened, small pleiomorphic, and small spherical synaptic vesicles, respectively.
AVCN (LENN & REESE [1966], and MCDONALD & RASMUSSEN[1971], in the cat and chinchilla; IBATA & PAPPAS [1976], in the cat; FELDMAN& PETERS[1972], GENTSCHEV& SOTELO [1973], and SOTELO, GENTSCHEV & ZAMORA [1976], in the rat; SCHWARTZ & GULLEY [1978], in the guinea-pig) have concentrated on the somata of the cells of the rostra1 AVCN and the associated synaptic terminals, especially the endbulbs of Held from the cochlea. That more than one
cell type can be identified in electron micrographs of the AVCN was recognized by MCDONALD & RA.sMUSSEN(1971), but detailed comparisons of the several types of cells are lacking in all available reports.
Moreover, -in previous studies, the subdivisions of the AVCN examined were not clearly identified nor were the dendrites and axons of any of the neuronal types described. Thus differences in the cell and fiber populations of the subdivisions, as demonstrated by lightmicroscopic methods, cannot usually be directly related to the electron-microscopic findings. CANT & MOREST (1979) demonstrated that the spherical cells of the anterior division of the AVCN described in Nissl-stained material (OSEN. 1969) corre-
1925
spond to the bushy cells described in Golgi imprcgnalions (BRAWER r/ trl., 1974). In the present study, the information derived from these light-microscopic studies was used to differentiate the somata and dendrites of bushy cells and their processes from those of other cell types. In each subdivision. the synaptic inputs to these cells were identified and. whenever possible, they were correlated with the endings described in <;olgi preparations (Rn,Mtix Y CAJAI., 1909: LORENTI: DE Nci. 1933, 1976: BR.~WFR & MOREST. 1975: CANT & MORESI, 1978).
EXPERIMENTAL
PROCEDURES
The cats used in this study were healthy adults. which had been raised in the laboratory. Normal marrriul. Seven cats were anesthetized with sodium pentobarbital and perfused through an intracardiac cannula with 50ml of a balanced salt solution (MTEWEN. 1956). followed by two aldehyde solutions. Five hundred milliliters of the first fixative. a mixture of l”,, formaldehyde. 1”; glutaraldehyde and lo, acrolein in 0.12 M phosphate or 0.12 M cacodylate buffer containing 0.008”/, calcium chloride. was followed by one liter of a more concentrated aldehyde solution containing 2”,, formaldehyde, 3”” glutaraldehyde and I’!,, acrolein in the same buffer. The solutions were titrated to pH 7.4 and warmed to 37°C immediately before the perfusion. The head was removed and kept overnight in the second fixative (without acrolem). The next day the brains were cut jnto blocks, sliced into transverse sections 1 mm thick on a macrotome, and postfixed in 2:, osmium tetroxide dissolved in 0.12 M phosphate buffer. Subdivisions of the AVCN and other auditory nuclei were located and dissected from the slices during osmication. Identification of a subdivision was subsequently verified on sections 1,um thick that were taken at the time of thin sectioning and stained with toluidine blue. The blocks of tissue were stained for I h in 2”, uranyl acetate in 0.05 M maleate buffer. dehydrated in increasing concentrations of methanol, and embedded in Epon 812. Thin sections were stained with O.lU,, lead citrate and 4”,, uranyl acetate in methanol. Expc~rimenral mufuriul. Unilateral labyrinthectomies were performed under aseptic conditions on six anesthetized cats. The right tympanic bulla was exposed from a ventral approach. and the cochlea was mechanically ablated with a blunt probe. After survivals of 4. I, 2. 4 (two cats), or 8 days, the animals were processed for electron microscopy as outlined above. Since the projection of the cochlear nerve is ipsilateral, the contralateral cochlear nucleus served as a control. The results from these experiments pertinent to the identification of the cochlear endings will be reported herz. RESULTS
The bushy cells Localization. The present study has been restricted to the anterior division (BRAWER et ul., 1974) of the AVCN, which, in the cat, makes up approximately the rostra1 half. The anterior division corresponds to the large and small spherical cell areas described by OSEN (1969). Features seen in Golgi preparations that distinguish the three subdivisions of the anterior division
are reiterated here to provide orientation for the electron-microscope study (see also CANT & MOREST, lY7Y). In the anterior part of the anterior division (AA). almost all of the neurons are bushy cells, and most of the dendrites in AA also belong to the bushy cells. The long dendrites of the few stellate and small cells in AA usually lie along the margins of the nucleus, although they may occasionally penetrate into the interior. Within the posterodorsal part of the anterior division (APD). almost all of the neurons are bushy cells. Stellate and small cells perch on the dorsal aspect of the subdivision. forming a cap and often sending long dendrites into the interior. In the posterior part of the anterior division (AP), there is a mixture of the three neuronal types and their dendrites appear to be intermingled as well. Thus. in AP. correlations of light- and electron-microscopic findings will be more difficult than in the other subdivisions. Our approach has been to describe the neurons and their processes in AA and in the marginal zones first and then to extrapolate those results to aid in analyzing AP and APD. In this report, the bushy cells are described. The appearance of stellate and small cells in electron micrographs will be described in a subsequent publication. Firru sttwturr of the cell body. In the following description. details which serve to distinguish the bushy cells from other cell types in the anterior division will be emphasized. The spherical cells present large. round profiles (Fig. I ). The cell bodies are closely packed (Fig. 3), although they do not appear to come into contact as in the rat (see SOTELOrt ul., 1976). Indeed. the cells, and the terminals covering their surfaces, are usually surrounded by lamellae of glial processes which separate them from components of the neuropil as well as from each other (Figs 3 and 5). A large, pale nucleus is usually centrally located in the cell body, although occasionally an eccentric nucleus may be seen. The contour of the nucleus is almost always very smooth (Figs I and 2), showing no infolding or indentations of the nuclear envelope. A single large nucleolus and small masses of chromatic and perichromatinic granules occupy the otherwise pallid karyoplasm typical of large neurons. The nuclei do not appear to contain intranuclear rods or sheets (cf. FELDMAN & PETERS. 1972). although such structures are frequently present in other neuronal types in the AVCN. A stereotyped arrangement of the organelles populating the perikaryon serves to distinguish the bushy cells. The nucleus is encompassed by cisterns of endoplasmic reticulum arranged in stacks extending out into the cytoplasm (Figs I and 2). The cisternae, which in some places may be continuous with the nuclear envelope (Fig. 2), are contacted by ribosomes along parts of their cytoplasmic surfaces. However. most of the polyribosomes associated with the endoplasmic reticulum form rosettes which lie in rows between the cisterns of the reticulum (Fig. 2). This nuclear cap of Nissl substance may be quite extensive
Bushy cells in anteroventral cochlear nucleus of cat
or made up of only a few layers. Additional stacks of endoplasmic reticulum and associated polyribosomes may be found throughout the perikaryon (Fig. 1). These layered structures correspond to the Nissl substance which defines the spherical cells in light micrographs (OSEN, 1969; CANT & MOREST,1979). Also surrounding the nucleus is an extensive Golgi apparatus consisting of tubular profiles, many vesicles with dark contents. and a few coated vesicles (Fig. 2). Less organized tubular profiles of endoplasmic reticulum are found throughout the perikaryal cytoplasm and are often continuous with subsurface cisterns, which are relatively common (Figs 3 and 5). The cytoplasm is given a somewhat dark and cluttered appearance by numerous free ribosomes, arranged in small rosettes (Figs 1, 2 and 5). The perikaryal cytoplasm is crowded with mitochondria, lysosomes, multivesicular bodies, microtubules and other organelles. We have not observed nematosomes or laminated inclusion bodies in these cells. Usually, the surface of the bushy cells appears relatively smooth (Fig. I), with only an occasional spicule or appendage. However, the smaller cells, particularly in the dorsal part of AP, are often studded with somatic spines which may contain membranous elements (Figs 5 and 6). Much of the surface of the cells is covered with synaptic terminals (Figs 1, 3 and 5), which form numerous axosomatic contacts. The terminals covering the surface of the soma are themselves blanketed by astrocytic processes. These processes also line the surface of the cell not covered by synaptic terminals (Fig. 3). The astrocytes contain bundles of glial filaments (Figs 3 and S), and their processes enter into numerous gap junctions with one another (Figs 3, 4 and 5). The axon hillock and initial segment. In a number of cases the profiles of an axon hillock and initial segment in continuity with identifiable bushy cells have been observed (e.g. Fig. 7). These are unremarkable except that many synaptic contacts occur on the axon hillock (Fig. 7). Much of the initial segment is surrounded by glial processes, most of which belong to astrocytes; however, a few synaptic contacts are found on this part of the axon as well. The proximal dendrites. Unlike the proximal dendrites of many large neurons, the cytoplasm of the bushy cell dendrites does not appear to represent a simple extension of the perikaryal cytoplasm. The primary dendrites, arising abruptly from the cell body (Fig. 8), like the dendrites of bushy cells seen in Golgi material, have a uniform diameter from the point of their origin. They are relatively straight and smooth, although an occasional appendage or spine may arise from the surface. The proximal dendrite is pale compared to the adjacent perikaryon, for very few polyribosomes or mitochondria invade the core of the dendrite (Figs 8 and 9). Stacks of granular endoplasmic reticulum accompanied by ribosomes may be found only at branch points (Fig. 8). Rosettes formed
1927
by free ribosomes and long straight mitochondria are huddled against the periphery of the dendrite, the interior being given over to parallel rows of microtubules accompanied at times by elements of smooth endoplasmic reticulum (Figs 8 and 9). A few neurofilaments line up with the microtubules. In cases in which the dendrites can be followed for a long distance from the soma, the mitochondria often increase in size distally. The Golgi apparatus of the perikaryon does not extend into the dendrite. Multivesicular bodies are occasionally seen in continuity with tubular profiles of the smooth endoplasmic reticulum (Fig. 10) and form another prominent component of the proximal dendrite. The surface of the dendrite, like that of the soma, is typically contacted by a number of synaptic endings (Figs 8 and 9). Along the surface, in between the synaptic terminals, bundles of small axons or loosely arranged glial processes are found (Fig. 8). The distal dendrites. In Golgi material, the proximal dendrites of the bushy cells give rise to a tuft of many long, thin appendages, the distal dendrites. It is clear that almost all dendritic profiles in AA must arise from this source (CANT & MOREST,1979). The proximal dendrites of the bushy cells, which would be the other major dendritic component, are rather short, and most of the long dendrites of the stellate and small cells appear to be excluded from the interior of AA. Not surprisingly, then, most of the dendritic profiles in electron micrographs from the center of AA appear to belong to one type (Fig. 11). In AA, small islands of neuropil are condensed around the densely packed cell bodies and fascicles of very large myelinated fibers. In electron micrographs of the neuropil, the most prominent dendritic profiles are very pale and contain numerous exceptionally large mitochondria (Figs 1l-l 5). Although microtubules are numerous and a few neurofilaments, polyribosomes and multivesicular bodies are also seen, cytoplasmic organelles are generally sparse in distal dendrites compared to proximal dendrites (compare Fig. 9 and the profiles in Fig. 11). Cut longitudinally, the dendrites exhibit an undulating shape with dilatations containing the mitochondria separated by narrow segments filled with microtubules and, often, neurofilaments (Fig. 12). These dilatations correspond to similar swellings in Golgi-impregnated cells. Indeed, the size, shape, and surface irregularities of these pale dendritic profiles correspond very closely to the morphology of the dendritic appendages of bushy cells observed in Golgi impregnations. In electron micrographs the dendrites often give rise to small spicules which contain only a fuzzy matrix, denser than the cytoplasm of the dendrites proper (Fig. 15). These spicules correspond t6 the spines identified in Golgi impregnations. The spicules rarely appear to be the site of synaptic contacts nor do they appear to form specialized contacts with any of the elements of the neuropil. The distal dendrites in AA are contacted by relatively few synaptic terminals and are often encased in
192x
FIG. 1. Bushy cell body in the anterior part of the anterior division of the anteroventral cochlear nucleus. Nut. nucleus: NB. Nissl body; AH. axon hillock; D. proximal dendrite; *. portions of an end-bulb that contact the dendrite (D) at its origin from the cell body. Scale = 10.0 /em. FIG 2. Portion
of the nucleus and perikaryon of a bushy cell. NB. Nissl body: Arrows indicate nuclear pores. Scale = 2.0 pm.
G. Golgi
apparatus.
FIG. 3. Axon terminals, including an end-bulb (EB). forming numerous synaptic contacts (s) on the surfaces of two bushy cells (BC) in the anterior part of the anterior division of the anteroventral cochlear nucleus. D. dendrite: f. glial filaments. Arrows indicate junctions between plial cell processes. Scale = I .(I kcm. FIG. 4. Gap junction
(arrow)
between
two astrocytic
processes
FIG. 5. Somatic spicules (sp) on a bushy cell (BC) in the posterior anteroventral cochlear nucleus. Ax. axon giving rise to a terminal Scale = I .O /urn. FIG. 6. Higher
magnification contact
(As). Scale = 0.005 ,Im.
part of the anterior division of the containing large. spherical vesicles.
of a somatic spine (sp) from Fig. 5. An axon terminal over the entire surface of the spicule. Scale = 0.25 ilrn.
forms
a synapttc
FIG. 7. Axon hillock (AH) and initial segment (IS) of a bushy cell. Axon terminals of heveral types form synapses (*) on the surface of both the axon hillock and initial segment. Scale = 2.0 pm. FIG. 8. Proximal FIG. 9. Dendritic
dendrite
(Den) of a bushy cell (BC). NB. Nissl body. Scale = 2.0 pm
profile (Den) in the anterior nucleus. mvb. multivesicular
FIG. IO. Multivesicular continuities
part of the anterior division of the antero\entral body: at. axon terminal. Scale = I .O pm.
bodies 111 the proximal dendrite of a bushy cell. Arrows indicate with the endoplasmic reticulum (ser) of the dendrite. Scale = 0.5 I’m.
FIG. I I. Neuropil in the anterior part of the anterior division of the anteroventral distal dendrite of a bushy cell. As. astrocyte: f, glial filaments; Ax. myelinated FIG I?. Distal dendrite
(Den) of a bushy
cell. m. microtubules.
cochlear
apparent
cochlear nucleus. Den. axon. Scale = 7.0 Mm.
Scale = 1.0 /cm
FIG. 13. Junction formed by the distal dendrites of bushy cells m the anterior part of the anterior division of the anteroventral cochlear nucleus. Arrows indicate increased cytoplasmic densities (d) in the dendrites at the point of contact. Scale = 0.5 irm. Fit;. 14. Distal
dendrite
(D) of a bushy cell which contacts the soma of a bushy tional cistern in the somatic cytoplasm. Scale = 0.5 Lrm.
Frti. 15. Distal dendrite
(D) of a bushy cell. *. spicules.
Scale =
cell (BC). c, subjunc-
I .OLrm
FIG. 16. End-bulb (EB) arising from a large myelinated axon and forming multlpie synaptic contacts (arrows) on the soma of a bushy cell (BC). 1, neurofilaments; *. spaces between the end-bulb and somatic membrane that typically contain glial processes. The arrowhead indicates points at which the end-bulb forms a synaptic contact with the dendrite of a bushy cell. Scale = 7.0 I’m. FIG. 17. Portion of an end-bulb vesicles in a terminal interposed FIG. 18. A synaptic
(EB) that forms contacts on a bushy cell soma. s\. small synaptic between processes of the end-bulb: g. glial processes. Scale = I.0 pm.
termmal containing large, spherical vesicles that forms an asymmetrical contact with the distal dendrite (D) of a bushy cell. Scale = I .O[cm.
synaptic
FIG. 19. Typical attachment plaque (ap) formed between an end-bulb and a bushy cell. Arrows indicate dense material that is connected by filamentous strands to a row of tubular elements; mit. mitochondrion. Scale = 0.2.5 pm.
--1929
FIG. 20. Several types of terminals contacting the soma of a bushy cell (BC) in the posterior part of the anterior division of the anteroventral cochlear nucleus. F, terminal with small, flattened vesicles; P, terminal with small, pleiomorphic vesicles; L, terminal with large, spherical vesicles; g, glial processes. Scale = 1.Opm. FIG. 21. Large terminal with many flattened synaptic vesicles (F) that forms synaptic contacts (*) on the soma of a bushy cell in the anterior part of the anterior division of the anteroventral cochlear nucleus. D, dendritic process that also contacts the bushy ceIi. Scale = 1.0 iurn. FIG. 22. Cluster of small terminals containing small, pleiomorphic vesicles that contact the soma of a bushy cell (BC). ax, axon of one of the terminals; *, terminal shown at higher magnification in Fig. 24. Scale = 1.0 pm. FIG. 23. The synaptic contacts (*) made by a terminal containing flattened vesicles. Scale = 0.5 gtn. FIG. 24. Higher magnification of the terminal indicated by an asterisk in Fig. 22. 1, 2, two types of contacts formed by these terminals; g, glial processes. Scale = 0.5 pm. FIG. 25. Terminals contacting the soma of a bushy cell in the posterior part of the anterior division of the anteroventral cochlear nucleus. P, terminal with small, pleiomorphic, dispersed vesicles: L, terminal with large, spherical vesicles. Scale = 1.0 pm. FIG. 26. Three types of terminals contacting dendrites ID). F, terminal with small, fattened vesicles; S, terminal with small, spherical vesicles; P, terminal with small. pleiomorphic vesicles. Scale = i.Opm. FIG. 27. Large terminal containing small, pleiomorphic vesicles (P) and making multiple, punctate contacts with dendritic processes I*) of bushy cells. Scale = 1.0 pm. FIG. 28. Proximal dendrite (D) of bushy cell that is contacted by terminals with small pleiomorphic vesicles (P). Scale = 1.0 pm. FIG. 29. Terminal with small, spherical vesicles (SS) that contacts the proximal dendrite of a bushy cell.
Scale = 0.5 pm. FIG. 30. Terminal containing small, spherical synaptic vesicles and dense-cored
vesicles (dc) that con-
tacts a distal dendrite (D) of a bushy cell. Scale = 1.0 pm. FIG. 31. Degenerating
end-bulb (EB) that contacts a bushy cell at the arrows. Four days after an ipsilateral cochlear ablation. Scale = I .Opm.
FIG. 32. Degenerating terminal bouton that contains swollen synaptic vesicles (*) and makes an arched synaptic contact (arrow) with a bushy cell soma. Four days after an ipsilateral cochlear ablation. Scale = 1.0 pm. FIG. 33. Surface of the soma of a bushy cell (BC) in the posterior part of the anterior division of the anteroventral cochlear nucleus 8 days after an ipsilateral cochlear ablation. The arrow indicates a vacant postsynaptic site covered by a glial process (g). Scale = 1.0 pm.
FIG. 34. Surface of the soma of a bushy cell (BC) in the anterior part of the anterior division of the anteroventral cochlear nucleus 8 days after an ipsilateral cochlear ablation. The arrow indicates a degenerating terminal. F, terminal with flattened vesicles; S, terminal with small, spherical vesicles. Scale = 1.0 pm. FIG. 35. Surface of the soma of a bushy cell in the posterior part of the anterior division of the anteroventral cochlear nucleus 8 days after an ipsilateral cochlear ablation. Asterisks mark evaginations of the cellular surface which contain synaptic densities (arrows). g, glial process; P. terminal containing small, pleiomorphic vesicles. Scale = 1.Opm. FIG. 36. Degenerating terminal (t) that forms synaptic contacts (arrows) on the surface of a bushy cell (BC). Scale = 0.5 pm. FIG. 37. Degenerating terminaf (t) that forms an asymmetrical contact (arrow) on a dendritic process (D). Scale = 0.5 pm. FIG. 38. Normal terminals, one containing flattened vesicles (F) and the other containing pleiomorphic vesicles (P), that contact the soma of a bushy cell in the posterior part of the anterior division of the anteroventral cochlear nucleus. Eight days after an ipsilateral cochlear ablation. Scale = 1.0 pm. FIG. 39. Normal terminals that contact the surface of a bushy cell (BC) and a dendrite (D) in the posterior part of the anterior division of the anteroventral cochlear nucleus. F, terminal containing flattened vesicles; P, terminal containing pleiomorphic vesicles. Eight days after an ipsilateral cochlear ablation. Scale = 1.Opm.
1935
I ‘)i(l
1937
1939
Bushy cells in
anteroventral cochlear nucleus of cat
wrappings of astrocytic processes for much of their length (Figs 11 and 12). At times, however, these dendrites may come into direct contact with one another. In some of these cases, there is no specialization of the opposing membranes, the membranes merely paralleling one another along a wavy course, whereas in other cases, junctions may occur (Fig. 13). At these junctions, the membranes straighten out in parallel to a layer of dense material in a widened extracellular space. The perijunctional cytoplasm of each dendrite contains a variable amount of dense material. Similar junctions may be formed between these distal dendrites and the somas or proximal dendrites of bushy cells (Fig. 14). Where this occurs, there is almost always a subsurface cistern situated opposite the dendrite in the cytoplasm of the bushy cell and the distal dendrite may contain a few vesicular profiles (Fig. 14). In AP and APD, the neuropil is considerably more complex than in AA. Along with elements not often found in the more rostra1 part of the nucleus, AP and APD also contain large pale dendritic profiles like those described above. Although still relatively naked the profiles in AP appear to receive more synaptic contacts than those in AA. Because of their size and shape, and by analogy with the dendrites in AA, it is likely that these profiles in AP and APD are the processes of bushy cells. Other types of dendritic profiles in AP may belong to the other types of neurons found there. Synapses on the bushy cells: normal morphology
The somatic and proximal dendritic surfaces of the bushy cells are contacted by numerous synaptic endings (e.g. Figs 1. 3, 5 and 8). The distal dendrites receive fewer contacts (e.g. Figs 11 and 12), but, for the most part, the fine structure of the endings contacting the dendrites and soma is similar. LENN & REESE(1966) demonstrated that two types of endings in the AVCN could be distinguished on the basis of the size of their synaptic vesicles, and, in the following description, terminals are divided into two groups on this basis. The first group contains terminals with large, spherical vesicles. The terminals in the second group contain small vesicles and can be further classified on the basis of the shape of the vesicles. Endings with large, spherical vesicles. The terminals with large, spherical vesicles (LV-S terminals) include the end-bulbs of Held (Figs 16 and 17) and smaller, bouton endings (e.g. Figs 3, 5 and 18). End-bulbs emerge from large meylinated axons and spread along the somatic surface, making multiple, punctate synaptic contacts which are arched toward the endbulb (Fig. 16). Large, spherical vesicles gather on the presynaptic side of the synapse. The postsynaptic cytoplasm contains some dense material giving the synapse a slightly asymmetrical appearance. The endbulbs also form attachment plaques with the cell surface (Fig. 19). In the end-bulb, these structures typically consist of two layers of dense material, one of
1941
which contacts the end-bulb membrane. The other is associated with a mitochondrion and the two layers are connected by strands of filamentous materials to a central row of vesicles or tubular structures (Fig. 19). In the present material, the end-bulbs are separated from the cell body at non-contact sites by variable spaces, which may contain glial processes or synaptic terminals with small vesicles (Figs 16 and 17). The central core of the end-bulbs is filled with microtubules and neurofilaments (Fig. 16). Mitochondria, lined up parallel to these elements, tend to separate them from the widely scattered vesicles which populate the periphery. Processes arising from the end-bulbs and small boutons containing large, spherical vesicles have fewer, if any, neurofilaments and more vesicles (Figs 3, 17 and 18), but the cytology is essentially the same. The smaller terminals also contact dendritic profiles in the neuropil, where the synaptic junctions are often appreciably more asymmetric (Fig. 15). Both end-bulbs and the smaller endings containing large, spherical vesicles contact the spherical cells throughout the anterior division. The end-bulbs also make synaptic contacts on the surface that faces the neuropil (Fig. 16). The postsynaptic components in these cases are usually very small processes which may arise from the soma or large, pale dendritic profiles like those of the distal dendrites of the bushy cells. End-bulbs are almost always found flanking the origins of the proximal dendrites of spherical cells (Fig. 1) and LV-S endings may also be found on the axon hillock (Fig. 7). Endings with small vesicles. Different types of endings with small vesicles (SV endings) are recognized on the basis of differences in vesicular shape and packing density (Fig. 20) and in the size and distribution of the terminals themselves. Although intermediate forms make absolute distinctions difficult, three basic types of endings, in addition to the LV-S endings, may be recognized in contact with bushy cells. All the types may be found in a single thin section. The three types are endings with many flattened vesicles, type SV-F; endings with small, round or pleiomorphic vesicles, type SV-P; and endings with small, spherical vesicles, type SV-S. Other types of terminals may be found in the anterior division, but, since their contacts with bushy cells are very rare or absent, they will not be considered here. Type W-F. The most common type of SV terminal in the anterior division contains numerous flattened vesicles (Figs 20, 21 and 23). The large terminals (up to 45 pm in dia.) arise from the thick axons which are unmyelinated in their preterminal portions and form several nearly symmetrical contacts with the soma or dendrites of bushy cells (Figs 21 and 23). The endings are almost filled with small vesicles of all shapes, many of which are extremely flattened. Only in areas in which mitochondria congregate do vesicles appear to be excluded. SV-F endings are found throughout the anterior division. Many of those in AP appear to have fewer vesicles, more of which are
flattened compared to those found in AA (compare Fig. 21. SV-F endings in AA, and Fig, 20, SV-F endings in AP). Type SV-P. Almost all the terminals on the bushy cells in AA are types LV-S or W-F. Most of the remaining terminals are small and contain a complement of small, pleiomorphic vesicles (W-P). none or very few of which are flattened. The SV-P terminals are not as large as the SV-F terminals. the largest seen being about l-1 5 pm in diameter (Figs 20, 22 and 24). The SV-P endings arise from thin, unmyelinated axonal segments and are almost always arranged in clusters (Fig. 22). They form two types of contacts with the bushy cells (Fig. 24). The first type (Fig. 24, 1) is a long straight. nearly symmetrical synaptic contact, the presynaptic side containing densities crowded by vesicles. The second type of contact (Fig. 24, 2) is perhaps slightly less symmetrical and is arched toward the endings while the vesicles on the presynaptic side are clustered a small distance away from the contact. Similar endings, forming synapses on dendrites, including the dendrites of bushy cells, may be considerably larger than the ones in the clusters on the cell body (Fig. 28). W-P terminals like those in AA are also found in AP. In addition. two subgroups of this type, which are rarely or never seen in AA, may be recognized. The first subgroup are also small endings and contain small, pleiomorphic vesicles (Figs 25 and 28). They are distinguished from the more common SV-P endings already described by their dark cytoplasm and the fact that the vesicles are not crowded together but are found lying loosely dispersed throughout the endings. They form a symmetrical contact with the bushy cell surface. The second subgroup of SV-P terminals consists of terminals found only very occasionally contacting bushy cells. These endings are distinguished from the other types by their much larger size, the relative paleness of the cytoplasm and the relatively looser packing of the synaptic vesicles (Fig. 27). They make contacts with many small processes. some of which arise from bushy cells (Fig. 27). Type SV-S. The third type of SV terminal contains small. densely packed spherical vesicles. These terminals very rarely contact the soma but are found on both proximal and distal dendrites of the bushy cells. They form moderately asymmetrical contacts (Figs 26 and 29). Occasionally, an SV-S terminal will also contain a number of dense-cored vesicles (Fig. 30).
Synupses on the bushy cells terminal
population
changes in the synaptic
after cochlear abkuion
The material from animals surviving cochlear ablations for 4 or 8 days could be used to seek answers to two questions raised by the study of normal material. First, do all LV-S terminals arise from the cochlea‘? And, second, are there other types of terminals which can be found to degenerate or to disappear after cochlear ablations’?
Four-day stm~ical. Four days after a cochlear ablation. there is not a conspicuous loss of endings from the ipsilateral cochlear nucleus However, definite signs of degeneration have appeared in LV-S terminals. both large and small (Figs 31 and 32). Often many of the synaptic vesicles have increased in size and there is an increased opacity in the cytoplasm of the terminal. That these are LV-S terminals is clear from the configuration of the synaptic sites with the multiple. arched contacts with asymmetrical densities. At this stage. as at the later stage described below. changes have not been observed in the other types of terminals. Ei&r-da!, surrical. Eight days alter cochlear ablation. large regions of the bushy cell surface are denuded of terminals (Figs 33. 34 and 35). Structures resembling the arching postsynaptic sites characteristic of LV-S terminals are sometimes still visible along this surface (Fig. 33). In other places the surface. still containing thickenings. presumably postsynaptic sites. may form evaginations (Fig. 35). An occasional electron-opaque degenerated terminal can be seen still attached to the surface of the cell (Figs 34 and 36) or engulfed by glial processes. Degenerating terminals found in the neuropil (Fig. 37) often display a markedly asymmetrical postsynaptic density. Normtrl LV-S c&inys rare not sew ut this .stuge, either on ,somus or in the neuropil.
Although it is not possible in a qualitative survey. to exclude the disappearance of a small percentage of one or more of the other terminal types, there IS no conspicuous loss, if any. None of the other types appears in recognizable stages of degeneration nor has any of the types been completely eliminated by the cochlear lesion. Indeed, ufter un 8-due surciwl, the SV endings in hot/l AA und AP uppeur normal (Figs 34. 35. 38 and 39). DISCUSSION The object of the present study was to provide a description of the fine structure of the bushy cells of the anterior division of the AVCN of the cat. Knowledge of the cytological characteristics of these particular cells and of their synaptic organization will allow their differential identification, and. therefore. comparisons with other cell types in the AVCN. As detailed descriptions of each of the neuronal types in the AVCN are obtained, correlations with equally detailed descriptions of the physiological properties of unit types (e.g. BOURK, 1976) may be expected to yield important insights into the relationships between structure and function in the mammalian central nervous system (MOREST,KIANG, KANE, GUINAN & GouFREY, 1973; KIANG, MOREST. GODFREY, GUNAN & KANE, 1973; MOREST. 1975). Since the anterior part of the anterior division of the AVCN is composed almost entirely of a single neuronal type, the spherical or bushy cell (OSEN. 1969: BRAWER et al.. 1974: CANT & MOREST, 1979). it
Bushy cells in anteroventral cochlear nucleus of cat
1943
shape of the vesicles and the size of the terminals. Although this classification scheme enables us to emphasize the variety of inputs to the AVCN, quantitation based only on these morphological characteristics is quite difficult because of intermediate types of endings. Clearly, the predominant non-cochlear ending is the type with flattened vesicles. These endings have been described in all studies using aldehyde fixation (MCDONALD & RASMUSSEN,1971; IBATA & PAPPAS,1976; SCHWARTZ& GULLEY,1978). We have not attempted to classify these endings further, although there is evidence that the population may be subdivided. For example, MCDONALD& RASMUSSEN (1971) found that some terminals with flattened vesicles stain for acetylcholinesterase, while others do not. Although possible differences in species and methods of preparation make comparisons of these classifications difficult, SCHWARTZ& GULLEY(1978) in their study of the AVCN of the guinea-pig, apparently subdivided this population further and showed two types of synaptic specializations in freeze-fracture preparations. In addition to the terminals with flattened vesicles, there is a significant number of endings on the bushy cells which contain pleiomorphic vesicles. These endings are further distinguished by their much smaller size. They are found clustered together in contact with bushy cell bodies and dendrites. A Cochlear input variety of these terminals with a dark cytoplasm and The major input to the bushy cells of the anterior more dispersed vesicles, which is not seen in AA, is division is the cochlea. As shown by LENN & REESE quite common in AP. This difference between AP and (1966), the large end-bulbs of Held from the cochlea AA perhaps relates to a differential innervation of the form synaptic junctions on the somatic surface. In two subdivisions (CANT& MOREST,1978). addition, the dendrites and axon hillock are contacted by smaller endings having an ultrastructure like that of the end-bulb. Eight days after cochlear ablation, all Synaptic organization of the anterior part of the anof these terminals with large, spherical vesicles have terior division disappeared from the anterior division, although Because the bushy cell is virtually the only neurdegenerating terminals making contacts typical of the onal type present in AA proper (CANT & MOREST, endings with large vesicles are still seen, especially on 1979), it provides the focus of the synaptic organizdendrites. The process of degeneration appears to in- ation of this particular nucleus. A major part of its volve a darkening reaction similar to that described input contacts the somas. About half of this axe by GENTSCHEV & SOTELO(1973) in the AVCN of the somatic input comes from the cochlea. Axdendritic rat and by KANE (1974) in the posteroventral and contacts and inputs to the axon hillock are similar to dorsal cochlear nuclei of the cat. The important point those seen on the cell soma. Axeaxonic contacts, for the present analysis is that all terminals with large other than those on the axon hillock, and dendre vesicles disappear after cochlear ablation. Thus, one dendritic synapses involving the bushy cells have not can identify these as cochlear inputs unambiguously been observed in the cat. Axon terminals contacting in normal material. On the other hand, no degenerthe end-bulb have been described in the rat (GENTSation of the terminals with small vesicles has been CHEV & SOTELO, 1973) and in the guinea-pig detected after cochlear ablation. With the present (SCHWARTZ& GULLEY,1978) but not on end-bulbs qualitative analysis over a limited range of survival which contact the bushy cells in the cat. The neuropil times, it is impossible to exclude the possibility that consists mainly of the dendrites of the spherical cells some of the small vesicle terminals arise in the coch- and the relatively few axon terminals that contact lea; however, there is as yet no evidence for this. them. Given this rather simple organization, one might expect that the output patterns of the spherical Non-cochlear endings cells in response to particular stimuli would be A large number of synaptic endings remain in condominated by the inputs to the cell body. These cells tact with the bushy cells after cochlear ablation. We might thus provide a simple system for studying inhave classified these endings, all of which have smaller put-output functions in the mammalian central nervesicles than the cochlear inputs, on the basis of the vous system. Knowledge of the source of the nonwas possible to identify and describe not only the cell bodies but also their dendritic and axonal processes in electron micrographs of this part of the nucleus. Similar cells were then identified in the more complex, posterior parts of the anterior division (AP and APD). Features of cellular morphology revealed by the electron microscope were correlated with the structure of the cells known from light microscopy (OSEN, 1969; BRAWER et al., 1974; CANT8~ MOREST,1979). The fine structure of the soma of the large cells in the anterior AVCN has been described in several species (LENN & REESE[1966], and MCDONALD& RASMUSSEN [1971], in the cat and chinchilla; IBATA & PAPPAS [1976], in the cat; FELDMAN& PETERS [ 19721, GENTSCHEV & S~TELO[1973], and SOTELOet al. [1976], in the rat; SCHWARTZ& GULLEY[1978], in the guinea-pig). The large, or principal, cells described in these studies correspond to the spherical, or bushy, cells as identified in light microscopy. The results of the present study provide an extension of these earlier studies in describing not only the cell body of carefully identified spherical cells but in identifying and describing the processes of the cells as well. In addition, it is shown that there are some differences in the inputs to these cells depending on their location in the anterior division.
cochlear terminals would allow cxperimcnts designed to stimulate them selectively. It is not yet possible to say whether the bushy cells in AP have the same organization or whether more complicated circuits are elaborated in conjunction with the other cell types present.
The bushy cells in AA undoubtedly correspond to the ‘primarylike’ units in the rostra1 AVCN described by BWRK (1976). In both cases, the overwhelming jireponderance of cells or units sampled fall into these categories. The primarylike units in AA have a distinctive spike waveshape. consisting of a small prepotential followed after approx. 0.5 ms by a larger spike. The pre-potentjal is thought to represent the depoiari~ation of the end-bulb, and the spike, the subsequent firing of the cell (BOIJRK. 1976). Since the prepotential is always accompanied by the spike, this appears to be a very secure synapse. In addition. spontaneous spike activity in the cells is always preceded by the pre-potential. implying that the excitatory stimulus is the cochtear input. These physiofogical properties conform to the morphological findings that suggest a preponderance ol an excitatory cochlear input to the bushy cells. The function of the noncochlear terminals is not known. The projections from the rostra1 AVCN to the medial and Iaterai superior olivary nuclei (WARR. 1966: OSEN. 1970) may implicate the bushy cells in binaural interactions and possibly provide a basis for localization or lateralization of sounds in space (see
EKULKAR. 1972). The security of a strongly excitatory synapse in the cochlear nucleus would tend to preserve the miniscule diotic temporal cues on which low frequency lateralization depends. The bushy cells may provide for this. If so, one might suppose that the non-cochlear synapses of bushy cells could introduce subtle modifications in the timing of bushy cell responses. Alternatively. the non-cochlear synapses could perform some other function within the temporal constraints set by the activity of the cochlear input. Other types of neurons in the cochlear nucleus also receive cochlear input and in turn project to different targets. Thus parallel pathways through the cochledr nucleus could participate in different functions, for example, in loudness or frequency discrimination. in sound locatization, or in auditory reflexes. On the other hand, interconnections between these pathways via the non-cochlear endings could provide for a measure of interaction between functional modalities. Morphological analysis of these pathways with precise identification of the relevant cell types and their inputs are a prerequisite to an understanding of the functional significance of the patterns of synaptic organization.
.4chnowirdgrmmt.s-This research was supported by USPHS grants 5 ROl NS 14347. I F22 NS 01220, and 2 R01 NS 06t15. We thank Ms PAT TIiOMPSOh. for typing the manuscript. The principal findings were presented at the Annual Meeting of the American Association of Anatomists, March, 1975 (CANT. 1975).
REFERENCES BOURK T.
R. (1976) Electrical responses of neural units in the anteroventral cochlear nucleus of the cat. Doctoral dissertation. MIT.. Cambridge. Mass. BRAWERJ. R. & MORESTD. K. (1975) Relations between auditory nerve endings and cell types in the cat’s anteroventral cochlear nucleus seen with the Golgi method and Nomarski optics. d. camp. Neural. 160,491-506. BRAWEKJ. R., MORESTD. K. & KANE E. COHEN(1974) The neuronal architecture of the cochlear nucleus of the cat. J. camp. sN~urol. 155, 251 300. CANT N. B. (1975) Organization of synaptic endings on the bushy cells of the rostra1 anteroventral cochlear nucleus (AVCN) of the cat. Anar. Rec. 181, 315. Cam N. B. & i%fORESI. D. K. (1978) Axons from non-cochlear sources in the anteroventral cochlear nucleus of the cat. A study with the rapid Golgi method. Neuroscience 3, 1003-1079. CANT N. B. & MOREST D. K. (1979) Organization of the neurons in the anterior division of the anteroventral cochlear nucleus of the cat. Light-microscopic observations. Neuroseiencr 4, 1909-1923. ERULKARS. D. (1972) Comparative aspects of spatial localization of sound. P~l~s~~~f. Rer. 52, 237 360. FELDMANM. L. & PETERSA. (1972) Intranuciear rods and sheets in rat cochlear nucleus. J. Neurorytctl. I, 109 I27. GENTSCHEVT. & SOTEI.OC. (1973) Degenerative patterns in the ventral cochlear nucleus of the rat after primary deafferentation. An ultrastructural study. Brain Res. 62, 37..-60. IRATA Y. & PAPPASG. D. (1976) The fine structure of synapses in relation to the large spherical neurons in the anterior ventral cochlear (sir-) of the cat. J. Neurocytol. 5, 395406. KANEE. C. (1974) Patterns of degeneration in the caudal cochlear nucleus of the cat after cochlear ablation. A!ruf. Rec. 179, 67-92. KIANC; N. Y. S., MORESTD. K., GODFREYD. A., GUINAN J. J. & KANE E. C. (1973) Stimulus coding at caudal levels of the cat’s auditory system -1. Response characteristic of single units. In Basic Mechtmisms in Hearing (ed. MOLLERA. R.). pp. 455478. Academic Press. New York. LENN N. J. & REESET. S. (1966) The fine structure of nerve endings in the nucleus of the trapezoid body and the ventral cochlear nucleus. Am. J. Annt. 118, 375.390. LORENTEDE Nd R. (1933) Anatomy of the eighth nerve--III. General plan of structure of the primary cochlear nuclei. Lar~n~os~o~e 43, 327-350.
Bushy cells in anteroventral cochlear nucleus of cat
1945
LORENTE DEN6 R. (1976) Some unresolved problems con~rning the cochlear nerve. Ann. Otol. Rhinos. Lary. Suppl. 34,8S, l-28. MCDONALDD. M. & RASMUSSEN G. L. (1971) Ultrastructural characteristics of synaptic endings in the cochlear nucleus having acetylcholinesterase activity. Brain Res. 28, l-18. MCEWEN L. M. (1956) The effect on the isolated rabbit heart of vagal stimulation and its modification by cocaine, hexamethonium and ouabain. J. PhysioL, Land. 131, 678-689. Mom D. K. (1975) Structural organization of the auditory pathways. In The Nervous System. Vol. 3: Human Communication and its Disorders (eds TOWERD. B. & EAGLESE. L.), pp. 19-29. Raven Press, New York. MORESTD. IL, KIANGN. Y. S., KANEE. C., GUINANJ. J. & GODFREYD. A. (1973)Stimulus coding at caudal levels of the cat’s auditory nervous system-II. Patterns of synaptic organization. In Basic Mechanisms in Hearing (ed. MILLER A. R.), pp. 479-504. Academic Press, New York. OSENK. K. (1969) Cytoarchitecture of the cochlear nuclei in the cat. J. camp. Neural. 136, 453-484. OSEN K. K. (1970) Afferent and efferent connections of three well-defined cell types of the cat cochlear nuclei. In Exci@atory Synaptic ~ec~unjs~ feds ANDERSNP. & JANSENJ. K. S.). pp. 295-300. Universitetsforlaget, Oslo. RAP&N Y CAJALS. (1909) ~~stologie du Sys&me Nbvew de i’~o~e et des VertPbrPs,Vol. I, pp. 774-838. Maloine, Paris. SOTELOC., GENTSCHEVT. & ZAMORAA. J. (1976) Gap junctions in ventral cochlear nucleus of the rat. A possible new example of electronic junctions in the mammalian central nervous system. Neuroscience I, 5-7. SCHWARTZA. M. & GULLEYR. L. (1978) Non-primary afferents to the principal cells of the rostra1 anteroventral cochlear nucleus of the guinea-pig. Am. J, hat. 153,489-508. WARRW. B. (1966) Fiber degeneration following lesions in the anterior ventral cochlear nucleus of the cat. Expl Neural. 14,453-474. (Accepted 26 June 1979)
Neuros
THE BUSHY CELLS IN THE ANTEROVENTRAL COCHLEAR NUCLEUS OF THE CAT. A STUDY WITH THE ELECTRON MICROSCOPE NELL B. CANT’ and D. K. MOREST Department of Anatomy, Harvard Medical School, Boston, MA 02115, U.S.A., and Department of Anatomy, The University of Connecticut Health Center, Farmington. CT 06032, U.S.A. bushy cells in the anterior division of the anteroventral cochlear nucleus of the cat were studied with the electron microscope. In the anterior part of the anterior division, profiles of bushy cells and their processes are easily identified, since few cells of other types are found in this region. In the posterior and posterodorsal parts of the anterior division, the bushy cells are intermingled with stellate and small cells but can be identified on the basis of light-microscopic descriptions and comparisons with the results from the anterior part. Bushy cells are large, spherical cells with a centrally located nucleus enveloped by sheets of rough endoplasmic reticulum. Thin proximal dendrites jut abruptly from the cell body and contain a relatively pale cytoplasm. The distal dendrites contain few organelles other than numerous, very large mitochondria. The cell soma and proximal dendrites, as well as the axon hillock, receive numerous synaptic terminals, but the distal dendritic processes are contacted by relatively few endings. At least four types of terminals form synaptic contacts with the bushy cells. Very large terminals, containing large, spherical synaptic vesicles and forming multiple asymmetrical contacts, correspond to the end-bulbs of Held from the cochlea. These terminals disappear after cochlear ablations, but the other three types remain. The most numerous of these is a large terminal that contains flattened synaptic vesicles and forms long, nearly symmetrical contacts with the soma and dendrites of bushy cells. The second type of non-cochlear terminal is smaller and contains small, pleiomorphic synaptic vesicles that are not flattened. The third type occurs mainly on bushy cell dendrites, contains small. spherical synaptic vesicles, and forms moderately asymmetrical contacts. The bushy cells probably correspond to the primarylike units described in electrophysiological studies of the anterior division. Primarylike units respond to activity in auditory nerve fibers in a one-to-one manner, a finding compatible with the observation that much of the surface of the soma and dendrites of the bushy cells is contacted by auditory nerve terminals (end-bulbs of Held). Neither the origins nor the functions of the several types of non-cochlear inputs to the bushy cells are known. Further analysis of these inputs and of the other neuronal types in the anterior division, when correlated with physiological and biochemical data from the same cell types, could clarify the functional significance of the observed patterns of synaptic organization. Abstract-The
THE ANTEROVENTRAL cochlear nucleus (AVCN) of the cat comprises a number of subdivisions, each with a characteristic population of neurons (OSEN, 1969; BRAWER, MOREST & KANE, 1974; CANT & MOREST, 1979). Light-microscopic studies have shown that the terminal patterns formed by the cochlear nerve vary among the subdivisions (RAM~N Y CAJAL, 1909; LORENTE DE N& 1933, 1976; BRAWER & MOREST, 1975). Moreover, inputs from non-cochlear source; are differentially distributed within the AVCN (CANT & MOREST, 1978). Electron-microscopic identification of the neuronal types and their inputs is necessary to
define principles of synaptic organization in the nucleus. Studies of the fine structure of neurons in the ’ Present address: Department of Anatomy, Duke University Medical Center, Durham, NC 27710, U.S.A. Abbreviations: AA, AP, APD, anterior, posterior and posterodorsal parts, respectively, of the anterior division of the AVCN; AVCN, anteroventral cochlear nucleus; LV-S, SV, SV-F, SV-P, SV-S, large spherical, small, small flattened, small pleiomorphic, and small spherical synaptic vesicles, respectively.
AVCN (LENN & REESE [1966], and MCDONALD & RASMUSSEN[1971], in the cat and chinchilla; IBATA & PAPPAS [1976], in the cat; FELDMAN& PETERS[1972], GENTSCHEV& SOTELO [1973], and SOTELO, GENTSCHEV & ZAMORA [1976], in the rat; SCHWARTZ & GULLEY [1978], in the guinea-pig) have concentrated on the somata of the cells of the rostra1 AVCN and the associated synaptic terminals, especially the endbulbs of Held from the cochlea. That more than one
cell type can be identified in electron micrographs of the AVCN was recognized by MCDONALD & RA.sMUSSEN(1971), but detailed comparisons of the several types of cells are lacking in all available reports.
Moreover, -in previous studies, the subdivisions of the AVCN examined were not clearly identified nor were the dendrites and axons of any of the neuronal types described. Thus differences in the cell and fiber populations of the subdivisions, as demonstrated by lightmicroscopic methods, cannot usually be directly related to the electron-microscopic findings. CANT & MOREST (1979) demonstrated that the spherical cells of the anterior division of the AVCN described in Nissl-stained material (OSEN. 1969) corre-
1925
spond to the bushy cells described in Golgi imprcgnalions (BRAWER r/ trl., 1974). In the present study, the information derived from these light-microscopic studies was used to differentiate the somata and dendrites of bushy cells and their processes from those of other cell types. In each subdivision. the synaptic inputs to these cells were identified and. whenever possible, they were correlated with the endings described in <;olgi preparations (Rn,Mtix Y CAJAI., 1909: LORENTI: DE Nci. 1933, 1976: BR.~WFR & MOREST. 1975: CANT & MORESI, 1978).
EXPERIMENTAL
PROCEDURES
The cats used in this study were healthy adults. which had been raised in the laboratory. Normal marrriul. Seven cats were anesthetized with sodium pentobarbital and perfused through an intracardiac cannula with 50ml of a balanced salt solution (MTEWEN. 1956). followed by two aldehyde solutions. Five hundred milliliters of the first fixative. a mixture of l”,, formaldehyde. 1”; glutaraldehyde and lo, acrolein in 0.12 M phosphate or 0.12 M cacodylate buffer containing 0.008”/, calcium chloride. was followed by one liter of a more concentrated aldehyde solution containing 2”,, formaldehyde, 3”” glutaraldehyde and I’!,, acrolein in the same buffer. The solutions were titrated to pH 7.4 and warmed to 37°C immediately before the perfusion. The head was removed and kept overnight in the second fixative (without acrolem). The next day the brains were cut jnto blocks, sliced into transverse sections 1 mm thick on a macrotome, and postfixed in 2:, osmium tetroxide dissolved in 0.12 M phosphate buffer. Subdivisions of the AVCN and other auditory nuclei were located and dissected from the slices during osmication. Identification of a subdivision was subsequently verified on sections 1,um thick that were taken at the time of thin sectioning and stained with toluidine blue. The blocks of tissue were stained for I h in 2”, uranyl acetate in 0.05 M maleate buffer. dehydrated in increasing concentrations of methanol, and embedded in Epon 812. Thin sections were stained with O.lU,, lead citrate and 4”,, uranyl acetate in methanol. Expc~rimenral mufuriul. Unilateral labyrinthectomies were performed under aseptic conditions on six anesthetized cats. The right tympanic bulla was exposed from a ventral approach. and the cochlea was mechanically ablated with a blunt probe. After survivals of 4. I, 2. 4 (two cats), or 8 days, the animals were processed for electron microscopy as outlined above. Since the projection of the cochlear nerve is ipsilateral, the contralateral cochlear nucleus served as a control. The results from these experiments pertinent to the identification of the cochlear endings will be reported herz. RESULTS
The bushy cells Localization. The present study has been restricted to the anterior division (BRAWER et ul., 1974) of the AVCN, which, in the cat, makes up approximately the rostra1 half. The anterior division corresponds to the large and small spherical cell areas described by OSEN (1969). Features seen in Golgi preparations that distinguish the three subdivisions of the anterior division
are reiterated here to provide orientation for the electron-microscope study (see also CANT & MOREST, lY7Y). In the anterior part of the anterior division (AA). almost all of the neurons are bushy cells, and most of the dendrites in AA also belong to the bushy cells. The long dendrites of the few stellate and small cells in AA usually lie along the margins of the nucleus, although they may occasionally penetrate into the interior. Within the posterodorsal part of the anterior division (APD). almost all of the neurons are bushy cells. Stellate and small cells perch on the dorsal aspect of the subdivision. forming a cap and often sending long dendrites into the interior. In the posterior part of the anterior division (AP), there is a mixture of the three neuronal types and their dendrites appear to be intermingled as well. Thus. in AP. correlations of light- and electron-microscopic findings will be more difficult than in the other subdivisions. Our approach has been to describe the neurons and their processes in AA and in the marginal zones first and then to extrapolate those results to aid in analyzing AP and APD. In this report, the bushy cells are described. The appearance of stellate and small cells in electron micrographs will be described in a subsequent publication. Firru sttwturr of the cell body. In the following description. details which serve to distinguish the bushy cells from other cell types in the anterior division will be emphasized. The spherical cells present large. round profiles (Fig. I ). The cell bodies are closely packed (Fig. 3), although they do not appear to come into contact as in the rat (see SOTELOrt ul., 1976). Indeed. the cells, and the terminals covering their surfaces, are usually surrounded by lamellae of glial processes which separate them from components of the neuropil as well as from each other (Figs 3 and 5). A large, pale nucleus is usually centrally located in the cell body, although occasionally an eccentric nucleus may be seen. The contour of the nucleus is almost always very smooth (Figs I and 2), showing no infolding or indentations of the nuclear envelope. A single large nucleolus and small masses of chromatic and perichromatinic granules occupy the otherwise pallid karyoplasm typical of large neurons. The nuclei do not appear to contain intranuclear rods or sheets (cf. FELDMAN & PETERS. 1972). although such structures are frequently present in other neuronal types in the AVCN. A stereotyped arrangement of the organelles populating the perikaryon serves to distinguish the bushy cells. The nucleus is encompassed by cisterns of endoplasmic reticulum arranged in stacks extending out into the cytoplasm (Figs I and 2). The cisternae, which in some places may be continuous with the nuclear envelope (Fig. 2), are contacted by ribosomes along parts of their cytoplasmic surfaces. However. most of the polyribosomes associated with the endoplasmic reticulum form rosettes which lie in rows between the cisterns of the reticulum (Fig. 2). This nuclear cap of Nissl substance may be quite extensive
Bushy cells in anteroventral cochlear nucleus of cat
or made up of only a few layers. Additional stacks of endoplasmic reticulum and associated polyribosomes may be found throughout the perikaryon (Fig. 1). These layered structures correspond to the Nissl substance which defines the spherical cells in light micrographs (OSEN, 1969; CANT & MOREST,1979). Also surrounding the nucleus is an extensive Golgi apparatus consisting of tubular profiles, many vesicles with dark contents. and a few coated vesicles (Fig. 2). Less organized tubular profiles of endoplasmic reticulum are found throughout the perikaryal cytoplasm and are often continuous with subsurface cisterns, which are relatively common (Figs 3 and 5). The cytoplasm is given a somewhat dark and cluttered appearance by numerous free ribosomes, arranged in small rosettes (Figs 1, 2 and 5). The perikaryal cytoplasm is crowded with mitochondria, lysosomes, multivesicular bodies, microtubules and other organelles. We have not observed nematosomes or laminated inclusion bodies in these cells. Usually, the surface of the bushy cells appears relatively smooth (Fig. I), with only an occasional spicule or appendage. However, the smaller cells, particularly in the dorsal part of AP, are often studded with somatic spines which may contain membranous elements (Figs 5 and 6). Much of the surface of the cells is covered with synaptic terminals (Figs 1, 3 and 5), which form numerous axosomatic contacts. The terminals covering the surface of the soma are themselves blanketed by astrocytic processes. These processes also line the surface of the cell not covered by synaptic terminals (Fig. 3). The astrocytes contain bundles of glial filaments (Figs 3 and S), and their processes enter into numerous gap junctions with one another (Figs 3, 4 and 5). The axon hillock and initial segment. In a number of cases the profiles of an axon hillock and initial segment in continuity with identifiable bushy cells have been observed (e.g. Fig. 7). These are unremarkable except that many synaptic contacts occur on the axon hillock (Fig. 7). Much of the initial segment is surrounded by glial processes, most of which belong to astrocytes; however, a few synaptic contacts are found on this part of the axon as well. The proximal dendrites. Unlike the proximal dendrites of many large neurons, the cytoplasm of the bushy cell dendrites does not appear to represent a simple extension of the perikaryal cytoplasm. The primary dendrites, arising abruptly from the cell body (Fig. 8), like the dendrites of bushy cells seen in Golgi material, have a uniform diameter from the point of their origin. They are relatively straight and smooth, although an occasional appendage or spine may arise from the surface. The proximal dendrite is pale compared to the adjacent perikaryon, for very few polyribosomes or mitochondria invade the core of the dendrite (Figs 8 and 9). Stacks of granular endoplasmic reticulum accompanied by ribosomes may be found only at branch points (Fig. 8). Rosettes formed
1927
by free ribosomes and long straight mitochondria are huddled against the periphery of the dendrite, the interior being given over to parallel rows of microtubules accompanied at times by elements of smooth endoplasmic reticulum (Figs 8 and 9). A few neurofilaments line up with the microtubules. In cases in which the dendrites can be followed for a long distance from the soma, the mitochondria often increase in size distally. The Golgi apparatus of the perikaryon does not extend into the dendrite. Multivesicular bodies are occasionally seen in continuity with tubular profiles of the smooth endoplasmic reticulum (Fig. 10) and form another prominent component of the proximal dendrite. The surface of the dendrite, like that of the soma, is typically contacted by a number of synaptic endings (Figs 8 and 9). Along the surface, in between the synaptic terminals, bundles of small axons or loosely arranged glial processes are found (Fig. 8). The distal dendrites. In Golgi material, the proximal dendrites of the bushy cells give rise to a tuft of many long, thin appendages, the distal dendrites. It is clear that almost all dendritic profiles in AA must arise from this source (CANT & MOREST,1979). The proximal dendrites of the bushy cells, which would be the other major dendritic component, are rather short, and most of the long dendrites of the stellate and small cells appear to be excluded from the interior of AA. Not surprisingly, then, most of the dendritic profiles in electron micrographs from the center of AA appear to belong to one type (Fig. 11). In AA, small islands of neuropil are condensed around the densely packed cell bodies and fascicles of very large myelinated fibers. In electron micrographs of the neuropil, the most prominent dendritic profiles are very pale and contain numerous exceptionally large mitochondria (Figs 1l-l 5). Although microtubules are numerous and a few neurofilaments, polyribosomes and multivesicular bodies are also seen, cytoplasmic organelles are generally sparse in distal dendrites compared to proximal dendrites (compare Fig. 9 and the profiles in Fig. 11). Cut longitudinally, the dendrites exhibit an undulating shape with dilatations containing the mitochondria separated by narrow segments filled with microtubules and, often, neurofilaments (Fig. 12). These dilatations correspond to similar swellings in Golgi-impregnated cells. Indeed, the size, shape, and surface irregularities of these pale dendritic profiles correspond very closely to the morphology of the dendritic appendages of bushy cells observed in Golgi impregnations. In electron micrographs the dendrites often give rise to small spicules which contain only a fuzzy matrix, denser than the cytoplasm of the dendrites proper (Fig. 15). These spicules correspond t6 the spines identified in Golgi impregnations. The spicules rarely appear to be the site of synaptic contacts nor do they appear to form specialized contacts with any of the elements of the neuropil. The distal dendrites in AA are contacted by relatively few synaptic terminals and are often encased in
192x
FIG. 1. Bushy cell body in the anterior part of the anterior division of the anteroventral cochlear nucleus. Nut. nucleus: NB. Nissl body; AH. axon hillock; D. proximal dendrite; *. portions of an end-bulb that contact the dendrite (D) at its origin from the cell body. Scale = 10.0 /em. FIG 2. Portion
of the nucleus and perikaryon of a bushy cell. NB. Nissl body: Arrows indicate nuclear pores. Scale = 2.0 pm.
G. Golgi
apparatus.
FIG. 3. Axon terminals, including an end-bulb (EB). forming numerous synaptic contacts (s) on the surfaces of two bushy cells (BC) in the anterior part of the anterior division of the anteroventral cochlear nucleus. D. dendrite: f. glial filaments. Arrows indicate junctions between plial cell processes. Scale = I .(I kcm. FIG. 4. Gap junction
(arrow)
between
two astrocytic
processes
FIG. 5. Somatic spicules (sp) on a bushy cell (BC) in the posterior anteroventral cochlear nucleus. Ax. axon giving rise to a terminal Scale = I .O /urn. FIG. 6. Higher
magnification contact
(As). Scale = 0.005 ,Im.
part of the anterior division of the containing large. spherical vesicles.
of a somatic spine (sp) from Fig. 5. An axon terminal over the entire surface of the spicule. Scale = 0.25 ilrn.
forms
a synapttc
FIG. 7. Axon hillock (AH) and initial segment (IS) of a bushy cell. Axon terminals of heveral types form synapses (*) on the surface of both the axon hillock and initial segment. Scale = 2.0 pm. FIG. 8. Proximal FIG. 9. Dendritic
dendrite
(Den) of a bushy cell (BC). NB. Nissl body. Scale = 2.0 pm
profile (Den) in the anterior nucleus. mvb. multivesicular
FIG. IO. Multivesicular continuities
part of the anterior division of the antero\entral body: at. axon terminal. Scale = I .O pm.
bodies 111 the proximal dendrite of a bushy cell. Arrows indicate with the endoplasmic reticulum (ser) of the dendrite. Scale = 0.5 I’m.
FIG. I I. Neuropil in the anterior part of the anterior division of the anteroventral distal dendrite of a bushy cell. As. astrocyte: f, glial filaments; Ax. myelinated FIG I?. Distal dendrite
(Den) of a bushy
cell. m. microtubules.
cochlear
apparent
cochlear nucleus. Den. axon. Scale = 7.0 Mm.
Scale = 1.0 /cm
FIG. 13. Junction formed by the distal dendrites of bushy cells m the anterior part of the anterior division of the anteroventral cochlear nucleus. Arrows indicate increased cytoplasmic densities (d) in the dendrites at the point of contact. Scale = 0.5 irm. Fit;. 14. Distal
dendrite
(D) of a bushy cell which contacts the soma of a bushy tional cistern in the somatic cytoplasm. Scale = 0.5 Lrm.
Frti. 15. Distal dendrite
(D) of a bushy cell. *. spicules.
Scale =
cell (BC). c, subjunc-
I .OLrm
FIG. 16. End-bulb (EB) arising from a large myelinated axon and forming multlpie synaptic contacts (arrows) on the soma of a bushy cell (BC). 1, neurofilaments; *. spaces between the end-bulb and somatic membrane that typically contain glial processes. The arrowhead indicates points at which the end-bulb forms a synaptic contact with the dendrite of a bushy cell. Scale = 7.0 I’m. FIG. 17. Portion of an end-bulb vesicles in a terminal interposed FIG. 18. A synaptic
(EB) that forms contacts on a bushy cell soma. s\. small synaptic between processes of the end-bulb: g. glial processes. Scale = I.0 pm.
termmal containing large, spherical vesicles that forms an asymmetrical contact with the distal dendrite (D) of a bushy cell. Scale = I .O[cm.
synaptic
FIG. 19. Typical attachment plaque (ap) formed between an end-bulb and a bushy cell. Arrows indicate dense material that is connected by filamentous strands to a row of tubular elements; mit. mitochondrion. Scale = 0.2.5 pm.
--1929
FIG. 20. Several types of terminals contacting the soma of a bushy cell (BC) in the posterior part of the anterior division of the anteroventral cochlear nucleus. F, terminal with small, flattened vesicles; P, terminal with small, pleiomorphic vesicles; L, terminal with large, spherical vesicles; g, glial processes. Scale = 1.Opm. FIG. 21. Large terminal with many flattened synaptic vesicles (F) that forms synaptic contacts (*) on the soma of a bushy cell in the anterior part of the anterior division of the anteroventral cochlear nucleus. D, dendritic process that also contacts the bushy ceIi. Scale = 1.0 iurn. FIG. 22. Cluster of small terminals containing small, pleiomorphic vesicles that contact the soma of a bushy cell (BC). ax, axon of one of the terminals; *, terminal shown at higher magnification in Fig. 24. Scale = 1.0 pm. FIG. 23. The synaptic contacts (*) made by a terminal containing flattened vesicles. Scale = 0.5 gtn. FIG. 24. Higher magnification of the terminal indicated by an asterisk in Fig. 22. 1, 2, two types of contacts formed by these terminals; g, glial processes. Scale = 0.5 pm. FIG. 25. Terminals contacting the soma of a bushy cell in the posterior part of the anterior division of the anteroventral cochlear nucleus. P, terminal with small, pleiomorphic, dispersed vesicles: L, terminal with large, spherical vesicles. Scale = 1.0 pm. FIG. 26. Three types of terminals contacting dendrites ID). F, terminal with small, fattened vesicles; S, terminal with small, spherical vesicles; P, terminal with small. pleiomorphic vesicles. Scale = i.Opm. FIG. 27. Large terminal containing small, pleiomorphic vesicles (P) and making multiple, punctate contacts with dendritic processes I*) of bushy cells. Scale = 1.0 pm. FIG. 28. Proximal dendrite (D) of bushy cell that is contacted by terminals with small pleiomorphic vesicles (P). Scale = 1.0 pm. FIG. 29. Terminal with small, spherical vesicles (SS) that contacts the proximal dendrite of a bushy cell.
Scale = 0.5 pm. FIG. 30. Terminal containing small, spherical synaptic vesicles and dense-cored
vesicles (dc) that con-
tacts a distal dendrite (D) of a bushy cell. Scale = 1.0 pm. FIG. 31. Degenerating
end-bulb (EB) that contacts a bushy cell at the arrows. Four days after an ipsilateral cochlear ablation. Scale = I .Opm.
FIG. 32. Degenerating terminal bouton that contains swollen synaptic vesicles (*) and makes an arched synaptic contact (arrow) with a bushy cell soma. Four days after an ipsilateral cochlear ablation. Scale = 1.0 pm. FIG. 33. Surface of the soma of a bushy cell (BC) in the posterior part of the anterior division of the anteroventral cochlear nucleus 8 days after an ipsilateral cochlear ablation. The arrow indicates a vacant postsynaptic site covered by a glial process (g). Scale = 1.0 pm.
FIG. 34. Surface of the soma of a bushy cell (BC) in the anterior part of the anterior division of the anteroventral cochlear nucleus 8 days after an ipsilateral cochlear ablation. The arrow indicates a degenerating terminal. F, terminal with flattened vesicles; S, terminal with small, spherical vesicles. Scale = 1.0 pm. FIG. 35. Surface of the soma of a bushy cell in the posterior part of the anterior division of the anteroventral cochlear nucleus 8 days after an ipsilateral cochlear ablation. Asterisks mark evaginations of the cellular surface which contain synaptic densities (arrows). g, glial process; P. terminal containing small, pleiomorphic vesicles. Scale = 1.Opm. FIG. 36. Degenerating terminal (t) that forms synaptic contacts (arrows) on the surface of a bushy cell (BC). Scale = 0.5 pm. FIG. 37. Degenerating terminaf (t) that forms an asymmetrical contact (arrow) on a dendritic process (D). Scale = 0.5 pm. FIG. 38. Normal terminals, one containing flattened vesicles (F) and the other containing pleiomorphic vesicles (P), that contact the soma of a bushy cell in the posterior part of the anterior division of the anteroventral cochlear nucleus. Eight days after an ipsilateral cochlear ablation. Scale = 1.0 pm. FIG. 39. Normal terminals that contact the surface of a bushy cell (BC) and a dendrite (D) in the posterior part of the anterior division of the anteroventral cochlear nucleus. F, terminal containing flattened vesicles; P, terminal containing pleiomorphic vesicles. Eight days after an ipsilateral cochlear ablation. Scale = 1.Opm.
1935
I ‘)i(l
1937
1939
Bushy cells in
anteroventral cochlear nucleus of cat
wrappings of astrocytic processes for much of their length (Figs 11 and 12). At times, however, these dendrites may come into direct contact with one another. In some of these cases, there is no specialization of the opposing membranes, the membranes merely paralleling one another along a wavy course, whereas in other cases, junctions may occur (Fig. 13). At these junctions, the membranes straighten out in parallel to a layer of dense material in a widened extracellular space. The perijunctional cytoplasm of each dendrite contains a variable amount of dense material. Similar junctions may be formed between these distal dendrites and the somas or proximal dendrites of bushy cells (Fig. 14). Where this occurs, there is almost always a subsurface cistern situated opposite the dendrite in the cytoplasm of the bushy cell and the distal dendrite may contain a few vesicular profiles (Fig. 14). In AP and APD, the neuropil is considerably more complex than in AA. Along with elements not often found in the more rostra1 part of the nucleus, AP and APD also contain large pale dendritic profiles like those described above. Although still relatively naked the profiles in AP appear to receive more synaptic contacts than those in AA. Because of their size and shape, and by analogy with the dendrites in AA, it is likely that these profiles in AP and APD are the processes of bushy cells. Other types of dendritic profiles in AP may belong to the other types of neurons found there. Synapses on the bushy cells: normal morphology
The somatic and proximal dendritic surfaces of the bushy cells are contacted by numerous synaptic endings (e.g. Figs 1. 3, 5 and 8). The distal dendrites receive fewer contacts (e.g. Figs 11 and 12), but, for the most part, the fine structure of the endings contacting the dendrites and soma is similar. LENN & REESE(1966) demonstrated that two types of endings in the AVCN could be distinguished on the basis of the size of their synaptic vesicles, and, in the following description, terminals are divided into two groups on this basis. The first group contains terminals with large, spherical vesicles. The terminals in the second group contain small vesicles and can be further classified on the basis of the shape of the vesicles. Endings with large, spherical vesicles. The terminals with large, spherical vesicles (LV-S terminals) include the end-bulbs of Held (Figs 16 and 17) and smaller, bouton endings (e.g. Figs 3, 5 and 18). End-bulbs emerge from large meylinated axons and spread along the somatic surface, making multiple, punctate synaptic contacts which are arched toward the endbulb (Fig. 16). Large, spherical vesicles gather on the presynaptic side of the synapse. The postsynaptic cytoplasm contains some dense material giving the synapse a slightly asymmetrical appearance. The endbulbs also form attachment plaques with the cell surface (Fig. 19). In the end-bulb, these structures typically consist of two layers of dense material, one of
1941
which contacts the end-bulb membrane. The other is associated with a mitochondrion and the two layers are connected by strands of filamentous materials to a central row of vesicles or tubular structures (Fig. 19). In the present material, the end-bulbs are separated from the cell body at non-contact sites by variable spaces, which may contain glial processes or synaptic terminals with small vesicles (Figs 16 and 17). The central core of the end-bulbs is filled with microtubules and neurofilaments (Fig. 16). Mitochondria, lined up parallel to these elements, tend to separate them from the widely scattered vesicles which populate the periphery. Processes arising from the end-bulbs and small boutons containing large, spherical vesicles have fewer, if any, neurofilaments and more vesicles (Figs 3, 17 and 18), but the cytology is essentially the same. The smaller terminals also contact dendritic profiles in the neuropil, where the synaptic junctions are often appreciably more asymmetric (Fig. 15). Both end-bulbs and the smaller endings containing large, spherical vesicles contact the spherical cells throughout the anterior division. The end-bulbs also make synaptic contacts on the surface that faces the neuropil (Fig. 16). The postsynaptic components in these cases are usually very small processes which may arise from the soma or large, pale dendritic profiles like those of the distal dendrites of the bushy cells. End-bulbs are almost always found flanking the origins of the proximal dendrites of spherical cells (Fig. 1) and LV-S endings may also be found on the axon hillock (Fig. 7). Endings with small vesicles. Different types of endings with small vesicles (SV endings) are recognized on the basis of differences in vesicular shape and packing density (Fig. 20) and in the size and distribution of the terminals themselves. Although intermediate forms make absolute distinctions difficult, three basic types of endings, in addition to the LV-S endings, may be recognized in contact with bushy cells. All the types may be found in a single thin section. The three types are endings with many flattened vesicles, type SV-F; endings with small, round or pleiomorphic vesicles, type SV-P; and endings with small, spherical vesicles, type SV-S. Other types of terminals may be found in the anterior division, but, since their contacts with bushy cells are very rare or absent, they will not be considered here. Type W-F. The most common type of SV terminal in the anterior division contains numerous flattened vesicles (Figs 20, 21 and 23). The large terminals (up to 45 pm in dia.) arise from the thick axons which are unmyelinated in their preterminal portions and form several nearly symmetrical contacts with the soma or dendrites of bushy cells (Figs 21 and 23). The endings are almost filled with small vesicles of all shapes, many of which are extremely flattened. Only in areas in which mitochondria congregate do vesicles appear to be excluded. SV-F endings are found throughout the anterior division. Many of those in AP appear to have fewer vesicles, more of which are
flattened compared to those found in AA (compare Fig. 21. SV-F endings in AA, and Fig, 20, SV-F endings in AP). Type SV-P. Almost all the terminals on the bushy cells in AA are types LV-S or W-F. Most of the remaining terminals are small and contain a complement of small, pleiomorphic vesicles (W-P). none or very few of which are flattened. The SV-P terminals are not as large as the SV-F terminals. the largest seen being about l-1 5 pm in diameter (Figs 20, 22 and 24). The SV-P endings arise from thin, unmyelinated axonal segments and are almost always arranged in clusters (Fig. 22). They form two types of contacts with the bushy cells (Fig. 24). The first type (Fig. 24, 1) is a long straight. nearly symmetrical synaptic contact, the presynaptic side containing densities crowded by vesicles. The second type of contact (Fig. 24, 2) is perhaps slightly less symmetrical and is arched toward the endings while the vesicles on the presynaptic side are clustered a small distance away from the contact. Similar endings, forming synapses on dendrites, including the dendrites of bushy cells, may be considerably larger than the ones in the clusters on the cell body (Fig. 28). W-P terminals like those in AA are also found in AP. In addition. two subgroups of this type, which are rarely or never seen in AA, may be recognized. The first subgroup are also small endings and contain small, pleiomorphic vesicles (Figs 25 and 28). They are distinguished from the more common SV-P endings already described by their dark cytoplasm and the fact that the vesicles are not crowded together but are found lying loosely dispersed throughout the endings. They form a symmetrical contact with the bushy cell surface. The second subgroup of SV-P terminals consists of terminals found only very occasionally contacting bushy cells. These endings are distinguished from the other types by their much larger size, the relative paleness of the cytoplasm and the relatively looser packing of the synaptic vesicles (Fig. 27). They make contacts with many small processes. some of which arise from bushy cells (Fig. 27). Type SV-S. The third type of SV terminal contains small. densely packed spherical vesicles. These terminals very rarely contact the soma but are found on both proximal and distal dendrites of the bushy cells. They form moderately asymmetrical contacts (Figs 26 and 29). Occasionally, an SV-S terminal will also contain a number of dense-cored vesicles (Fig. 30).
Synupses on the bushy cells terminal
population
changes in the synaptic
after cochlear abkuion
The material from animals surviving cochlear ablations for 4 or 8 days could be used to seek answers to two questions raised by the study of normal material. First, do all LV-S terminals arise from the cochlea‘? And, second, are there other types of terminals which can be found to degenerate or to disappear after cochlear ablations’?
Four-day stm~ical. Four days after a cochlear ablation. there is not a conspicuous loss of endings from the ipsilateral cochlear nucleus However, definite signs of degeneration have appeared in LV-S terminals. both large and small (Figs 31 and 32). Often many of the synaptic vesicles have increased in size and there is an increased opacity in the cytoplasm of the terminal. That these are LV-S terminals is clear from the configuration of the synaptic sites with the multiple. arched contacts with asymmetrical densities. At this stage. as at the later stage described below. changes have not been observed in the other types of terminals. Ei&r-da!, surrical. Eight days alter cochlear ablation. large regions of the bushy cell surface are denuded of terminals (Figs 33. 34 and 35). Structures resembling the arching postsynaptic sites characteristic of LV-S terminals are sometimes still visible along this surface (Fig. 33). In other places the surface. still containing thickenings. presumably postsynaptic sites. may form evaginations (Fig. 35). An occasional electron-opaque degenerated terminal can be seen still attached to the surface of the cell (Figs 34 and 36) or engulfed by glial processes. Degenerating terminals found in the neuropil (Fig. 37) often display a markedly asymmetrical postsynaptic density. Normtrl LV-S c&inys rare not sew ut this .stuge, either on ,somus or in the neuropil.
Although it is not possible in a qualitative survey. to exclude the disappearance of a small percentage of one or more of the other terminal types, there IS no conspicuous loss, if any. None of the other types appears in recognizable stages of degeneration nor has any of the types been completely eliminated by the cochlear lesion. Indeed, ufter un 8-due surciwl, the SV endings in hot/l AA und AP uppeur normal (Figs 34. 35. 38 and 39). DISCUSSION The object of the present study was to provide a description of the fine structure of the bushy cells of the anterior division of the AVCN of the cat. Knowledge of the cytological characteristics of these particular cells and of their synaptic organization will allow their differential identification, and. therefore. comparisons with other cell types in the AVCN. As detailed descriptions of each of the neuronal types in the AVCN are obtained, correlations with equally detailed descriptions of the physiological properties of unit types (e.g. BOURK, 1976) may be expected to yield important insights into the relationships between structure and function in the mammalian central nervous system (MOREST,KIANG, KANE, GUINAN & GouFREY, 1973; KIANG, MOREST. GODFREY, GUNAN & KANE, 1973; MOREST. 1975). Since the anterior part of the anterior division of the AVCN is composed almost entirely of a single neuronal type, the spherical or bushy cell (OSEN. 1969: BRAWER et al.. 1974: CANT & MOREST, 1979). it
Bushy cells in anteroventral cochlear nucleus of cat
1943
shape of the vesicles and the size of the terminals. Although this classification scheme enables us to emphasize the variety of inputs to the AVCN, quantitation based only on these morphological characteristics is quite difficult because of intermediate types of endings. Clearly, the predominant non-cochlear ending is the type with flattened vesicles. These endings have been described in all studies using aldehyde fixation (MCDONALD & RASMUSSEN,1971; IBATA & PAPPAS,1976; SCHWARTZ& GULLEY,1978). We have not attempted to classify these endings further, although there is evidence that the population may be subdivided. For example, MCDONALD& RASMUSSEN (1971) found that some terminals with flattened vesicles stain for acetylcholinesterase, while others do not. Although possible differences in species and methods of preparation make comparisons of these classifications difficult, SCHWARTZ& GULLEY(1978) in their study of the AVCN of the guinea-pig, apparently subdivided this population further and showed two types of synaptic specializations in freeze-fracture preparations. In addition to the terminals with flattened vesicles, there is a significant number of endings on the bushy cells which contain pleiomorphic vesicles. These endings are further distinguished by their much smaller size. They are found clustered together in contact with bushy cell bodies and dendrites. A Cochlear input variety of these terminals with a dark cytoplasm and The major input to the bushy cells of the anterior more dispersed vesicles, which is not seen in AA, is division is the cochlea. As shown by LENN & REESE quite common in AP. This difference between AP and (1966), the large end-bulbs of Held from the cochlea AA perhaps relates to a differential innervation of the form synaptic junctions on the somatic surface. In two subdivisions (CANT& MOREST,1978). addition, the dendrites and axon hillock are contacted by smaller endings having an ultrastructure like that of the end-bulb. Eight days after cochlear ablation, all Synaptic organization of the anterior part of the anof these terminals with large, spherical vesicles have terior division disappeared from the anterior division, although Because the bushy cell is virtually the only neurdegenerating terminals making contacts typical of the onal type present in AA proper (CANT & MOREST, endings with large vesicles are still seen, especially on 1979), it provides the focus of the synaptic organizdendrites. The process of degeneration appears to in- ation of this particular nucleus. A major part of its volve a darkening reaction similar to that described input contacts the somas. About half of this axe by GENTSCHEV & SOTELO(1973) in the AVCN of the somatic input comes from the cochlea. Axdendritic rat and by KANE (1974) in the posteroventral and contacts and inputs to the axon hillock are similar to dorsal cochlear nuclei of the cat. The important point those seen on the cell soma. Axeaxonic contacts, for the present analysis is that all terminals with large other than those on the axon hillock, and dendre vesicles disappear after cochlear ablation. Thus, one dendritic synapses involving the bushy cells have not can identify these as cochlear inputs unambiguously been observed in the cat. Axon terminals contacting in normal material. On the other hand, no degenerthe end-bulb have been described in the rat (GENTSation of the terminals with small vesicles has been CHEV & SOTELO, 1973) and in the guinea-pig detected after cochlear ablation. With the present (SCHWARTZ& GULLEY,1978) but not on end-bulbs qualitative analysis over a limited range of survival which contact the bushy cells in the cat. The neuropil times, it is impossible to exclude the possibility that consists mainly of the dendrites of the spherical cells some of the small vesicle terminals arise in the coch- and the relatively few axon terminals that contact lea; however, there is as yet no evidence for this. them. Given this rather simple organization, one might expect that the output patterns of the spherical Non-cochlear endings cells in response to particular stimuli would be A large number of synaptic endings remain in condominated by the inputs to the cell body. These cells tact with the bushy cells after cochlear ablation. We might thus provide a simple system for studying inhave classified these endings, all of which have smaller put-output functions in the mammalian central nervesicles than the cochlear inputs, on the basis of the vous system. Knowledge of the source of the nonwas possible to identify and describe not only the cell bodies but also their dendritic and axonal processes in electron micrographs of this part of the nucleus. Similar cells were then identified in the more complex, posterior parts of the anterior division (AP and APD). Features of cellular morphology revealed by the electron microscope were correlated with the structure of the cells known from light microscopy (OSEN, 1969; BRAWER et al., 1974; CANT8~ MOREST,1979). The fine structure of the soma of the large cells in the anterior AVCN has been described in several species (LENN & REESE[1966], and MCDONALD& RASMUSSEN [1971], in the cat and chinchilla; IBATA & PAPPAS [1976], in the cat; FELDMAN& PETERS [ 19721, GENTSCHEV & S~TELO[1973], and SOTELOet al. [1976], in the rat; SCHWARTZ& GULLEY[1978], in the guinea-pig). The large, or principal, cells described in these studies correspond to the spherical, or bushy, cells as identified in light microscopy. The results of the present study provide an extension of these earlier studies in describing not only the cell body of carefully identified spherical cells but in identifying and describing the processes of the cells as well. In addition, it is shown that there are some differences in the inputs to these cells depending on their location in the anterior division.
cochlear terminals would allow cxperimcnts designed to stimulate them selectively. It is not yet possible to say whether the bushy cells in AP have the same organization or whether more complicated circuits are elaborated in conjunction with the other cell types present.
The bushy cells in AA undoubtedly correspond to the ‘primarylike’ units in the rostra1 AVCN described by BWRK (1976). In both cases, the overwhelming jireponderance of cells or units sampled fall into these categories. The primarylike units in AA have a distinctive spike waveshape. consisting of a small prepotential followed after approx. 0.5 ms by a larger spike. The pre-potentjal is thought to represent the depoiari~ation of the end-bulb, and the spike, the subsequent firing of the cell (BOIJRK. 1976). Since the prepotential is always accompanied by the spike, this appears to be a very secure synapse. In addition. spontaneous spike activity in the cells is always preceded by the pre-potential. implying that the excitatory stimulus is the cochtear input. These physiofogical properties conform to the morphological findings that suggest a preponderance ol an excitatory cochlear input to the bushy cells. The function of the noncochlear terminals is not known. The projections from the rostra1 AVCN to the medial and Iaterai superior olivary nuclei (WARR. 1966: OSEN. 1970) may implicate the bushy cells in binaural interactions and possibly provide a basis for localization or lateralization of sounds in space (see
EKULKAR. 1972). The security of a strongly excitatory synapse in the cochlear nucleus would tend to preserve the miniscule diotic temporal cues on which low frequency lateralization depends. The bushy cells may provide for this. If so, one might suppose that the non-cochlear synapses of bushy cells could introduce subtle modifications in the timing of bushy cell responses. Alternatively. the non-cochlear synapses could perform some other function within the temporal constraints set by the activity of the cochlear input. Other types of neurons in the cochlear nucleus also receive cochlear input and in turn project to different targets. Thus parallel pathways through the cochledr nucleus could participate in different functions, for example, in loudness or frequency discrimination. in sound locatization, or in auditory reflexes. On the other hand, interconnections between these pathways via the non-cochlear endings could provide for a measure of interaction between functional modalities. Morphological analysis of these pathways with precise identification of the relevant cell types and their inputs are a prerequisite to an understanding of the functional significance of the patterns of synaptic organization.
.4chnowirdgrmmt.s-This research was supported by USPHS grants 5 ROl NS 14347. I F22 NS 01220, and 2 R01 NS 06t15. We thank Ms PAT TIiOMPSOh. for typing the manuscript. The principal findings were presented at the Annual Meeting of the American Association of Anatomists, March, 1975 (CANT. 1975).
REFERENCES BOURK T.
R. (1976) Electrical responses of neural units in the anteroventral cochlear nucleus of the cat. Doctoral dissertation. MIT.. Cambridge. Mass. BRAWERJ. R. & MORESTD. K. (1975) Relations between auditory nerve endings and cell types in the cat’s anteroventral cochlear nucleus seen with the Golgi method and Nomarski optics. d. camp. Neural. 160,491-506. BRAWEKJ. R., MORESTD. K. & KANE E. COHEN(1974) The neuronal architecture of the cochlear nucleus of the cat. J. camp. sN~urol. 155, 251 300. CANT N. B. (1975) Organization of synaptic endings on the bushy cells of the rostra1 anteroventral cochlear nucleus (AVCN) of the cat. Anar. Rec. 181, 315. Cam N. B. & i%fORESI. D. K. (1978) Axons from non-cochlear sources in the anteroventral cochlear nucleus of the cat. A study with the rapid Golgi method. Neuroscience 3, 1003-1079. CANT N. B. & MOREST D. K. (1979) Organization of the neurons in the anterior division of the anteroventral cochlear nucleus of the cat. Light-microscopic observations. Neuroseiencr 4, 1909-1923. ERULKARS. D. (1972) Comparative aspects of spatial localization of sound. P~l~s~~~f. Rer. 52, 237 360. FELDMANM. L. & PETERSA. (1972) Intranuciear rods and sheets in rat cochlear nucleus. J. Neurorytctl. I, 109 I27. GENTSCHEVT. & SOTEI.OC. (1973) Degenerative patterns in the ventral cochlear nucleus of the rat after primary deafferentation. An ultrastructural study. Brain Res. 62, 37..-60. IRATA Y. & PAPPASG. D. (1976) The fine structure of synapses in relation to the large spherical neurons in the anterior ventral cochlear (sir-) of the cat. J. Neurocytol. 5, 395406. KANEE. C. (1974) Patterns of degeneration in the caudal cochlear nucleus of the cat after cochlear ablation. A!ruf. Rec. 179, 67-92. KIANC; N. Y. S., MORESTD. K., GODFREYD. A., GUINAN J. J. & KANE E. C. (1973) Stimulus coding at caudal levels of the cat’s auditory system -1. Response characteristic of single units. In Basic Mechtmisms in Hearing (ed. MOLLERA. R.). pp. 455478. Academic Press. New York. LENN N. J. & REESET. S. (1966) The fine structure of nerve endings in the nucleus of the trapezoid body and the ventral cochlear nucleus. Am. J. Annt. 118, 375.390. LORENTEDE Nd R. (1933) Anatomy of the eighth nerve--III. General plan of structure of the primary cochlear nuclei. Lar~n~os~o~e 43, 327-350.
Bushy cells in anteroventral cochlear nucleus of cat
1945
LORENTE DEN6 R. (1976) Some unresolved problems con~rning the cochlear nerve. Ann. Otol. Rhinos. Lary. Suppl. 34,8S, l-28. MCDONALDD. M. & RASMUSSEN G. L. (1971) Ultrastructural characteristics of synaptic endings in the cochlear nucleus having acetylcholinesterase activity. Brain Res. 28, l-18. MCEWEN L. M. (1956) The effect on the isolated rabbit heart of vagal stimulation and its modification by cocaine, hexamethonium and ouabain. J. PhysioL, Land. 131, 678-689. Mom D. K. (1975) Structural organization of the auditory pathways. In The Nervous System. Vol. 3: Human Communication and its Disorders (eds TOWERD. B. & EAGLESE. L.), pp. 19-29. Raven Press, New York. MORESTD. IL, KIANGN. Y. S., KANEE. C., GUINANJ. J. & GODFREYD. A. (1973)Stimulus coding at caudal levels of the cat’s auditory nervous system-II. Patterns of synaptic organization. In Basic Mechanisms in Hearing (ed. MILLER A. R.), pp. 479-504. Academic Press, New York. OSENK. K. (1969) Cytoarchitecture of the cochlear nuclei in the cat. J. camp. Neural. 136, 453-484. OSEN K. K. (1970) Afferent and efferent connections of three well-defined cell types of the cat cochlear nuclei. In Exci@atory Synaptic ~ec~unjs~ feds ANDERSNP. & JANSENJ. K. S.). pp. 295-300. Universitetsforlaget, Oslo. RAP&N Y CAJALS. (1909) ~~stologie du Sys&me Nbvew de i’~o~e et des VertPbrPs,Vol. I, pp. 774-838. Maloine, Paris. SOTELOC., GENTSCHEVT. & ZAMORAA. J. (1976) Gap junctions in ventral cochlear nucleus of the rat. A possible new example of electronic junctions in the mammalian central nervous system. Neuroscience I, 5-7. SCHWARTZA. M. & GULLEYR. L. (1978) Non-primary afferents to the principal cells of the rostra1 anteroventral cochlear nucleus of the guinea-pig. Am. J, hat. 153,489-508. WARRW. B. (1966) Fiber degeneration following lesions in the anterior ventral cochlear nucleus of the cat. Expl Neural. 14,453-474. (Accepted 26 June 1979)