Projections of auditory nerve in the cat as seen by anterograde transport methods

Neurorrence Vol. 4, pp. 1299 ID 1313 Pergamon Press Ltd. 1979. Printed in Grea: Britain

PROJECTIONS OF AUDITORY NERVE IN THE CAT AS SEEN BY ANTEROGRADE TRANSPORT METHODS D. R. JONESand J. H. CA~SEDAY Laboratories of ~to~ary~gology and Department of Psychology, Duke University, Durham, NC 27710, U.S.A. A~~ct-Asc~ndiog projections of the auditory nerve of the cat were studied by autoradiographi~ methods after injection of labeled precursors into the cochlea. In animals in which [3H]leucine was injected, transport to the cochlear nucleus was observed after survival periods of 2-7 days, but there was no evidence of transport to more central structures in the auditory pathways. In these cases the pattern of labeling around cell bodies depended on the type of cell and on the location of the cell within subdivisions of the cochlear nucleus. For example, there was dense perisomatic labeling around spherical cells in the anteroventrai cochlear nucleus and around octopus cells in the posteroventral cochiear nucleus. Some other types of cells in subdivisions of both anteroventral and posteroventral cochlear nucleus had little perisomatic labeling. In the dorsal cochlear nucleus, the most densely labeled area consisted of a band just central to the layer of fusiform cells. Labeled fibers were not found peripheral to this band in the granular layer. molecular layer and peripheral part of the layer of fusifotm cells, In the dorsal nucleus there was no evidence of dense perisomatic labeling such as was found around some cells in the ventral divisions of the cochlcar nucleus. Following injections of a mixture of [?Tjproline and C3H]fucose into the cochlea, labeling was Seen in the superior olivary complex. TransneuronaI transport was suggested as the explanation for this finding because of (1) the choice of the labeled precursors, (2) the length of the survival periods (14 or 20 days), and (3) the evidence that isotope was within neurons in the cochlear nucleus. We concluded that under appropriate conditions, unique patterns of silver grains are found around different types of cells in the various subdivisions of the cochfear nucleus, apparently because fibers and terminals of the auditory nerve become saturated with the radioisotope, As a result, a highly detailed survey of projections of the auditory nerve to each subdivision of the cochlear nucleus is revealed at the light-microscopic level. Under different experimental conditions labeled fibers are found in the superior oiivary complex, but these conditions are those most conducive to transneuronal Iran+ port and so such eliperiments cannot be used as evidence of projection of auditory nerve fibers beyond the cochiear nucleus.

Tm mm nucleus in mammals consists of three major divisions, each of which receives a branch of each auditory nerve fiber (RAM~N Y CAJAL, 1909; I..~RENTEDE NC& f933h; LEWY & K~BRAK, 1936; SANDO, 1965; FELDMAN& HARRWN, 1967; @EN, 1970). Several features of this innervation are well known. For example, there is clear evidence from Go&i and electron-microscopic studies that the shape and size of terminals of the auditory nerve are distinctly different in the various subdivisions of the cochlear nucleus (RAM~N Y CAJAL, 1909 ; LOREWEDE N6, g933a,b, %9?6; MORES, KI.WG, KANE, GUINAN & GODFREY, 1973; BRAWER& MOREST,1975; KANE, 1973, 19743); these differences are related to the morphology of cells in the cochlear nucleus and seem to have functional significance (KIANG, MORE.%GODFREY,GUINAN & KANE, 1973). Other aspects of ascending projections of the auditory nerve remain unclear. Studies in which degeneration techniques were used have yielded conflicting evidence concerning the organization of primary Ab~reui~t~~n~.AVa, AVp, anterior and posterior subdivisions, respectively, of the anteroventral nucleus; RVa, PVp, anterior and posterior subdivisions, respectively, of the posteroventral cochiear nucleus.

afferents to the faminae of the dorsal cochlear nucleus (LORESTEDE N6, 19336; RA~~~EN, 1957; POWELL & COWAN, 1962; POWELL& ERULKAR,1962; W, 1970; COHEN, BRAWER & MOREST, 1972; KANE, I974a. b). Recently, a~t~ra~o~aphic studies have yielded results which have revived the question af whether or not fibers from the cochlea project to auditory. structures central to the cochlear nucleus (CARPENTER, BATTON & PETER,1978). In this report we will present autoradiographic evidence of auditory nerve projections after injection of different Iabeled precursors in the cochlea. Pretiminary accounts of some of these results have appeared elsewhere (CASERAY& JONES,1977; JONES& CASSEDAY, f 978).

Tritiated precursors of proteins and giycoproteins were injected into the cochlea in 6 cats. In all surgical procedures aseptic precautions were taken. The animals were anesthetized with sodium pentobarbitai, the bulia on one side was opened, and the labeled precursors were injected, via a SOili Hamilton syringe, through the round window. In order to retain the labeled material within the cochtea, the round window was covered first by gelfoam and then by a layer of dental cement, following the injection.

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D. K. JONESand J. H.

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TO obtain a high density of labeling in the cochlear nucleus, we attempted to ‘saturate’ cochlear nerve fibers with C3H]leucine. In the absence of evidence regarding the site or mode of incorporation of amino acids into cochlear fibers and because axonal transport may be as slow as 1 mm/day (COWAN, GOTTLXEB,HENDRICKSON,PRICE & WCIOLSEY, 1972; GRAYBIEL,1975), we assumed in the first few cases that injections of large volumes of the isotope with high levels of radioactivity (up to 1SmCi) would maximize the labeling of cochlear nerve fibers and terminals. At first, long survival times were used for the same reasons. As the experiment progressed we found labeling in the cochlear nucleus was virtually the same with lower radioactivity and shorter survival times. The following amounts of isotope and length of survival were used: 1.5 mCi, 7 days; 1.5 mCi, 5 days; 750&i. 2 days; 250 &i, 6 days. To investigate the possibility of transneuronal transport we used a mixture of C3H]proline and C3H]fucose, because of the demonstration of transneuronal’transport of this mixture in the visual system (e.g. WIESEL,HUBEL& LAM, 1974). One cat received l.OmCi of this mixture and was allowed to survive 14 days; the second animal received 3.0 mCi and survived 20 days. At the end of the survival periods the animals were killed by administration of a lethal dose of sodium pentobarbital and then perfused through the aorta with 0.9% saline, followed by 10% formalin. The brains were sectioned at 30pm intervals on a freezing microtome. Brains from 2 of the cats injected with C3H]leucine were sectioned in the parasagittal plane, and the remaining four brains were sectioned in the frontal plane. The sections were placed on glass slides and prepared for autoradiography by routine methods (COWANet al., 1972). The sections were coated with Kodak NTB-2 photographic emulsion and then exposed 5 weeks for the cases injected with C3H]leucine and 12 weeks for the cases injected with E3H]proline and C3H]fucose. The emulsion was developed in Kodak D-19 developer, and the sections were stained through the emuision with cresyl violet. To describe patterns of labeling in the cochlear nucleus. it was necessary first to analyze the cytoarchitecture of the cochlear nucleus. In this analysis we followed the descriptions of OSEN(1969) and of BRAWER,MOREST& KANE (1974), of cell types in the cat’s cochlear nucleus; our description of cytoar~hitectonic areas is derived from that of BRAWERet at. (1974). Then we examined the distribution of silver grains around cells in each of the cytoarchitectonic areas, using light microscopy with light-field and dark-field illumination. In all cases we examined the entire brain stem, with dark-field illumination, to determine whether there were silver grains over auditory pathways central to the cochlear nucleus. The location of grains in the brain stem was mapped with the aid of an X-Y recorder which was coupled to the stage of the microscope. Materials. The following radioactive substances were used: L-[4,5-“H(N)]-Leucine (specific activity: &6OCi/ mmolf; L-[6-3H]-Fucose (specific activity: 10-15 Ci/mmol): L-[2,3-3H]-Proline ~s~ci~~ activity: 2@-4O Ci/mmol); obtained from New England Nuclear.

C’ASSEDAY

FIG. I. Line drawings of frontal (a) and parasagittal (b) sections through the cochlear nucleus to indicate cytoarchitectonic subdivisions of the sections shown in the photomicrographs of Fig. 2. In (a) section 177 is most rostral; section 37 is most caudal. To relate the subditisions shown here to the su~ivisions of BRAWERrf ul. (1974). in the anteroventral cochlear nucleus our ‘AVa’ corresponds to their anterior subdivision, which they further subdivide into anterior, posterior and posterodorsal parts; our ‘AVp’ corresponds to their posterior subdivision, which they further subdivide into dorsal and ventral parts. In the posteroventral cochlear nucleus, our ‘PVa’ consists of their anterior, anterodorsal, lateral and ventral parts; our ‘PVp’ consists of their central (octopus cell area), dorsal and posterior parts. In the dorsal nucleus we have indicated the base of the fusiform cell layer by the dashed line. Abhreoiations:AVa, anteroventral nucleus, anterior subdivision; AVp, anteroventral nucleus, posterior subdivision; PVa, posteroventral nucleus, anterior subdivision; PVp, posteroventral nucleus, posterior subdivision; DCN. dorsal cochlear nucleus; g, granule cell layer; IAS, intermediate acoustic stria; DAS, dorsal acoustic stria.

nucleus, and the pattern of labeling was essentially the same in all of these cases. The density of labeling appeared to be similar in all but the animal in which the least activity of the isotope (250&i) was used. In this case the overall density of the label was less than in the other animals, but the pattern of labeling, to be described below, was the same. Only in the animals injected with [‘HJproline and [3H]fucose was there evidence of transport central to the cochlear nucleus. Transport to the cochlear nucleus after injection of

RESULTS In the animals injected with [3H]ieucine, labeling in auditory pathways was restricted to the cochlear

[“H]leucine The relation of the overall density of silver grains to cytoarchitectonic areas of the cochlear nucleus is

Auditory nerve projections

shown in Figs 1 and 2. The cytoarchite~tonic division illustrated in Fig. 1 is simifar to the scheme of BRAWERet al. (1974). However, the principal distinctions in our experimental material are related to broader cytoarchitectonic subdivisions than the smallest subdivisions used by BRAWERet al. (1974). For the purpose of this report it is only necessary to subdivide each major division of the cochlear nucleus into two regions: anterior and posterior subdivisions of the anteroventral nucleus (AVa, AVp); anterior and posterior subdivisions of the posteroventral cochlear nucleus (PVa, PVp); peripheral layers and deep polymorph layer of the dorsal cochlear nucleus (see caption to Fig. 1). Where it is relevant to our results, we will refer to the finer subdivisions of BRAWERet al. (1974). There are systematic variations in the density of the label between subdivisions, and some of these variations are apparent even in the low-power photomi~ro~aphs of Fig. 2. Labeling is densest around the root of the auditory nerve (see ‘AVp’, sections 97 and 137, Figs la and 2a) but decreases greatly in the most anterior part of the anteroventral cochlear nucleus (Figs 1 and 2). In frontal sections, the posteroventral nucleus appears very densely labeled in the area of convergence of descending fibers of the auditory nerve (see ‘PVp’ in Figs la and 2a). In parts of the anterior subdivision of the posteroventral nucleus (‘PVa’, Fig. 2a). labeling is not dense, and this appearance may be a consequence of the plane of the section which is perpendicular to descending branches of the auditory nerve fibers. In the parasagittal section, shown in Fig. 2(b), labeling is dense throughout the posteroventral nucleus; in this figure the plane of the section is nearly parallel to the descending branches of auditory nerve fibers. In the dorsal cochlear nucleus, the densest labeling is in the deep layers. Peripheral to the deep layers, the density of labehng decreases abruptly. We will show next that the silver grains are distributed i: such a way as to form unique patterns around specific types of cells, Anteroventral co&ear nucleus. Round and oval cells in the anterior subdivision of the anteroven~al nucleus are encircled by dense ag~egations of silver grains, as can be seen in the dark-field and light-field photomicrographs in Figs 3(a) and 4(a). This labeling pattern is most obvious in the anterior and dorsal parts of the anterior subdivision (‘AA’ and ‘APD’ of BRAWERet al., 1974), even though the overall density of label in these areas is light (cf. Fig. 2a, section 177). Most of the cells in these parts resemble the spherical cells described by OSEN (1969). In the posterior part of the anterior subdivision (‘AP’ of BRAWERet al., 1974) there appears to be greater variation in the density of grains adjacent to cell bodies than in the anterior parts. While the somata of many cells in the posterior part are silhouetted by many silver grains, other cells have few grains around them. In the posterior subdivision of the anteroventral

1301

nucleus, cells are interspersed among densely labeled fascicles of the auditory nerve (Fig. 3b). Yet these cell bodies are seldom surrounded by dense aggregations of silver grains. The two main types of cells in this subdivision appear to have different patterns of grains around their cell bodies. Globular cells (&EN, 1969) are often completely encircled by a thin band of silver grains. The density of grains around the somata of these cells is considerably less than around spherical cells, a difference which may reflect the differences in the innervation of these two types of cells, as we will discuss later. Other cells, which are ovoid in shape and many of which we could identify as multipolar cells, are not totally encapsulated by silver grains and have only a few clusters of silver grains adjacent to their somata. A distinctive pattern of label is seen around cells which are elongated in shape and which appear to be a type of multipolar cell, judging from the clumped appearance of their Nissl substance (OSEN, 1969). These cells are never totally surrounded by grains, but often silver grains appear in clusters at the poles of the soma, as in Figs 3(b) and 4(b). Posteroventral cochlear nucleus. In the posterior subdivision (‘PVp’ in Fig 1) of the posteroventral nucleus a striking pattern of labeling is seen around octopus cells. These cells are so heavily encrusted with silver grains that silhouettes are formed around the somata and proximal dendrites, as shown in Figs 3(c) and 4(d). Such dense labeling is not seen around the other types of cells found in this subdivision. In the anterior subdivision of the posteroventral nucleus (‘PVa’ in Fig. I), there appears to be an even distribution of silver grains in all extracellular spaces with little tendency for the grains to be clustered around ceil bodies (Fig. 3d). There is an obvious difference in the density of grains around cells in the anterior subdivision of the posteroventral nucleus and the density around spherical or octopus cells (see Figs 3 and 4). Elongated cells in the posteroventral nucleus have very few silver grains adjacent to their son-rata. When grains are present, they are found near the poles of the soma, a labeling pattern that is also seen around elongate cells in the anteroventral nucleus. The &ation of one such cell in the posteroventral nucleus, just across the border from the descending root of the auditory nerve, is indicated in Fig. 3(c). Dorsal cochlear nucleus. The distribution of silver grains seen in Fig. 2 reveals a projection that appears to be densest at the base of the fusiform cells. Figure 5 shows that the labeling of fusiform cells is confined to the region of the basal dendrites, and even here is very light when compared to spherical cells in the anteroventral cochlear nucleus, for example. Peripheral to these basal dendrites, i.e. in the remainder of the fusiform cell layer and in the molecular layer, labeling is not above the level of background, such as is seen in the contralateral cochlear nucleus. ln

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D. R. JONES and J. H.

CASSLDA~

the deepest part of the polymorph layer, the density of the label is somewhat less than in the region of the basal dendrites of the fusiform cells (Fig. 2). Occa-

this nucleus ipsi~terai to the injection; Iabel is found in the medial nucleus of the trapezoid body only contralateral to the injection. In one case (O-109), as

sionally a few silver grains can be seen adjacent to the somata of cells in the deep layer.

Fig. 7(a) shows, transport occurred only to the highfrequency regions of the superior olive, that is, to the medial limb of the lateral superior olive and to the ventral part of the medial superior olive (TSUCHITANI & BOUDREAU. 1966; GIGNAN. NORRIS & GUNAN, 1971). No label above the level of background activity was found in the periolivary cell groups, the dorsal or intermediate striae, the lateral lemniscus, or the inferior colliculus. In the ‘Discussion’ we will propose an explanation for the absence of label in some pathways from the cochlear nucleus.

Labeling in the cochlear nucleus and the superior olioary complex after injections qf [3H]proline and

[‘Hlfucose There are clear differences between the results just described and the results of the cases in which the cochleas were injected with a mixture of [3H]proline and [3H]fucose. First, there is extremeiy dense labeling in the cochlear nucleus (Figs 6a and 6b) after injections of C3H]proline and [3H]fucose. In most areas of the cochlear nucleus, silver grains completely cover the cell bodies (Figs 6c and d). The fact that many of these cells were sectioned through the central part, so that the nucleolus was visible, indicates that the isotope was present within these neurons, not just within terminals at their surfaces. Only in the following areas is there a low density of label: peripheral layers of the dorsal cochlear nucleus, granular cell layers, and a narrow strip of small cells interposed between the anteroventral border of the dorsal cochlear nucleus and the anteroventra~ cochlear nucleus. Figure 6b illustrates the contrast between the density of labeling in these areas and other parts of the cochlear nucleus. Those areas in which only light labeling is seen, in the cases injected with r3H]fucose, correspond to the areas in which no labeling is seen in the cases injected with C3H]leucine. Second, there is evidence of transport to the superior olivary complex only in the animafs injected with the mixture of [3H]proiine and [3H]fucose (Fig. 7). In both of these cases the labeling in the superior olive is not dense, but clear differences can be seen between superior olivary nuclei on each side. Label is found in the lateral superior olive only on the side ipsilateral to the injection; label is found in both medial superior olives but is confined to the half of

DISCUSSION

Two major points are raised by the results of this study. First, the results after injection of C3H]leucine into the cochlea reveal unique patterns of grains around specific types of cells in the cochlear nucleus; we will discusss the significance of this finding for projections of the auditory nerve to the cochlear nucleus. Second, the finding that labeled fibers are seen in the superior olivary complex alter injection of the mixture of C3H]proline and E3Hffucose has significance for the question of whether or not fibers of the auditory nerve project beyond the cochlear nucleus. Projections to the co&ear

nucleus

The most striking finding was the magnitude of the differences in the pattern and density of labeling around different types of cells. In the follo~ng, when we refer to silver grains around cell bodies, we mean to exclude reference to dendrites, which are not usually seen with Nissl stains. Of course, light-microscopic observations do not reveal the presence or absence of nerve terminals; however, comparison of our results with evidence from other studies suggests that the pattern and density of grains around cells

FIG. 2. Low-power, dark-field photomicrographs of autoradiographs of cochlear nucleus in frontal (a) and parasigattal (b) sections following injection of [‘Hlleucine into the cochlea. The lightest areas show the highest density of labeled fibers. In (a) section 177 is most rostral; section 37 is most caudal. These photomi~rographs have been trimmed to show the borders of the cochiear nucteus. FIG. 3. Dark-field photomicrographs of autoradiographs to show differences in labeling in different subdivisions of the cochlear nucleus. (a) Dense labeling which outlines spherical cells in the anterior subdivision of the anteroventral cochlear nucleus. (b) Labeling in the posterior subdivision of the anteroventral cochlear nucleus. The most densely labeled parts in this photomicrograph do not occur around cell bodies. The ‘n’ near the center of the photograph indicates the location of an elongated cell (see text). The ‘n’ in the lower right of the Figure marks the location of a globular cell. (c) Labeling in the octopus cell area in the posterior subdivision of the posteroventral cochlear nucleus. This Figure illustrates the manner in which sifver grains silhouette somata and proximal dendrites of octopus cells. An octopus cell located near the center of this photomicro~aph is shown in Fig. 4d. The unlabeled area at the upper left of 3c marks the border between the octopus cell area and the anterior subdivision of the posteroventral nucleus. The ‘n’ marks the location of an elongate cell in the anterior subdivision. (d) Labeling in the anterior subdivision of the posteroventral cochlear nucleus. This area appears lightly labeled, and there is a conspicuous lack of any tendency for silver grains to form aggregates around cells: the locations of several cells are marked (‘n’).

dorsal

anterior

b

posterior

lmm ventral FIG. 2. 1303

1304

FIG. 4. High power light-field photomicrographs to show difference in distribution of silver grains around neurons from various subdivisions of the ventral cochlear nucleus. (a) Two spherical cells in the anterior subdivision of the anteroventral nucleus; (b) an elongate cell in the posterior subdivision of the anteroventral nucleus, (c) a round cell in the anterior subdivision of the posteroventral nucleus; (d) the same octopus cell as shown at the upper center of Fig. 3~. Portions of this Figure have been published elsewhere (JONES& CASSEDAY, 1978) but are presented again here for comparison with Fig, 3.

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FIG. 5. (a) Dark-field photomicrograph of the superficial layers and the superficial part of the deep layers of the dorsal cochlear nucleus. A white line has been drawn to indicate the lateral surface of the nucleus. Labeling in the deep layers, to the right of the Figure, is markedly more intense than in superficial layers, approximately between the point of the arrow and the lateral surface. The arrow indicates the location of the fusiform cell shown in (b). (b) and (c) Silver grains near the basal dendrites (at right) of two fusiform cells. These cells are oriented with their long axes roughly parallel to the length of the arrow in (a).

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FIG. 6. Photomicrographs to show transport to the cochlear nucleus 14 days after injection of [‘HIproline and C3H]fucose in the cochlea. In (a) and (b) dark-field photomicrographs show labeling in frontal sections of the anterior (a) and the posterior (b) cochlear nucleus. Labeling is very dense in all areas of the cochlear nucleus except the granular layer and the molecular layer of the dorsal cochlear nucleus (b). In (c) and (d) light-field photomicrographs show dense accumulations of silver grains over cells in the anteroventral cochlear nucleus (c) and over an octopus cell in the posteroventral nucleus (d). Abbreviations: AV, anteroventral cochlear nucleus; Ce, cerebellum; DCN, dorsal cochlear nucleus; g, granular layer; PV, posteroventral cochlear nucleus; V, fifth nerve; VIII, eighth nerve: VN, vestibular nuclei. 1307

Auditory nerve projections

1309

FIG. 7. Drawings to illustrate the distribution of silver grains in sections of the medulla of two animals in which [3H]proline and [3H]fucose were injected in the cochlea. The survival periods were 20 days for the animal shown in (a) and 14 days for the animal shown in (b). In both cases the labeled structures in the superior olivary complex were the targets of the anteroventral cochlear nucleus. Abbreviations: AVa, anterior subdivision of the anteroventral cochlear nucleus; AVp, posterior subdivision of the anteroventral cochlear nucleus; BC, brachium conjunctivum; LSO, lateral superior olive; MLF, medial longitudinal fasciculus; MNTB, medial nucleus of the trapezoid body; MSO, medial superior olive; Py, pyramidal tract; V. fifth nerve; VII, seventh nerve; VIII, eighth nerve; VN, vestibular nuclei.

in the cochlear nucleus reflect the innervation of the somata of these cells by the auditory nerve. The two clearest examples for such comparisons are seen in the distribution of silver grains around spherical and octopus cells. The encapsulation of spherical cells by dense aggregations of silver grains clearly reflects the very dense innervation of these cells by fibers of the auditory nerve. The auditory nerve terminates on spherical cells in two types of endings, bulbs of Held and boutons (OSEN, 1970; LBRENTE DE N6, 19336, 1976; BRAWER& MOREST, 1975), so that a large part of the soma of each of these cells is covered with endings of the auditory nerve. Likewise, the picture of densely packed grains around the somata and proximal dendrites of octopus cells corresponds to other evidence of the innervation of these cells. Using electron-microscopic techniques, KANE (1973) estimated that up to 70% of the soma and proximal dendrites of the octopus cell are covered by endings of the auditory nerve (cf. also OSEN, 1970). KANE (1977) has recently supported this estimate with light-microscopic observations of projections to caudal parts of the cochlear nucleus, shown by autoradiography after injection of C3H]leutine into the cochlea.

Another observation that is probably also related to known patterns of innervation is the finding that the density of grains around globular cells, at the cell body, is almost always less than that around spherical cells. Globular cells are innervated by modified bulbs of Held which are smaller and less ramified than the bulbs found in more rostra1 parts of the anteroventral nucleus (RAM~N Y CAJAL, 1909; L~RENTE DE N6, 1933a,b; 1976; OSEN, 1970; HARREON & IRVING, 1965; TOLBERT& MOREST,1977). Since the presence of silver grains around the somata of some cells accurately reflects the pattern of innervation of these cells by the auditory nerve, the absence of grains around the somata of other cells may signify a different pattern of innervation, especially when the presence or absence of grains is related to cell types or architectonic areas. For example, the finding that very few silver grains are seen around the cell bodies of elongated cells suggests that the soma of this type of neuron receives few terminals from the auditory nerve. This conclusion does not exclude the possibility of innervation of dendrites of elongated cells. Indeed, clustering of grains in the vicinity of the proximal dendrites suggests this possibility.

ln the anterior subdivision of the posteroventral cochlear nucleus, the absence of dense clusters of silver grains around cell bodies suggests that cell bodies in this area are not densely innervated by auditory nerve fibers. Studies with Golgi or axonai degeneration techniques have not revealed bulbous endings but do show a dense plexus of auditory nerve fibers in the anterior part of the posteroventral cochlear nucleus (RAM~N Y CAJAL. 1909; LORENTEDE N6, 19336; OSEN, 1970). Taken together these findings raise the hypothesis that the principat termination of auditory nerve fibers in this area is upon dendrites rather than cell bodies. The pattern of labeled fibers and terminals in the dorsal cochlear nucleus is of special interest in view of differing reports concerning the termination of auditory nerve fibers here. There is agreement among most investigators that the dorsal cochlear nucleus receives a less dense projection than the ventral divisions. There is disagreement regarding the innervation of the laminae of the dorsal nucleus and regarding innervation of different cell types. The principal differences, which are based mainly on studies of axonal degeneration after cochlear damage. concern the following issues: whether or not the deep layers receive a dense projection from the auditory nerve (POWELL & COWAN, 1962; OSEN, 1970; COHEN rt al., 1972); whether or not the cefl bodies of fusiform cells are innervated by the auditory nerve (POWELL & COWAN, 1962; OSEN, 1970; COHEN er al.. 1972; KANE, 1974u.b; 1977): and whether or not the molecular layer receives a projection from the auditory nerve (OSEN, 1970; COHEN et al., 1972; KANE. 1974a,h). Our tindings support the hypothesis that the principal projection of the auditory nerve in the dorsal nucleus is to the basal dendrites of fusiform cells (cf. also WEXLER & GULLEY, 1978). Of course, the absence of silver grains near the apical dendrites and cell bodies of fusiform cells or in the molecular layer. in our experiments, can not be taken as proof that this external part of the dorsal nucleus receives no projection from the auditory nerve. Indeed projections from the auditory nerve to these layers has been reported in both light- and electron-microscopic studies of degeneration following cochlear ablation in the cat (COHEN rt al., 1972; KANE, 1974~). Our point is that the projection of the auditory nerve to the outer layers must be very slight, especially when compared to other sources of input. For example, in our studies of auditory pathways in the tree shrew, the densest innervation of the outer layers appears to arise from the anteroventral cochlear nucleus, while the auditory nerve projects to the deep layers (JONES & CASSEIIAY, 19793, as in the present observations on the cat. Projections

beyond

the cochlear

nucleus

Interest in the issue of whether or not auditory nerve fibers project beyond the cochlear nucleus has been renewed by a report showing labeling of auditory structures central to the cochlear nucleus after

injections of [3H]proline into the cochlea or into the’ ampulla of one semicircular canal of Macaque monkeys (CARPENTERet ui., 1978). Our observations arc relevant to this issue in that they suggest that labeling in the superior olivary complex depends on which labeled precursor is used. on length of survival or both. We will argue that our findings, as well as those of CARPENTERet (~1.(197Q are most consistent with the view that labeling central to the cochlear nucleus. after injection of labeled precursors in the cochlea. is the consequence of transneuronai transport. First, examination of the variables relevant to transneuronal transport support this interpretation. Experiments to investigate axonal transport from the oye have shown that the use of [“Hlproline and [“H]fucose provides the most clear-cut cvldence of transneuronal labeling (GKAFSTEIN,197 1; GKAFSTEIN & LAURENO. 1973; WEISELet ~1.. 1974). that [“H]leube transported transneuronally tine may also (HENDRICKSON, 1972; GRAFSTEIN& LAUREN<). 1973). and that long post-injection survival periods increase the density of transneuronal labeling (GRAFSTEIN & LAURENO, 1973). Little is known about transneuronal transport in auditory pathways, but it has been reported to occur in the avian auditory system after long survival periods but not after short survival periods, following injection of [3H]proline in the cochlea (PARKS & RUBEL. 1978). In the present experiments, as well as those of CARPENTER c’f (II. (19781, labefing occurred central to the cochlear nucleus only under conditions that would be considered optimal for transneuronal transport according to the evidence of studies cited above. Furthermore, when we used conditions that. by this same evidence must be CORsidered less than optimal for t~Ilsneurona1 transport. we failed to see labeling central to the cochlear nucleus. To be sure. we have not investigated, systematically, the relationship between survival time and choice of labeled precursor. For example, the survival time necessary for transneuronai transport after the injection of [3H]leucine remains unclear. Likewise, the survival period for maximum labeling of auditory pathways central to the cochlear nucleus, after injection of C3H]proline and [3H]fucose, is not established by the present study. However, the level of radioactivity injected does not, by itself. seem to determine the presence of label in the superior oiivary complex : in our experience label was found here only after injections of [3H]proline plus C3H]fucose, even though the total radioactivity injected in one of these cases (1.0 mCi) was less than the radioactivity of [‘Hlleutine injected in two other cases (I .5 mCi each). Our second point concerns the finding that labeling in the superior olivary complex was accompanied by dense accumulations of silver grains overlying cell bodies in the cochlear nucleus (Fig. 6) of cwzs injected with C3H]proline and C3H]fucose. The labeling of these cells, especially those sectioned near their center, leaves little question that the isotope gained access to cells in the cochlear nucleus and was avail-

Auditory nerve projections

able for transport from there to the superior oiivary complex. We conclude first, that labeling in the superior olivary complex, following injection of labeled precursors in the cochlea, is seen only under conditions most conducive to transneuronai transport and second, under these conditions, labeling over cells in the co&lear nucleus indicates that transneuronal transport did occur. It is worth examining the logical grounds on which CARPENTERet ai. (1978) rejected the possibility of transneuronal transport, because their argument is based on an observation that we also have made. In the present study, as well as that of CARPENTERet ai. (I978), not all pathways from the cochlear nucleus were labeled. In the report by CARPENTERet at. (1978), the labeled structures in the superior olivary complex, lateral lemniscus and inferior colliculus, were precisely those to which the anteroventral cochlear nucleus has been shown to project in the cat (WAR& 1966; 1969; 1972) and monkey (STROMINGER& STROMINGER,1971). But the intermediate acoustic stria and the pericentral region of the inferior colliculus were also labeled, while the dorsal acoustic stria was not. This difference, between the labeled areas and the pathways known to project from the cochlear nucleus, provided the basis for the proposal, by CARPENTERet al. (1978), that the cochlea projects beyond the cochlear nucleus. We will offer an explanation to account for most of these results by a process of selective transneuronai transport from the cochlear nucleus. The starting point for this explanation is the observation that in the visual system, not all pathways in the geniculocortical system are labeled by transneuronal transport after injection of tritiated precursors in the eye (WEISEL et al., 1974; WEBER, CASAGUNDE & HARTING,1977). These experiments suggest that there is no reason to expect that all pathways from the cochlear nuclei would be revealed by transneuronal transport. To account for selective transneuronal labeling via the cochlear nucleus requires the assumption that only some types of cells are responsible for transneuronal transport. ~R~s~~ & LAURFNO(1973) and GRAYLUEL (1975) have suggested that, while transneuronal transport is not confined to areas of synaptic contact, the transported isotope can be 1ocaIized in areas adjacent to the terminal or preterminal part of the axon. Therefore, it is not unreasonable to assume that those cells which have closest contact with auditory nerve fibers would also have closest access to the isotope and would be most likely to contribute to transneuronal transport. Except for the pericentral inferior colliculus, all labeled structures found by CARPENTERet al. (1978) in the monkey can be accounted for by assuming that transport occurred via two pathways from the cochlear nucleus: (I) from cells in the anteroventral nucleus and (2) from octopus cells in the posteroventral nucleus, which is

1311

the origin of the ~te~e~ate acoustic stria WARR, 1972). The present study shows that it is these neurons which are in the most intimate contact with labeled auditory nerve fibers following cochlear injections (see Fig. 4). There is no known projection from the cochlear nuclei to the pericentrai inferior colliculus, but it should be noted that virtually all published information on projections from the cochlear nucleus to the inferior colliculus has been obtained from studies of anterograde degeneration. It is not unlikely that additional pathways to the inferior colliculus may be revealed with the more sensitive autoradiographic technique. In the present study labeling central to the cochlear nucleus was much less extensive than in the study by CARPENTERet al. (1978). We found label only in some of the targets of the anteroventral cochlear nucleus, and in one case even the projections to the medial and lateral superior Olives were incomplete. Perhaps the survival periods used for these animals were not long enough to reveal all pathways through which transneuronal transport may occur in the cat. Implications of present study

Some, although not necessarily all, fibers in the auditory nerve degenerate after damage to hair cells (e.g. SWENDLIN, 1971; 1975; KIANG, LIBERMAN& LEVINE,1976), and the solution to several problems in auditory function may depend on assessing which of these fibers remain viable after co&ear damage. Anterograde transport methods, of course, reveal viable fibers only and, as the present work has shown, can provide a highly detailed survey of projections from the cochlea to the cochlear nucleus in normal animals. Thus, in the case of partial damage to the cochlea, it may now be possible to discover how the location of intact hair cells corresponds to patterns of projection of those auditory nerve fibers that maintain their connection with the cochlear nucleus. Such studies should not only reveal new details of topographic projections from the cochlea but, in addition, may reveal the source of puzzling discrepancies between the location of hair cell loss and behavioral measures of changes in absolute thresholds for pure tones {e.g. BREDBERG& ~u~~R-D~~~, 1975; BLMESLEE,HYNSON, HAMERNIK & HENDERSDN, 1978). When there has been complete destruction of hair cells, it should be possible to ask which, if any, fibers in the auditory nerve still project to the cochlear nucleus. The answer is of obvious practical significance for attempts to stimulate the auditory nerve electrically for prosthetic purposes after hair ceil loss. The answer may also be relevant to one of the most interesting unsolved problems in the study of hearing, the functional difference between inner and outer hair cells. SRXNDLIN(1971; 1975) has suggested that Type II cells in the spiral ganglion innervate outer hair cells. Part of his evidence for this connection is that Type II cells are resistant to degeneration after transection of the auditory nerve and that agerent endings

1312

D. R. JONESand J. H. CASEDAY

outer hair ceils remain intact after this transection. to degeneration after hair cell damage, it may be possible to use autoradiographic methods, after hair cell damage, to discover the central projections of Type II cells and in at

As Type II cells are also resistant

this way to infer ascending

connections

cochlear damage. If projections can be traced across at least one synapse, it may be possible to discover which connections to the superior olive and the inferior colliculus remain functionally connected to auditory nerve fibers after partial damage to the cochlea.

of outer hair

C&.

Likewise, the demons&ration of ~ransneuronal transport holds promise for finding pathways that remain viable in the central auditory system following

AcknowledgementsWe thank KATHRYNRAINEYand GAIL. BOYARSKY for assistance in this project. This research reported here was supported by Grant NS 12322 from the National Institutes of Health.

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