Hearing Research, 51 (1991) 11-28 0 1991 Elsevier Science Publishers B.V. (Biomedical

HEARES

11 Division)

0378-5955/91/$03.50

01481

Glycine immunoreactive

projections from the dorsal to the anteroventral cochlear nucleus

Richard L. Saint Marie, Christina G. Benson *, E.-Michael Ostapoff and D. Kent Morest Department

of Anatomy and Center for Neurological Sciences, Uniuersity of Connecticut Health Center, Farmington, (Received

2 May 1990; accepted

Connecticut,

U.S.A.

9 July 1990)

The aim of the present study was to investigate whether projections from the dorsal cochlear nucleus (DCN) to the anteroventral cochlear nucleus (AVCN) use either of two inhibitory transmitters, glycine or GABA. Retrograde HRP labeling of DCN-to-AVCN projection neurons was combined with postembedding immunocytochemistry in the DCN of guinea pigs. Following injections of HRP in the anterior or posterior divisions of AVCN, large numbers of neurons were labeled in the DCN. All of these were located in the deep layer, except for a few granule cells. Nearly all (96%) of the projection neurons were immunoreactive for glycine and most had dendritic and somatic morphologies corresponding to those of elongate neurons (so-called ‘corn’ cells); only a few resembled small stellate neurons. Few (3%) retrogradely labeled neurons were immunoreactive for GABA. The results suggest that projections from the deep DCN to the AVCN are formed primarily by glycinergic elongate neurons. These projections could have a substantial inhibitory influence on the output of neurons in the AVCN. Cochlear

nucleus;

Glycine;

GABA;

Immunocytochemistry;

Guinea

Introduction Glycine is an important inhibitory transmitter in the anteroventral cochlear nucleus (AVCN). Iontophoresis of glycine reduces spontaneous and tone-evoked activity in AVCN neurons, and this inhibition is mediated by strychnine-sensitive postsynaptic receptors (Caspary et al., 1979; Martin et al., 1982). Glycine levels and the density of glycine receptors are high in the cochlear nucleus, including AVCN (Godfrey et al., 1978, 1988; Frostholm and Rotter, 1986; Sanes at al., 1987; Araki et al., 1988; Glendenning and Baker, 1988) and mechanisms for the high-affinity uptake and calcium-dependent release of glycine have been demonstrated in AVCN (Staatz-Benson and

Correspondence to: Richard Saint Marie, Department of Anatomy, University of Connecticut Health Center, Farmington, CT 06030, U.S.A. * Dr. Benson has previously published under the name of C. Staatz-Benson.

pig; Auditory

pathways;

Horseradish

peroxidase

Potashner, 1987, 1988). Finally, glycinergic postsynaptic receptors and presynaptic endings are numerous along the somata and dendrites of AVCN neurons (Schwartz, 1983, 1985; Altschuler et al., 1986; Wenthold et al., 1987, 1988; Aoki et al., 1988; Araki et al., 1988). Several sources for glycinergic endings in AVCN have been proposed. These include intrinsic connections (Oertel, 1983; Wu and Oertel, 1984, 1986; Wenthold et al., 1987; Staatz-Benson and Potashner, 1988) commissural projections from the contralateral cochlear nucleus (Wenthold, 1987; Benson and Potashner, 1990), and descending projections from the ipsilateral and contralateral superior olivary complex (Adams and Wenthold, 1987; Godfrey et al., 1988; Ostapoff et al., 1988; Staatz-Benson and Potashner, 1988; Benson and Potashner, 1990). Accumulating evidence also suggests that neurons in the deep dorsal cochlear nucleus (DCN) may use glycine as a transmitter and provide inhibitory projections to the AVCN (Adams and Wenthold, 1987;

12

Wenthold et al., 1987; Wickesberg and Oertel, 1990). Neurons located in the deep layer of the DCN have axons that ramify in the three principal subdivisions of the cochlear nucleus. As a result they are ideally situated to influence the output of each of the principal cell types and, consequently, to influence monaural and binaural processing at all levels of the auditory brainstem. Projections from the dorsal to the ventral cochlear nucleus were first described by Lorente de No (1933) in Golgi material. He described the association tracts that connect the ventral and dorsal cochlear nuclei as mixed bundles with axons projecting in both directions. He later called these the ventrotubercular tracts (Lorente de No, 1981). More recent studies have shown that projections from DCN to the AVCN and posteroventral cochlear nucleus (PVCN) are substantial and tonotopically organized (Oliver, 1984; Feng and Vater, 1985; Snyder and Leake, 1988; Wickesberg and Oertel, 1988). Golgi impregnations and intracellular injections have demonstrated that neurons in the deep DCN have axons with local ramifications in the deep and fusiform cell layers and elaborate arborizations in the AVCN and PVCN (Lorente de No. 1933, 1981; Cant and Morest, 1978; Oertel and Wu, 1989). In the present study, we have localized inhibitory transmitters in neurons of the DCN that project to AVCN. using postembedding immunocytochemistry together with retrograde transport of HRP. Our previous studies of projections from the lateral superior olive to the inferior colliculus suggest that this is an accurate and reliable method for identifying projection neurons that may be glycinergic or GABAergic (Saint Marie et al., 1989).

oxidase (HRP) that was confined to the AVCN. Animals, pretreated with atropine sulfate (0.05 mg/kg, i.p.), were surgically anesthetized with sodium pentobarbital (Nembutal, Abbott: 32 mg/kg, i.p.) supplemented as necessary with diazepam (Valium, Hoffmann LaRoche: l-2 mg/kg, i.p.). For the HRP injections, a partial craniotomy was performed and the lateral aspect of the right cerebellum was removed by aspiration to expose the cochlear nucleus on that side. Once exposed, a single injection of HRP was made in the right AVCN with glass micropipets (20-40 pm tip diameters) attached to a calibrated air-pressure injection system (Picospritzer II, General Valve Corp.). To ensure that the micropipet did not enter the DCN, the AVCN was approached vertically by driving the micropipet down through the cerebellar peduncles, rostra1 to the DCN. HRP in sterile saline (20-40 nl) was injected as a 20% solution (w/v) in its unconjugated form (Sigma, Type VI) or as a 2% solution (w/v) when conjugated to wheat germ agglutinin (WGA) (Sigma). The composition, volume, and position of the injection sites are summarized in Table I along with the survival times for each of the cases. Following the injections, animals survived for 24 to 48 h. They were then re-anesthetized and sacrificed by vascular perfusion at 37°C. The perfusion consisted of a brief washout with 50 ml of 0.1 M phosphate buffer (pH 7.2j, followed by perfusion with 500-750 ml of phosphate buffered

TABLE

I

SUMMARY OF GUINEA PIGS

INJECTIONS

IN THE

AVCN

Survival time (h)

HRP (cone)

(nl)

GP1010489 GP1013189 GP2013189

AP. E PD. PV

48 48

20% 20%

20 30

PD

48

20%

GP1022789

PD. PV

24

2% (WGA)

40

GP2022789

PD, PV

24

2% (WGA)

35

Materials and Methods

OF

Volume

Injection site *

Case

Five adult albino guinea pigs of either sex (Buckberg, 450-550 g) were used in this study. This species was selected since it had previously been used for neurochemical and cytochemical studies of glycine uptake and release in the cochlear nucleus (Staatz-Benson and Potashner, 1987, 1988; Benson and Potashner, 1990). Each animal received an injection of horseradish per-

HRP

20

* Position of the core of the inJection site in AVCN: AP. posterior part of the anterior division: E, external granular layer: PD. dorsal part of the posterior division; PV. ventral part of the posterior division; WGA. wheat germ agglutininHRP complex.

13

(pH 7.2) 1% paraformaldehyde and 2.5% glutaraldehyde. Brains were stored overnight at 4°C in the aldehyde fixative and transversely sectioned the following day with a Vibratome (Sorval) at 60 pm. To visualize the HRP, sections were processed with tetramethylbenzidine (Mesulam, 1978) (Sigma) or metal-intensified diaminobenzidine tetrahydrochloride (Adams, 1981) (Sigma) as substrates. Sections processed with tetramethylber&dine were counterstained with neutral red and mounted on glass slides. Those sections processed with diaminobenzidine were either counterstained with cresyl violet and mounted on glass slides or postfixed with osmium tetroxide (1%) and flat-embedded in Epon (Polybed 812, Polysciences) for semi-thin sectioning. To label neurons innnunocytochemically, semithin plastic sections of the injected cochlear nuclei were cut at 1.5 pm. The plastic and the osmium were removed from the sections, as described elsewhere (Saint Marie et al., 1989). Sections were then hydrated and incubated overnight at room temperature with affinity-purified, polyclonal rabbit antisera to BSA-glutaraldehyde conjugates of glycine or GABA (Wenthold et al., 1986, 1987). The working dilutions for the antisera were 1 : 300 or 1: 500 for the anti-glycine and 1: 500 for the anti-GABA. Control sections, in which the primary antiserum was eliminated or replaced by non-immune rabbit serum or in which the primary antiserum was pre-adsorbed with antigen, showed no labeling above background, as described previously (Saint Marie et al., 1989). To identify DCN neurons that projected to AVCN, semi-thin sections of the DCN were initially examined unstained at 300 X . The positions and cell types of perikaryal profiles containing a nucleus and retrogradely transported HRP granules were noted in camera lucida drawings of the sections. To determine which of the retrogradely labeled neurons were immunoreactive for glycine or GABA, somatic profiles were then located in the immediately adjacent sections which had been immunostained with antibodies to glycine or GABA (after Saint Marie et al., 1989). In many cases retrogradely transported HRP granules were also visible in the immunoreacted sections. To determine the average diameter of retro-

gradely labeled neurons in semi-thin sections, labeled somatic profiles that contained a nucleolus were drawn at 800 x and digitized, as described previously (Saint Marie et al., 1989). Rt3SUItS

Glycine immunocytochemistry Many elements in the DCN were stained by the glycine antibody, and the use of semi-thin plastic sections and postembedding immunocytochemistry revealed sharp differences in the character of the staining of the different layers and cell types (Fig. 1). For example, the molecular layer contained the most intensely stained neurons in the DCN; these were the cartwheel and small stellate neurons. Cartwheel neurons were recognized by their prominent, unstained nucleolus and deeply invaginated nucleus (Fig. 2A). Nissl substance, which filled the nuclear invaginations and usually formed a narrow rim around the rest of the nucleus, stained lighter than the rest of the perikaryon. Small stellate neurons, on the other hand, were smaller than cartwheel neurons. They had indented nuclei, were usually horizontally elongated, and contained little perinuclear cytoplasm (Figs. 2A and C). Glycine immunoreactive [glycine( +)] neurons with similar morphology were also observed among the granule cells of the external granular layer of the ventral cochlear nucleus. We estimate that all cartwheel neurons in our material were glycine( +). It is not clear, however, if all small stellate neurons were glycine( + )_ Because of their small size, unstained examples could have been mistaken for granule cells, which were also unstained. Most of the non-somatic staining in the molecular layer could be attributed to the dendrites and dendritic spines of the cartwheel neurons. The resolution of the present method was such that stained synaptic spines could routinely be observed on the dendrites of cartwheel neurons (Fig. 2B). Moderate numbers of glycine( +) axons and boutons (‘puncta’) occurred in the molecular layer and some of these contacted the somata and dendritic shafts of the cartwheel and small stellate neurons (Figs. 2A-C). Other characteristic differences were that glycine( + ) axons and boutons were much more abundant in the deeper layers of the DCN than in

ML

FL

PL

-

AS

15

the molecular layer. Deep to the molecular layer, a broad band of cells, composed largely of unstained fusiform and granule cells, defined the fusiform cell layer (Fig. 1). Fusiform cells were typically contacted by many glycine( + ) boutons, and these often occurred in densely packed clusters along the somatic and dendritic surfaces (Fig. 2C). Many fewer glycine(+) boutons contacted granule cell somata (Fig. 2C). Deeper still, the polymorphic (or deep) cell layer contained many glycine( + ) neurons (Fig. 1). The dorsal and intermediate acoustic striae are relatively cell sparse and few of the neurons there were glycine( +). Some thick glycine( +) axons (3-6 pm diam.) were evident in the acoustic striae, but the great majority of strial axons were unstained. The deepest third of the polymorphic cell layer contained the only other thick glycine( +) axons in the DCN. Finer glycine( + ) axons, however, were evident throughout the DCN. These were especially conspicuous in the superficial two-thirds of the polymorphic cell layer, where many axons and their swellings resembled the axonal nests previously described in Golgi and reduced silver preparations (Cohen et al., 1972; Kane, 1974). The quality of the immunocytochemistry in semi-thin sections was sufficiently good that many previously described cell types could be identified in the polymorphic cell layer of the DCN, and some of these were glycine( +). By far the largest population were small neurons which had somatic profiles of 12-24 pm in average diameter and were glycine( + )_ Most of these small glycine( + ) profiles were elongated, with their principal dendrites directed apically toward the fusiform cell layer or basally toward the acoustic striae (Fig. 1, E). Other glycine( +) profiles were elongated parallel to the surface and layers of the DCN (Fig. 1, H), especially in the deeper regions of the polymorphic cell layer. Together such profiles appear to correspond to the small elongate neurons

of Brawer et al. (1974), which were referred to as ‘corn’ cells by Lorente de No (1933). Other small glycine( +) profiles were round or polygonal in appearance (Fig. 1, R). It was not clear, however, if these latter represented transversely cut elongate neurons or a separate population of small glycine( + ) neurons. For example, small stellate neurons have also been reported in the polymorphic cell layer (Brawer et al., 1974). There were also a few large glycine( + ) somatic profiles (up to 28 pm average diameter) in the deep DCN, which appeared to be large elongate neurons. We estimate that between 90% and 95% of the small-tolarge neurons (i.e., non-granule and non-giant cell types) in the deep DCN were glycine immunoreactive. In general, elongate neurons (large and small) in the deep DCN stained more lightly with the glycine antiserum than did cartwheel and small stellate neurons in the molecular layer (Fig. 1). Their perikarya were uniformly stained and, in most cases, their nuclei were stained only slightly darker than their perikarya. As with the cartwheel and small stellate neurons of the superficial layers, their nucleoli did not stain. Examples of glycine (+) elongate neurons are presented in Figs. 2D and 3A. The neuron in Fig. 2D has its dendrite oriented parallel to the layers of the DCN, whereas the one in Fig. 3A, which is one of the largest elongate neurons that we encountered in the deep DCN, is oriented perpendicular to the layers of the DCN. Usually, only a few glycine( +) boutons could be found along the somata of elongate neurons, but many glycine( +) swellings gathered around their dendrites. In a number of cases, the glycine( +) dendrites of elongate neurons could be traced into axonal nests, which themselves contained glycine( +) boutons and many fine glycine( +) axons (Fig. 2D). This feature is also typical of small elongate neurons in Golgi impregnated material (Kane, 1974: Fig. 8).

Fig. 1. Anti-glycine stained plastic section (1.5 pm) of the guinea pig DCN showing the differential distribution of stained neurons among the three layers; molecular layer (ML), fusiform cell layer (FL) and polymorphic cell layer (PL). Most of the neurons in the ML, i.e., cartwheel (C) and small stellate (S) neurons, are darkly stained, whereas in the FL most neurons are unstained, including fusiform cells (F) and granule cells (under g). In the PL most of the neurons have vertically elongated somata (E) and are glycine( +). Other glycine( +) neurons in the PL have horizontally elongated (H) or round (R) somata. A few unstained giant (G) neurons are also apparent in the PL. AS = acoustic striae. Scale bar = 200 pm.

16

17

Only a very few of the glycine( + ) neurons in the polymorphic layer of the DCN were very large (> 35 pm average diameter), and some of these may represent a subcategory of the giant neurons described previously (Osen, 1969; Brawer et al., 1974; Kane et al., 1981; Moore, 1986). Except for their size, these cells were similar to the smaller glycine( + ) neurons in the deep DCN in their staining characteristics and in the low density of their glycine( + ) perisomatic boutons (Fig. 3B). Non-reactive [glycine(-)] neurons were also common in the polymorphic cell layer of the DCN. For example, unstained granule cells were found throughout this layer, and many of the small cells in and near the acoustic striae were unstained. Most noteworthy were the many unstained giant neurons which were common in the deeper half of the polymorphic cell layer in rostra1 sections of the DCN. Aside from the few glycine( + ) giant cells described above, the great majority (> 90%) of giant cells in the deep DCN were glycine(-). Their somata and dendrites were contacted by many glycine( + ) boutons and, in some cases, were nearly completely covered with these contacts (Fig. 3C).

GABA Zmmunocytochemistry Staining with the GABA antiserum was similar to that reported by others (Thompson et al., 1985; Peyret et al., 1986; Wenthold et al., 1986). GABA immunoreactive [GABA( + )] axons and boutons were present throughout the DCN, but were most abundant in the molecular and fusiform cell layers. In the superficial layers, small stellate neurons were moderately-to-darkly stained, while cartwheel neurons were moderately stained. No stained fusiform or granule cells were observed. GABA( + )

TABLE II GLYCINE AND GABA IMMUNOREACTIVITY DCN OF GUINEA PIGS Cell type

Immunoreactivity a

Pericellular boutons b

Glycine

GABA

Glycine( + )

GABA( + )

+ ++

** *

Superficial layers: ++ Cartwheel Small stellate + + Fusiform 0 Granule 0 Polymorphic cell layer: + Elongate Giant

lglycine(-)I

o

0

***

0

*

*** * ** *

0

*

**

0

****

*

0 0

* *

* *

Giant [glycine( + Granule

IN THE

)I + 0

a Symbols represent the relative intensity of the immunoreactivity for the various cell types: + = stained, + + = darkly stained, 0 = unstained. b Symbols represent the estimated proportion of somatic and proximal dendritic surface in contact with stained boutons: * = 50%.

boutons were evident along the somata of all neuronal types in the superficial layers, but were less numerous on the somata and proximal dendrites of fusiform cells than glycine( +) boutons. Cartwheel somata and dendrites, on the other hand, were contacted by many more GABA( + ) boutons than glycine( + ) ones. Table II summarizes the immunoreactivity of the principal cell types of the DCN and the prevalence of immunoreactive boutons along their somata and proximal dendrites.

Fig. 2. Examples of glycine( +) and glycine(-) neurons in plastic sections of the DCN. A: The perikaryon and nucleus of a cartwheel neuron are darkly stained. The nucleus contains an unstained nucleolus and several deep invaginations. Nissl substance, which fills the nuclear invaginations and forms a narrow ring around the nuclear envelope, is lightly stained. A prominent cap of lightly stained Nissl substance is apparent adjacent to the nuclear invaginations. The small darkly stained cell to the left may be a small stellate neuron. B: The secondary and tertiary dendrites of a cartwheel neuron contain many glycine( + ) dendritic spines and a few examples are indicated (arrowheads). C: Fusiform cells (F) are glycine(-); their somata and dendrites are contacted by many glycine(+) boutons. A compact nest of glycine(+) boutons (arrowheads) appears to surround the emerging basal dendrite of one of these fusiform cells. Granule cell somata (g) also are glycine(-) and occasionally contacted by glycine( +) boutons. The small glycine(+) cell in the upper right-hand comer is a small stellate neuron. D: Glycine( + ) elongate neuron in the deep DCN has a dendrite that projects into a nest of glycine( +) axons and boutons. The neighboring gIycine( + ) dendrite to the left may be from another elongate neuron and also is contacted by a cluster of glycine( + ) boutons. Scale bar = 25 pm.

18

19

In the deep layers, only a handful of small neurons stained with the GABA antiserum (Fig. 3D), but these were too few to allow a categorical identification. Some resembled elongate neurons, others may have been small stellate neurons. Few GABA( +) perisomatic contacts were found on giant neurons or elongate neurons (Table II). The difference in the number of glycine( + ) and GABA( + ) contacts on the somata of giant neurons was quite dramatic (compare Figs. 3C and 3D). Many fewer axons and boutons stained in the deep DCN with the GABA antiserum compared with the glycine antiserum. Consequently, it was not clear if GABA( + ) axons contributed to the axonal nests of the deep DCN. Retrograde transport Injections of HRP into the AVCN retrogradely filled neurons bilaterally in the inferior colliculi, superior olivary complex, and cochlear nuclei. Described below are the neurons labeled from the ipsilateral DCN. Details of retrogradely labeled neurons in the superior olivary complex, inferior colliculi, and contralateral cochlear nucleus will be presented elsewhere. In each case, the injection site was confined to the AVCN, except possibly for some spread into the inferior cerebellar peduncle. A more detailed description of the injec- . tion sites is presented in the following section. In the DCN, neurons that retrogradely transported HRP from the ipsilateral AVCN were found almost exclusively in the polymorphic cell layer. A few small cells sometimes labeled in the acoustic striae. In the molecular and fusiform cell layers, no neurons were labeled except for a few granule cells in one case in which the injection site included the external granular layer (Fig. 8, inset). In that case a few parallel fibers with their typical hook-like appendages also were labeled in the molecular layer. These fibers may have been

damaged and filled by the injection pipet penetrating the external granular layer. Retrogradely labeled neurons in the deep DCN were predominantly small with elongated, multipolar somata (Fig. 4). In most cases, their dendritic fields were elongated perpendicularly or obliquely relative to the surface and layers of the DCN (Cells 2-4, 6, 8-lo), but a small proportion had dendritic fields oriented approximately parallel to the layers (Cell 5). A very few may have been small stellate neurons (Cell 7). Although smaller than typical giant neurons, one large retrogradely labeled cell (Cell 1) resembled a vertical giant neuron with respect to its shield-shaped cell body and ascending dendrites (compare Brawer et al., 1974: Fig. 20). Otherwise, there was no labeling of giant or granule cells in the DCN. Average somatic diameter was calculated for retrogradely labeled neurons in the deep DCN from somatic profiles in semi-thin sections (see Methods). The range and distribution of cell sizes are presented in Fig. 5 for all of the cases that received injections in AVCN. 94% of the 169 neurons labeled in the 5 cases were small (< 24 pm average diameter): the population had a mean average diameter of 18.4 pm (k3.2 SD). The average size of the population projecting to the anterior (mean = 19.1 pm average diameter f 2.9 SD) and posterior (mean = 18.2 pm average diameter f 3.2 SD) divisions of the AVCN was similar. The remaining neurons labeled from injections of the AVCN were large (up to 28 pm average diameter), but these may represent the upper end of a continuum of sizes rather than a distinct population. Combined retrograde transport and immunocytochemistry Postembedding immunocytochemistry of semithin plastic sections was used to determine which

Fig. 3. Immunoreactivity of neurons in plastic sections of the deep DCN. A and B: Large elongate neuron in the deep DCN (A) and a horizontahy elongated giant neuron near the acoustic stria (B) are both stained with the glycine antiserum. In both neurons the nucleus is stained more darkly than the perikaryon, while the nucleolus is unstained. The adjacent neuropil in both examples contains many darkly stained axons and boutons, but few of these contact the stained somata. C and D. The cell body and proximal dendrites of a giant neuron appear in two adjacent 1.5 pm sections stained for glycine or GABA, resp. In C the cell body and dendrites are contacted by many glycine(+) boutons. In D many fewer GABA( +) boutons contact the cell body and dendrites. A small GABA( +) neuron stained lightly with the anti-glycine in the adjacent section C. Scale bar = 25 urn.

20

of the retrogradely labeled neurons were glycinergic and which were GABAergic. An example of a neuron doubly labeled with retrograde HRP and the glycine antiserum is presented in Fig. 6. Nearly all of the DCN neurons that labeled retrogradely from the AVCN were glycine( +) (96% of 296 neurons examined). A very few (3%) were

GABA( + ) and some of these were also glycine( + ). Only 3% of the retrogradely labeled neurons did not stain with either antiserum. In four of the five cases, injections of HRP were placed in the posterior division of the AVCN (Table I). Three of these were large injections that included a large part of the dorsal and ventral

Fig. 4. Camera lucida drawings of neurons in DCN retrogradely labeled from injections of HRP in AVCN. These neurons were from sections treated with tetramethylbenzidine as the substrate for HRP. The locations of the neurons are noted in drawings of the tissue sections (Inset) and their orientations and proportions are the same as in the indicated tissue sections. All of the neurons are located in the polymorphic cell layer (PL). Most (2-4, 6, 8-10) are elongated with apical dendrites extending toward the fusiform cell layer (FL) and basal dendrites extending toward the acoustic striae (AS). Others (5, 7) have dendrites parallel to the AS. Although smaller, one large neuron (1) has dendrites that resemble those of a vertical giant neuron (see Brawer et al.. 1974, Fig. 20). E = external granular layer. Case No. GP1010489. Scale bar = 50 pm.

21 rn

li

60,

MEAN

10.4k3.2

N=169

11

13

15

17

AVERAGE

19

21

23

25

27

29

DIAMETER

Fig. 5. Distribution of average diameters for neurons in the deep DCN that transported HRP from injections in AVCN. Diameters were measured in semi-thin sections from somatic profiles that contained a nucleolus and HRP granules. Mean = mean average diameter + SD.

parts of the posterior division. The fourth was a smaller injection which was confined to the dorsal part of the posterior division. The latter case, and one other, produced small numbers of retrogradely labeled neurons in the DCN. Nevertheless, the proportion, types, and immunoreactivity of the projection neurons were similar in all four of the cases with injections in the posterior division of AVCN. Almost all of the retrogradely labeled neurons were glycine( + ). The range for the four cases was 95-1001. Only 2% of the retrogradely labeled neurons were GABA( + ) and each GABA( + ) neuron also stained with the glycine antiserum. Fig. 7 illustrates the types, distribution, and immunoreactivity of the retrogradely labeled DCN neurons for a typical case that received a large injection of HRP in the posterior division of AVCN. The injection site is illustrated as a dark ‘core’ of intense labeling that extended from the ventral part (PV) of the posterior division, caudally, to the dorsal part (PD) of the posterior division, rostrally. The core is surrounded by a ‘halo’ (hatched) of more diffuse staining. Caudally the halo was confined to the posterior division of the AVCN, but rostrally it extended into the posterior part (AP) of the anterior division. All of the retrogradely labeled neurons were located in the polymorphic cell layer, and these neurons extended across most of the transverse axis of the DCN. The overwhelming majority of the retrogradely labeled cells were glycine( + ). One projec-

tion neuron in these sections was both glycine( + ) and GABA( + ). Only a few retrogradely labeled cells were not immunoreactive with either antiserum. One case in this study had an injection of HRP that was restricted to the anterior division of AVCN (Fig. 8). The injection site was located laterally in the posterior part (AP) of the anterior division and included part of the external granular layer (E). The injection in this case was small and, consequently, a correspondingly restricted projection from the DCN was retrogradely labeled. Fig. 8 illustrates that projections to the posterior part of the anterior division are similar to those described for the posterior division. Nearly all (93%) of the projection to the anterior division was glycine( + ). Approximately 5% of the retrogradely labeled neurons were GABA( + ) but, unlike those projecting to the posterior division, these did not also stain with the glycine antiserum.

Fig. 6. Neuron in the deep DCN doubly labeled with the glycine antiserum and with retrogradely transported HRP. This cell was lightly stained with the anti-glycine so that the HRP granules (arrows) would not be obscured. The dendrite of this neuron is oriented toward the fusiform cell layer. Scale bar = 25 pm.

22

.* f t

l*

.*

3

l

.

GLY+ cell A GLY+, GABA+ o GLY’, GABA’

l

cell cell

i

E

23

Discussion was immunocytochemistry Postembedding combined in the present study with retrograde transport of HRP to identify and characterize neurons in the DCN that project to the AVCN and may be inhibitory, i.e., glycinergic or GABAergic. The principal findings were that: 1) DCN neurons projecting to the AVCN reside almost exclusively in the polymorphic cell layer of the DCN; 2) DCN-to-AVCN projection neurons are, for the most part, small-to-large elongate neurons; 3) nearly all of the DCN-to-AVCN projection neurons were glycine immunoreactive; 4) very few of these projection neurons were GABA immunoreactive; and 5) DCN neurons that projected to the posterior part of the anterior division of the AVCN were similar in morphology and glycine immunoreactivity to those that projected to the posterior division of AVCN. General immunocytochemical observations As reported previously (Saint Marie et al., 1989), postembedding immunocytochemistry for glycine and GABA in semi-thin Epon sections produces consistently low background staining and good discrimination between immunoreactive and non-immunoreactive cell profiles. Resolution is improved by the thinness of the sections to the point that stained dendritic spines could be observed routinely on the dendrites of cartwheel neurons in the DCN in the present study. The present observations on the immunoreactivity of cartwheel and small stellate neurons in the superficial layers confirm those of earlier reports (Thompson et al., 1985; Peyret et al., 1986, 1987; Wenthold et al., 1986, 1987; Aoki et al., 1988). We estimate that the great majority, if not all, of the cartwheel and small stellate neurons in the DCN contain both GABA and glycine im-

munoreactivities. Moreover, these cell types, which have locally ramifying axons (Wouterlood and Mugnaini, 1984; Wouterlood et al., 1984; Kane, 1974), probably contribute to the many GABA and glycine immunoreactive boutons observed on cartwheel and fusiform somata and dendrites in the superficial layers of the DCN. The overwhelming majority of the giant cells encountered in the present study (i.e., those with somata > 35 pm) did not stain with either antiserum. Only a few giant neurons in the deep DCN were glycine( +) (see also Wenthold et al., 1987). These two cytochemically distinct types of giant neurons may have structural differences as well. For example, those giant cells which were glycine( + ) had few glycine( + ) perisomatic boutons, while those which were glycine(-) were virtually covered by glycine( + ) perisomatic boutons. Kane et al. (1981) described types of large and giant cells in the deep DCN, some of which had few axosomatic synapses, whereas others were nearly covered with such synapses. However, it could not be determined with semi-thin sections which of the previously described morphological types (e.g., Brawer et al., 1974; Kane et al., 1981; Moore, 1986) correspond to the glycine( +) and glycine(-) giant cells.

Morphological characterization of DCN-to-A VCN projection neurons The most numerous neuronal type in the deep DCN (except for granule cells) is a small neuron, which was first described in the cat and referred to as the corn cell (Lorente de No, 1933) or small elongate neuron (Brawer et al., 1974). These neurons typically have elongated dendritic fields which extend toward the fusiform cell layer and acoustic striae. Elongate neurons in the deep DCN have also been observed in Golgi material of mouse

Fig. 7. Plot showing glycine immunoreactivity of DCN neurons which were retrogradely labeled after injection of HRP in the posterior division of AVCN. All retrogradely labeled neurons are located in deep DCN and are distributed nearly evenly across the dorsomedial-to-ventrolateral and rostra-to-caudal extents of the deep DCN. Nearly all retrogradely labeled neurons are glycine( +). Inset (lower right) illustrates the darkly stained core (solid) and more lightly stained halo (hatched) of the injection site. The injection is centered in the posterior division of AVCN including its dorsal (PD) and ventral (Pv) parts. Rostrally the halo extends slightly into the anterior division of the AVCN, including parts of the anterior (AA) and posterior (AP) parts. The injection site did not extend into the external granular layer (E), PVCN, or DCN. Section numbers (when multiplied by the section thickness of 60 pm) indicate distance from the caudal pole of DCN. AS = acoustic striae. Orientation bars (upper left): D = dorsal; R = rostra]; L = lateral. Case No. GP2022789. Scale bar = 1 mm (3.6 mm for the inset).

cell A GABA+ Cell o GLY’; GABA’ l

GLY+

cell

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(Browner and Baruch, 1982; Webster and Trune, 1982), rabbit (Disterhoft et al., 1980), and guinea pig (Moore, 1986). Elongate neurons project locally to the deep and fusiform cell layers (Lorente de No, 1933, 1981) and distally to the AVCN and PVCN via the ventrotubercular tracts of Lorente de No (1933, 1981). Cant and Morest (1978) suggested that the axonal projections of these neurons could form elaborate pericellular nests in the AVCN of cats. Oertel and Wu (1989) showed with intracellular injections that individual elongate neurons can have axonal arborizations in all three subdivisions of the cochlear nucleus (AVCN, PVCN, and DCN). They refer to these elongate neurons as tuberculoventral neurons, based on the destination of their axons. The great majority of neurons labeled with HRP in the present study appear to be elongate neurons. Nearly all were located in the deep DCN and labeled neurons were found throughout this region (see also Wickesberg and Oertel, 1988). Most had elongated cell bodies and, when dendrites were visible, they conformed to the descriptions of elongate neurons given by Brawer et al. (1974) in cats and ‘fan’ cells given by Moore (1986) in guinea pigs. As described from Golgi impregnated material (Cohen et al., 1972; Kane, 1974) the dendrites of elongate neurons are sometimes associated with axonal nests. The previous studies indicate that cochlear nerve fibers contribute to these axonal nests. The present results suggest that glycine( + ) axons also contribute. Several observations suggest that tuberculoventral projections originate predominantly from a single cell type. These are the uniform distribution of cell size (Fig. 5) the location in the deep DCN of nearly all DCN-to-AVCN projection neurons, the moderate immunoreactivity to the glycine but not the GABA antiserum, and the

relatively low numbers of glycine( +) and GABA( + ) boutons on the somata of tuberculoventral neurons. Although most tuberculoventral neurons appear to be elongate neurons, we cannot rule out the possibility that some small stellate neurons in the deep DCN also may be glycine( + ) and project to the AVCN. Also, a small number of retrogradely labeled neurons were GABA( + ), were immunoreactive for both glycine and GABA, or were immunoreactive for neither. Some of these may represent separate cell types. While there is evidence that some superficial granule cells project to the external granular layer of the AVCN, we found no evidence to suggest that fusiform or giant cells project to AVCN ipsilaterally. The role of inhibitory projections from the DCN to A VCN Several lines of evidence, including the present study, suggest that glycine may be the transmitter used by neurons projecting from the DCN to AVCN. The deep layer of the DCN in guinea pigs is filled with glycine immunoreactive neurons (Wenthold et al., 1987) and brief reports indicate that the same is true in cats (Adams and Wenthold, 1987) rats (Osen et al., 1987) gerbils (Schwartz et al., 1987), and mice (Wickesberg et al., 1990). Moreover, many of these glycine( + ) neurons have projections to AVCN in guinea pigs (present study) and in cats (Adams and Wenthold, 1987). Stimulation of the DCN produces trains of monosynaptic IPSPs in bushy and stellate neurons in the AVCN in slice preparations of mouse cochlear nucleus (Wickesberg and Oertel, 1990). These monosynaptic IPSP’s are blocked by strychnine, an antagonist of the glycine receptor, suggesting that this inhibition is mediated by glycine. Cytochemical studies suggest that the somata of bushy and stellate cells in AVCN are contacted

Fig. 8. Plot showing glycine immunoreactivity of neurons in the deep DCN which were retrogradely labeled after injection of HRP in the anterior division of AVCN. Retrogradely labeled neurons are located mostly in the middle region of the deep DCN, and nearly all are glycine( +). Inset (lower right) illustrates the darkly stained core (solid) and more lightly stained halo (hatched) of the injection site. The injection is centered laterally in the anterior division of AVCN, including its posterior part (AP), and extends into the external granular layer (E). Ventrally the halo extends slightly into the dorsal part (PD) of the posterior division. Caudally the injection site extends as far as, but does not include, the anterior part (A) of PVCN. A few retrogradely labeled granule cells appeared in the ML and FL (not plotted here), and these probably resulted from the injection of E. Section numbers (when multiplied by the section thickness of 60 pm) indicate distance from the caudal pole of DCN. AS = acoustic striae. Orientation bars (upper left): D = dorsal; R = rostral; L = lateral. Case No. GP1010489. Scale bar = 1 mm (3.6 mm for the inset).

26

by mauy glycinergic ladings (Schwartz, 1983, 1985; Altschuler et al., 1986; Wenthold et al., 1987, 1988; Aoki et al., 1988; Saint Marie, unpublished observations). The latter observations are consistent with Golgi findings (Cant and Morest, 1978; Lorente de No, 1981) which indicate that axons from the DCN form extensive synaptic nests around neuronaI somata in the AVCN, e.g., type IV and possibly type V axons of Cant and Merest (1978). Other studies show that synaptic uptake and release of glycine in the AVCN are reduced only by half after knife cuts which Iesioned the centrifugal projections to the rostra1 coehlear nucleus (Staatz-Benson and Potashner, 1988). What aetivity remained after these lesions was attributed to intrinsic glycinergic connections, including projections from DCN. The content of glycine in the latter projections, however, could not be confirmed by lesions thou~t to isoIate AVCN from DCN (Godfrey et al., 1988). It is not yet clear how inhibitory inputs from DCN neurons might affect auditory signal processing in the AVCN, but they could explain some of the response characteristics of AVCN neurons. Neurons in the deep DCN are monosynaptically excited by stimulation of the co&ear nerve (Oertel and Wu, I989), and some of these probably belong to the type II units which predominate in the deep layer of the DCN (Young and Brownell, 1976; Voigt and Young, 1980; Young, 1980; Young and Voigt, 1982). As would be expected of tuberculoventral projection neurons, type II units can sometimes be, antidromitally activated by electrical stimulation of AVCN. Type II units typicalfy have Iow spontaneous activities, respond vigorously to best-frequency tones, and respond poorly to broadband noises. In~bito~ inputs with these response characteristics could explain noise/to.ne differences in the response areas of certain “ phase-locked” neurons in AVCN (Caspary et al,, 1988). Also, since the pathway from DCN to the ventral cochlear nucleus is short, inhibitory projections from type II, tuberculoventral neurons may explain certain rapid inhibitory phenomena observed in the AVCN (e.g., transient choppers - Bl~~kbu~ and Sachs, 1989; Feng, 1989) and PVCN (e.g., onset choppers Smith and Rhode, 1989).

I~bition of bushy and stellate neurons in AVCN by tuberculoventral projections could influence signal processing at many levels of the auditory pathway. AVON neurons are a critical first link in several monaural and binaural pathways which ascend ultimately to the auditory midbrain, thalamus, and cortex. Spherical and globular bushy cells, for example, provide necessary inputs to b~~au~aI pathways in the superior olivary complex. These pathways carry important information about interaural time and level differences to the midbrain. Stellate neurons, on the other hand, may contribute monaural inputs directly to the auditory midbrain Tuberculoventral neurons could also have widespread in~bitory in~uen~es on other areas of the cochlear nucleus and their respective outputs. Golgi impregnation (Lorente de No, 1933, 1981) and intracellular injection studies (Oertel and Wu, 1989) have shown that the axons of elongate neurons can also form local plexuses in the deep and fusiform cell layers of DCN and collateral plexuses in the PVCN. Projections to the FVCN may contribute to the many ~lycinergi~ perisomati~ and peridendritic endings found on octopus and stellate neurons in this region (Altschuler et al., 1986; Wenthold et al., 1987, 1988; Saint Marie, unpublished ob~e~ations~~ Local connations, on the other hand, may contribute to the many glycinergic boutons that we find along the somata and dendrites of f~siform cells, elongate neurons, and giant cells. For example, lateral in~b~to~y interactions between neighboring elongate neurons could explain their poor responses to broadband noise. Young and his colleagues have proposed that elongate neurons (type II units) are responsible for the inhibitory regions found in the response maps of fusiform cells (type IV units) (Young and Brownell, 1976; Voigt and Young, 1980; Young and Voigt, 1982; Young et al., 1988). The somata and basal dendrites of fusiform cells are in the local terminal fields of the elongate neurons (Lorente de Nlii, 1933. 1981). Moreover, Caspary et al. (1987) have shown that the non-monotonic rate intensity functions of fusiform cells are strychnine sensitive and probably the result of gIy~iner~~ inputs. Fusiform and giant Negroes in the DCN, as well as octopus and stellate newrons in PVCN, represent the principal monaural inputs

27

from the caudal cochlear nucleus to the auditory midbrain (inferior colliculus and nuclei of the lateral lemniscus) that could be inhibited by projections from elongate neurons. Acknowledgements The authors are especially grateful to Dr. Steven J. Potashner for his support of these studies and for his critical review of the manuscript. We would also like to thank Dr. Duck 0. Rim for his helpful comments on the manuscript. Antisera for glycine and GABA were kindly provided by Dr. Robert J. Wenthold. This investigation was supported by NIH Grants ROl DC00127 and ROl DCO0199. References Adams, J.C. (1981) Heavy metal intensification of DAB-based HRP reaction product. J. Histocbem. Cytochem. 29, 775. Adams, J.C. and Wenthold, R.J. (1987) Immunostaining of GABA-ergic and glycinergic inputs to the anteroventral cochlear nucleus. Sot. Neurosci. Abstr. 13, 1259. Altschuler, R.A., Betz, H., Parakkal, M.H., Reeks, K.A. and Wenthold, R.J. (1986) Identification of glycinergic synapses in the cochlear nucleus through immunocytcchemical localization of the postsynaptic receptor. Brain Res. 369, 316-320. Aoki, E., Semba, R., Keino, H., Kato, K. and Kashiwamata, S. (1988) Glycine-like immunoreactivity in the rat auditory pathway. Brain Res.442, 63-71. Araki, T., Yamano, M., Murakami, T., Wanaka, A., Betz, H. and Tohyama, M. (1988) Localization of glycine receptors in the rat central nervous system: An imrnunocytochemical analysis using monoclonal antibody. Neuroscience 25, 613624. Benson, C. and Potashner, S.J. (1990) Retrograde transport of [3H]glycine from the cochlear nucleus to the superior olive in the guinea pig. J. Comp. Neurol., 296, 415-426. Blackbum, C.C. and Sachs, M.B. (1989) Classification of unit types in the anteroventral cochlear nucleus: PST histograms and regularity analysis. J. Neurophys. 62, 1303-1329. Brawer, J.R., Morest, D.K. and Kane, E.C. (1974) The neuronal architecture of the cochlear nucleus of the cat. J. Comp. Neurol. 155, 251-300. Browner, R.H. and Baruch, A. (1982) The cytoarchitecture of the dorsal cochlear nucleus in the 3-month- and 26-monthold C57BL/G mouse: A Golgi impregnation study. J. Comp. Neurol. 211, 115-138. Cant, N.B. and Morest, 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, 10031029. Caspary, D.M., Havey, D.C. and Faingold, C.L. (1979) Effects of microiontophoretically applied glycine and GABA on neuronal response patterns in the cochlear nuclei. Brain Res. 172, 179-185.

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Glycine immunoreactive projections from the dorsal to the anteroventral cochlear nucleus.

The aim of the present study was to investigate whether projections from the dorsal cochlear nucleus (DCN) to the anteroventral cochlear nucleus (AVCN...
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