Multiple projections from the ventral nucleus of the trapezoid body in the rat

HmlUI RESMKH ELSEVIER

Heating Research 93 (1996) 83-101

Multiple projections from the ventral nucleus of the trapezoid body in the rat W. Bruce Warr *, Jo Ellen Beck Boys Town National Research Hospital, 555 North 30th Street, Omaha, NE 68131, USA

Received 11 April 1995; revised 19 September 1995; accepted 27 September 1995

Abstract

An analysis of the central projections of the ventral nucleus of the trapezoid body (VNTB) in the rat, a region of the superior olivary complex known for its neuronal heterogeneity, was made using two anterograde axonal tracers, [3H]leucine and biotinylated dextran amine (BDA). A mixture of these tracers was injected iontophoretically into the VNTB and the results analyzed by first assessing magnitudes of autoradiographic signal in nuclei receiving projections and then identifying the axons and terminals responsible for this signal in parallel sets of sections processed for BDA. Our analysis showed that in addition to its projections to each cochlea via the olivocochlear bundle, the VNTB has 3 major central sites of axonal terminations: (1) the cochlear nucleus, particularly the molecular layer of the contralateral dorsal cochlear nucleus, (2) the contralateral lateral superior olive, and (3) the ipsilateral inferior colliculus. Other sites receiving projections from the VNTB included the VNTB itself and the nuclei of the lateral lemniscus. Significantly, the relative magnitudes of labeling within the nuclei receiving inputs from the VNTB varied consistently as a function of the dorsoventral location of the injection site, confirming previous work showing that there is a partial segregation within this nucleus of neurons according to their projections. Our data also revealed an orderly topographic pattern of projections to the cochlear nuclei, lateral superior olive and the inferior colliculus which is consistent with the known tonotopic organization both of the VNTB and these projection targets. Methodologically, the co-injection of two tracers was advantageous in that patterns of silver grains in autoradiographs could be used to confirm whether axons and terminals labeled with BDA had originated from labeled somata at the injection site or were the result of uptake of BDA by fibers of passage. Keywords: Auditory pathway; Cochlear nucleus; Olivocochlear bundle; Superior olivary complex; Inferior colliculus

1. I n t r o d u c t i o n

The ventral nucleus of the trapezoid body (VNTB), or medioventral periolivary nucleus, is a heterogeneous cell group of the superior 01ivary complex (SOC) situated strategically at the intersection of ascending projections from the cochlear nucleus and descending projections from the inferior colliculus (IC) (Huffman and Henson, 1990; Spangler and Warr, 1991; Schwartz, 1991). Its importance as a site of interaction between ascending and descending auditory pathways is indicated by the fact that, of all the nuclei of the SOC, the VNTB is the sole recipient of a major descending projection from the ipsilateral IC (Anderson et al., 1980; Caicedo and Herbert, 1993; Vetter et al., 1993). Neuroanatomical and physiological evidence

* Corresponding author. Tel.: (402) 498-6528; Fax: (402) 498-6351; E-mail: [email protected]. 0378-5955/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 3 7 8 - 5 9 5 5 ( 9 5 ) 0 0 1 9 8 - 0

suggests that this descending projection may be involved in the activation of olivocochlear neurons (Glenn and Oatman, 1980; Rajan, 1990; Vetter et al., 1993), but its primary target would appear to be the larger group of VNTB neurons projecting to the cochlear nucleus (FayeLund, 1986; Thompson and Thompson, 1993). Ascending input to VNTB originates primarily in the contralateral ventral cochlear nucleus (VCN) (Warr, 1972; Vater and Feng, 1990) and likely represents axon collaterals of multipolar cells (Warr, 1982; Thompson and Thompson, 1991), which are probably excitatory (Guinan et al., 1972; Oliver, 1987). In addition, VNTB receives crossed excitatory input from globular bushy cells which send a collateral to it before terminating as calyces of Held on the principal cells of the medial nucleus of the trapezoid body (MNTB) (Spirou et al., 1990; Kuwabara et al., 1991; Smith et al., 1991). The MNTB principal cells, in turn, also project to the VNTB of the same side (Spangler et al., 1985; Kuwabara and Zook, 1991) and are presum-

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W.B. Warr, J.E. Beck / Hearing Research 93 (1996) 83-101

ably inhibitory (Moore and Caspary, 1983). Because sounds occurring in one hemifield are represented in the opposite IC (Irvine, 1986; Glendenning et al., 1992), it can be reasoned that the descending projections from IC to the ipsilateral VNTB modulate neuronal activity driven by sounds occurring in the same auditory field. The present study was motivated in part by a desire to learn more precisely where, within the central auditory pathway, information processed in the VNTB is transmitted. Knowledge of the projections of the VNTB, particularly in rodents, is based largely on retrograde labeling, which provides little information about the course and terminations of axons. Retrograde labeling studies demonstrate that the VNTB is a populous, longitudinally oriented, cell column comprised of several different, partially overlapping cell groups with either ascending or descending projections, including those to the cochlea (Warr, 1975; White and Warr, 1983; Aschoff and Ostwald, 1988; Vetter and Mugnaini, 1992), cochlear nucleus (Adams, 1983a; Osen et al., 1984; Faye-Lund, 1986; Spangler et al., 1987; Winter et al., 1989; Shore et al., 1991; Sherriff and Henderson, 1994), and inferior colliculus (Beyerl, 1978; Adams, 1979; Nordeen et al., 1983; Faye-Lund, 1986; Aschoff and Ostwald, 1988). Based on the selective retrograde labeling of VNTB and lateral lemniscal neurons in the chinchilla from injections of [3H]glycine in the ipsilateral IC, it is likely that these ipsilateral projections are glycinergic and therefore inhibitory (Saint Marie and Baker, 1990). Finally, anterograde transport studies in the cat (Spangler et al., 1987) found a pattern of ascending and descending projections from VNTB which are very similar to those we report here. In this study, we analyzed the projections of the VNTB to structures of the central auditory pathway. The projections of medial olivocochlear neurons, which form a part of the VNTB in the rat, will be described in a subsequent report. In carrying out this study, we employed a novel combination of axonal labeling techniques: the anterograde transport of radiolabeled protein after injection of [3H]leucine and the primarily, but not strictly, anterograde transport of biotinylated dextran amine (BDA) (Veenman et al., 1992). Since BDA labeling of axons can result from uptake by 'fibers of passage', the availability of parallel sets of autoradiographs enabled us to determine whether or not axonal labeling with BDA was an artefact or the genuine result of uptake of the tracer by somata at the injection site.

2. Materials and methods

2.1. Animals and surgery Experimental animals were 22 adult male SpragueDawley rats weighing 275-350 g. Prior to surgery, animals were anesthetized by injection (0.75 m l / 3 0 0 g, i.m.)

of a mixture containing 10 ml of ketamine (100 mg/ml), 1 ml of acepromazine (t0 m g / m l ) and 1 ml of atropine (0.008 grains/ml) and mounted in a stereotaxic instrument. Observing sterile procedures, the scalp was reflected, the interparietal bone opened with a diamond burr, the dura mater pierced, and an iontophoretic injection made through a micropipet guided to the target using coordinates obtained from a stereotaxic atlas (Paxinos and Watson, 1986). We aimed the pipet at the rostral end of the VNTB because at this location its dorsoventral extent is greatest and thus the injectate was less likely to spread to adjacent cell groups. 2.2. lontophoresis of injectates In this study, we made co-injections of radio-labeled amino acid (Hendrickson and Edwards, 1978) and dextran amine (Veenman et al., 1992). Glass micropipets with tips measuring 26-50 /zm inner diameter (ID) were filled with a mixture containing tritiated leucine and either BDA or fluorescein-labeled dextran amine (Molecular Probes, Eugene, OR; Product #D-1956 or #D-1820, both 10000 MW). The mixtures were prepared by drying 200 /zl of [ 3H]L-leucine, specific activity 170 mCi/mmol (NEN-460, NEN Research Products), and redissolving it in 4/zl of 5% aqueous BDA or fluorescein-labeled dextran amine, for a final concentration of radioactivity of 50 /zCi//xl. Iontophoretic injections were made with 1 /zA positive current (Midgard Instruments) passed intermittently, 7 s o n / 7 s off, for 15 min. We noted that, during iontophoretic delivery, the mixture containing the fluorescein-labeled dextran amine (Animals 1-4) formed a fine precipitate and later histological examination revealed no evidence of fluorescein labeling. For this reason, in subsequent animals the injectate consisted of the mixture containing BDA (Animals 15-21). A 0.5 /zA negative holding current was applied during advance and withdrawal of the pipet. After delivery, the pipet was left in place for 5 min before being withdrawn. The skull opening was filled with Gelfoam and the incision closed with wound clips. Animals were returned to their cages and closely monitored until they had recovered from anesthesia and had begun eating and grooming normally. 2.3. Fixation and histology Following 7 - 1 0 days post-operative survival, animals were anesthetized as described above and perfused through the heart with an aldehyde mixture containing 1.0% paraformaldehyde and 1.25% glutaraldehyde in 0.01 M phosphate buffer, pH 7.4. The brain was removed and blocked in a coronal plane with the aid of a sectioning guide (Activational Systems) from the caudal end of the CN through the rostral end of the IC. Following 24 h in 30% sucrose, the brain was sectioned at 30 /xm on a freezing microtome and the sections collected sequentially in 6

W.B. Warr, J.E. Beck/Hearing Research 93 (1996) 83-101

compartments. Sections from 2 equally spaced compartments (e.g., 1 and 4, or 2 and 5, etc.) were processed on slides for autoradiography (AR) in diluted (1:1) Kodak NTB2 nuclear track emulsion, stored with desiccant at 4°C for 6 - 1 2 weeks, and developed in Dektol (Hendrickson and Edwards, 1978). After drying, the sections were counterstained with cresyl violet and coverslipped. BDA was demonstrated in the sections in 1 or 2 compartments adjacent to the AR series by incubation for 3 h in ABC solution (ABC Elite, Vector Laboratories) containing 1% Triton-X100. A black reaction product was produced in a solution containing diaminobenzidine-HCL and cobalt-nickel (Adams, 1981). The sections were then

85

mounted and counterstained with cresyl violet. A parallel series of sections was stained for acetylcholinesterase (ACHE) using a 90 min incubation (Mathisen and Blackstad, 1964) and counterstained with cresyl violet as an aid in identifying nuclear boundaries and cell types.

2.4. Data collection and analysis The material was analyzed with a Zeiss research microscope equipped with a digitized stage attached to an MDPIot Data Acquisition and Plotting System (Minnesota Datametrics). In order to assess whether the AR injection had spread beyond the VNTB, we determined whether

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silver grains overlying neuronal somata in adjacent cell groups exceeded background, using a 25 × objective and bright-field illumination. In order to compare the magnitude of AR labeling observed in different structures within a given animal and also within the same structure in different animals, we made judgments of the relative silver grain density (AR signal) in each auditory structure from the CN through the IC in each animal using a Wild M420 Macroscope with a dark-field light source and set to 80 × magnification (see Table 1). Each structure was assigned a score ranging from + + + , which indicated the greatest AR signal density, to zero (0), which indicated that AR signal level was not consistently above background. Intermediate scores were defined as follows: + + , AR signal was consistently above background but less than the maximum found in other auditory structures in that animal; + , AR signal was consistently above background; + / - , AR signal was minimally or inconsistently above background. If AR signal varied in magnitude across a given structure, ratings were made on that part containing the greatest amount. The care and use of animals in this study was approved by the Creighton University Animal Care and Use Committee.

87

represent classes of neurons presumed to be responsible for the projections. These projections may be either ipsilateral (IC), contralateral (LSO), or both (cochlea and CN). A more detailed summary of the results is provided in Table 1 which contains a quantitative summary of the distribution and relative magnitudes of AR signal in the main projection targets of the VNTB in the 7 experimental animals analyzed in this study. Because inspection of these data suggested that the dorsoventral location of the injection site accounted for most of the variation in the relative magnitude of projections to its several target nuclei, we have listed the animals in Table 1 according to injection location, with the animal having the most dorsal injection at the top. By comparing the projections arising from a progression of discrete injection sites spanning the dorsoventral axis of VNTB (Fig. 1C, animals 16, 15 and 17), several trends can be seen in Table 1 . First, the most dorsal injection of VNTB (animal 16) produced relatively stronger

3. Results

3.1. Injection sites In 7 animals we obtained small (0.4-0.7 mm diameter) injections in the VNTB (Fig. 1C,D, asterisks), all of which were centered at least as far rostral as the rostral tip of the MSO. In some animals, somatic AR signal had spread to various extents to adjacent nuclei, as listed in Table 1, thus compromising straightforward interpretation of the results. However, in 3 cases, animals 15-17 (Fig. 1C), the injection site was confined to the target, as illustrated for animal 15 (Fig. 2A). The BDA injection site in this and other cases in which BDA was injected (animals 15-21), was somewhat smaller than the corresponding site indicated by AR signal (Fig. 2B). Control injections (Fig. 1C, animals 19 and 21), placed in the medial nucleus of the trapezoid body (MNTB) and the superior paraolivary nucleus (SPN), did not spread to VNTB.

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3.2. Overview of findings Because the results of this study are complex and difficult to visualize, we refer the reader to Fig. 3 which is a schematic diagram showing the general organization of projections from the VNTB of the rat. Fig. 3 depicts the left VNTB as being comprised of four partially overlapping territories, each of which has a primary projection target indicated by the arrows. Four types of symbols

Fig. 2. Comparison of AR (A) and BDA (B) labeling at injection sites in animal 15. Labeling with BDA is generally more restricted than with [3H]leucine. The small spot dorsal to the injection site in A is gliosis along the pipet track. Sections in A and B are separated by 60 ~ m .

W.B. Warr, J.E. Beck~Hearing Research 93 (1996) 83-101

88

projections to the IC than to the contralateral DCN or LSO, whereas the opposite was true following the most ventral selective injection (animal 17). Second, a relatively low level of AR signal in the ipsilateral IC was a characteristic feature in animals with injections confined to VNTB, but was in sharp contrast to those in which the injection affected the SPN or MSO (animals 1, and 21). Third, AR labeling of the olivocochlear bundle (OCB) was heaviest after injections in intermediate dorsoventral position (animals 15 and 16), which would directly label the somata of medial olivocochlear neurons (visualized with AChE staining and indicated by dots in Fig. 1), but OCB labeling was relatively weak or absent following the most dorsal and ventral injections (animals 1 and 17) and the most medial injection (animal 3), which would have labeled these neurons only weakly, if at all.

portion (Fig. 4C,D). Although the rostral part of the anteroventral cochlear nucleus (RAV) appears to be innervated by fibers that enter it directly from the ventral acoustic stria (Figs. 4D,E and 6A), the remainder of the VCN is supplied by fibers that travel caudally along its medial margin. The fibers innervating the DCN of either side continue caudally and dorsally between the inferior cerebellar peduncle and DCN (Fig. 4B, right) and enter the deep DCN. 3.5. Terminations within the DCN The contralateral DCN was one of the major terminal sites of VNTB projections, whereas ipsilaterally there was only a relatively low level of AR signal which appeared to be distributed irregularly in the various layers, but most commonly in the deep layer (Fig. 4A,B). In the ipsilateral DCN, there was no clear evidence that terminal labeling conformed to isofrequency bands, which extend rostrocaudally, perpendicular to the coronal plane (Ryan et al., 1988; Kaltenbach and Lazor, 1991; Caicedo and Herbert, 1993). In most animals there was a dense patch of AR signal in the deep DCN adjacent to the granular cell lamina (Fig. 4B). In the contralateral DCN, there was a heavy projection to the molecular and fusiform cell layers, which, in animal 15 was restricted to the ventral half of the nucleus, reflecting the topography and, presumably, the tonotopic map of VNTB projections (e.g., Fig. 4A). The projection was most intense from the ependymal surface of the DCN to a depth of approximately 110-120 /xm, which includes the entire molecular layer, but there was also significant labeling in the fusiform layer (Fig. 5A,B). The AR signal in the molecular layer consisted mostly of large clusters of silver grains which, in adjacent sections processed for the demonstration of BDA, corresponded to nests of terminal axons and boutons (Fig. 5B). These nests appeared to arise either from a single axon or from the coalescence of

3.3. Projections from VNTB The full range of axonal projections from the VNTB are best exemplified in animal 15 because the injection was centered dorsoventrally in the nucleus and labeled all the projection pathways characteristic of the nucleus (Fig. 2). The injection site was in the low frequency portion of VNTB, based both on its lateral location (Friauf, 1992; Caicedo and Herbert, 1993) and the resulting distribution of AR signal in the contralateral DCN and LSO (Ryan et al., 1988; Sommer et al., 1993). Fig. 4 illustrates how in animal 15 AR-labeled axons radiate in various directions from the injection site, including laterally to the ipsilateral CN, medially to the contralateral LSO and CN, dorsally in the OCB, caudally within the VNTB itself, and rostrally in the ipsilateral lateral lemniscus (LL). 3.4. Pathways to the cochlear nuclei AR-labeled fibers reach the CN by way of the ventral acoustic stria of both sides, but only within its most rostral

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Fig. 3. Schematicdiagram of the main fmdings of the present study, presented as an aid to visualizing the complexpattern of projections of the VNTB to be described and documented below. Not shown is an intrinsic projection which extends longitudinally within the VNTB, from rostral to caudal.

W.B. Warr. J.E. Beck/Hearing Research 93 (1996) 83-101

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Fig. 4. Course and distribution of AR labeling in animal 15 plotted by means of the MDPIot System. Each dot represents at least 6 silver grains. See separate list for key to abbreviations.

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W.B. Warr, J.E. Beck/Hearing Research 93 (1996) 83-101

several varicose axons which terminate in tufts of boutons. Occasionally, boutons and varicosities appeared to contact neuronal somata in the molecular or fusiform layers, but more typically they terminated freely in the neuropil. In several instances, we noted that AR signal stopped abruptly at the edges of clusters of granule cells in the molecular layer, creating obvious vacant patches in the autoradiograph (Fig. 5A, asterisk). 3.6. Terminations within V C N

As was typical in other cases, AR signal levels in animal 15 were low in the octopus cell area (OCA) and the rostral posteroventral CN (RPV). Moreover, there was little evidence of projections to the cochlear root nucleus (RN) as shown in Fig. 4A,C. There was a noticeable increase in AR signal in the overlying caudal part of the anteroventral CN (CAV) at the level of the RN, however, and this signal increased still further rostrally through RAV. AR signal tended to be stronger in the more ventral parts of the VCN, consistent with the fact the injection in animal 15 was placed in the lateral part of the VNTB, which represents lower frequencies (Friauf, 1992). AR signal levels in the VCN of animal 15 were more or less bilaterally symmetrical, both in location and magnitude, but in other animals there was noticeable variability in their relative magnitudes (see Table 1). In the VCN of either side, the pattern of AR signal consisted of dense clusters of silver grains distributed on a ground of diffuse labeling (Fig. 6A). The corresponding B D A material contained varicose fibers of a range of diameters, the thicker of which often appeared to give rise to large boutons that terminated on neuronal somata, whereas the thinner fibers appeared to contribute to a more or less diffuse terminal plexus (Fig. 6B). There were no obvious differences in size or morphology between fibers innervating the ipsilateral versus the contralateral VCN. It is worth noting that both control animals confirm previous work showing that there is a significant ipsilateral descending projection to rostral VCN originating in the medial part of the SOC (see Table 1, RAV), including at least the MNTB (Faye-Lund, 1986; Winter et al., 1989; Benson and Potashner, 1990; Schofield, 1994). This finding was a caution against interpreting the AR signal in the ipsilateral RAV in animals 1, 2 and 3 as arising exclusively from the VNTB. 3.7. OCB and projections to granule cells

The OCB was strongly labeled in animal 15 and could be traced bilaterally from the injection to its exit from the brain with the vestibular root (Fig. 4C). However, because of the large projection to the VCN via the ventral acoustic stria, it was usually not possible to ascertain with complete certainty whether the OCB was the source of any AR signal observed within the VCN. Nevertheless, in animal

Fig. 5. Comparison of patterns of AR (A) and BDA (B) labeling in the contralateral DCN in nearby sections in animal 17. Layers of the DCN are numbered 1-3, corresponding to the molecular, fusiform cell and deep layers, respectively. A: silver grains are concentrated in the molecular layer, where they form more or less discrete clusters throughout its depth. The area lacking silver grains ( * ) was a cluster of granule cells. B: several dense nests of varicose fibers and terminal boutons, corresponding to the clusters of silver grains shown in A.

W.B. Wart, J.E. Beck~Hearing Research 93 (1996) 83-101

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Fig. 6. Comparison of patterns of AR (A) and BDA (B) labeling in the ipsilateral rostral AVCN at a level 9 0 / z m caudal to Fig. 3D in animal 15. A: AR signal consists of coarse patches and diffuse, scattered silver grains. Boxed area indicates the location of B, below. B: Thick fibers terminate in coarse, claw-like endings which may encircle single neurons. Thin fibers meander across the nucleus forming a diffuse terminal plexus made up of beaded axons.

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W.B. Warr, J.E. Beck/Hearing Research 93 (1996) 83-101

Fig. 7. Comparison of AR (A) and BDA (B) labeling in adjacent sections of the contralateral LSO in animal 17. A: AR signal forms dense patches oriented parallel to the radial isofrequency laminae. B: Thin beaded axons follow the ventral hilus of the LSO to terminate in nests of terminal boutons which appear to be situated between the somata of large neurons.

W.B. Warr, J.E. Beck/Hearing Research 93 (1996) 83-101

93

15 and several others with clear OCB labeling, low to moderate AR signal levels were observed in the subpeduncular comer granular cell regions of either side which appeared most likely to come from the OCB (see Table 1, GCA). In contrast, however, we never observed AR signal in the superficial granule cell layer which overlies the dorsolateral surface of the AVCN. Study of material stained for BDA produced some examples of fibers that might have been collateral branches of the OCB diverging to the subpeduncular comer, but efforts to confirm these observations were limited by the lack of serial sections stained for BDA. 3.8. Projections to the LSO In animal 15 labeled fibers traveling to the contralateral LSO passed through the ipsilateral MNTB, and crossed the midline in the dorsal half of the trapezoid body at a level mostly rostral to their eventual destination. The fibers then traversed the MNTB of the opposite side (Fig. 4D) where there was some evidence of sparse terminals in both AR and BDA material. Similarly, there were sparse terminations in lateral periolivary cell groups both rostral and ventral to the contralateral LSO (Fig. 4D, LNTB). The majority of AR-labeled fibers appeared to enter the LSO from either its ventral or rostral surface. Within the LSO, dense bands of AR signal were observed to span the full width of the neuropil, parallel to the isofrequency planes of the nucleus (Fig. 7A), and extending its full anteroposterior extent. Inspection of BDA material with oil immersion objectives revealed that this projection was composed of thin, beaded axons which, in places, coalesced to form dense, elongated arbors which occupied the full thickness of the tissue section (Fig. 7B). These arbors did not form perisomatic nests, but rather occurred in the neuropil occupying the space between neuronal somata. The ipsilateral LSO in animals 15 and 16 contained some scattered, sparse AR signal which could not be attributed to spread of injectate to the MNTB. However, animals 4 and 17, which had heavy input to the contralateral LSO, exhibited little or no input ipsilaterally.

Fig. 8. Comparison of patterns of AR (A) and BDA (B) labeling in the ipsilateral VNTB in animal 17. A: arrows indicate a narrow band of AR signal. B: a nearby section shows a corresponding band of thin, beaded axons which form terminal nests. Both photomicrographs are of equal magnification.

closely corresponding region containing longitudinally oriented axons, the branches of which coalesce into terminal nests. No clear perisomatic relation between these terminal nests and the somata of the local neuronal population was apparent.

3.9. Intrinsic plexus of the VNTB 3.10. Projections to the lateral lemniscus and its nuclei The ipsilateral VNTB in animal 15 contained a tow to moderate level of AR signal that extended from the injection site caudally to the end of the SOC (Fig. 4C,D), an observation which was confirmed in all experimental animals of this study, but not in the controls. It was not possible to determine if this intrinsic projection extended in the rostral direction also because the injections were placed extremely rostrally in the VNTB and this part of the nucleus was traversed by labeled ascending axons destined to join the LL. In Fig. 8A, arrows indicate a small region of AR signal in the VNTB of animal 17 approximately 1.5 mm caudal to the injection site, and in Fig. 8B, there is a

In animal 15, AR-labeled fibers proceeded rostrally from the injection site to join the ipsilateral LL proper, but few if any coursed along its medial margin (Fig. 4F-I). There was low to moderate AR signal ipsilaterally in all of the nuclei of the lateral lemniscus, including VNLL, INLL and DNLL in all of the animals with selective injections of the VNTB. The INLL typically appeared to receive slightly greater inputs than did either the VNLL or DNLL, but in most cases the ratings in Table 1 were not sensitive to subtle differences. It should be noted, however, that compared to those of the MNTB (Table 1, animals 19 and 21),

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W.B. Warr. J.E. Beck~Hearing Research 93 (1996) 83-101

the projections of the V N T B relatively minor.

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3.11. Projections to the IC A s w a s true in all animals with injections c o n f i n e d to V N T B , the injection in animal 15 produced a s o m e w h a t

sparse but remarkably extensive projection to the ipsilateral IC (Fig. 4 F - J ) . The vast majority of the A R signal w a s confined to parts of the IC which, in a parallel series stained for ACHE, had an A C h E - p o s i t i v e neuropil (Fig. 9B), including C N I C and layer 2 o f ECIC, but largely e x c l u d i n g layer 3 o f ECIC (Paxinos and Watson, 1986; Herbert et al., 1991). Thus, the projection did not extend

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Fig. 10. Semi-schematic representation of the topographic organization of projections to the contralateral CN (B), the contralateral LSO (C) and the ipsilateral CNIC (D) observed in 3 rats with injections in different mediolateral locations within VNTB (A).

into the dorsal cortex, which forms a superficial layer over caudal CNIC (not shown in Fig. 4), but did extend to the rostral extremity of ECIC, deep to the superior colliculus (Fig. 4H-J). There was always some minor AR signal in that part of the deepest layer of ECIC (layer 3 of Faye-Lund and Osen, 1985) which is located laterally adjacent to CNIC (Fig. 4G,H) and in some animals there were a few AR-labeled fibers in the contralateral LL and IC; however, they did not produce a recognizable terminal pattern. A detailed examination of the projection to IC in animal 15 showed that it consisted of a broad band of AR signal in the dorsolateral, low-frequency region of the CNIC (Ryan et al., 1988; Friauf, 1992), which was made up of thin as well as a few thicker tracks of silver grains (Fig. 9C). BDA-staining revealed that the dorsolateral portion of CNIC contained the terminal arbors of both thin beaded axons and a smaller number of thick fibers and terminals (Fig. 9D). We could find no clear evidence in any of our animals, experimental or control, of multiple bands of AR signal in the CNIC similar to those observed after tracer injections in the CN of the rat (Oliver and Beckius, 1993) and the MSO or LSO of the cat (Henkel and Spangler, 1983; Shneiderman and Henkel, 1987). The distribution of AR signal in the IC of experimental animals differed in several ways from that found after a control injection in the SPN (Table l). In the latter case, the level of AR signal was noticeably greater and occupied only the medial, high-frequency region of CNIC and the rostrally adjacent ECIC. In addition, the injection in SPN

produced clear projections both laterally to the adjacent, AChE-negative layer 3 of ECIC and to the dorsomedial cortex (DCIC), both quite unlike injections confined to VNTB.

3.12. Evidence of tonotopic organization We observed that the mediolateral position of injection sites within the VNTB was related to the specific location of its terminal projections. These findings are consistent with the known tonotopic organization of both the VNTB, and the nuclei receiving projections from it, namely the CN (Ryan et al., 1988; the LSO (Sommer et al., 1993); and the IC (Osen, 1972; Ryan et al., 1988). In order to illustrate this tonotopy, plots of AR signal from 3 rats, animals 4, 2, and 3, which respectively had injections in lower, middle and high-frequency regions of the VNTB (Fig. 10A), were superimposed on a standard series of sections (Fig. 10B-D). The resulting pattern of projections to each target consisted of broad terminal fields and there was considerable overlap between the fields produced by adjacent injection sites.

4. Discussion

4.1. Injection sites An important asset of the present study was that the injection sites obtained by iontophoretic delivery of

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[3H]L-leucine were, in 3 out of 7 experimental animals, entirely confined to this nucleus, thus making interpretation of the resulting AR findings fairly straightforward. Most of the injections were also placed in such a way as to label primarily one quadrant or another of the VNTB, which analysis revealed to have differential projections. On the other hand, such small injection sites were obtained at the cost of limiting the study to the rostral end of the VNTB, where the nucleus is largest. As a result, we obtained no data pertaining to the projections of the caudal VNTB, and thus the generality of any conclusions drawn from these data must be limited. A further limitation on the present findings is that none of the injections sampled the high-frequency portion of the dorsal VNTB which contains and mixture of cells projecting either to the cochlea or to the IC.

4.2. Co-injections of anterograde tracers Methodologically, the simultaneous injection of [3H]leucine and BDA was a valuable technical innovation because, in contrast to the data obtainable from each tracer when used alone, labeled axons and terminal arborizations revealed by BDA could be corroborated in adjacent autoradiographs as having originated from the injection site. The injection of BDA also had the advantage of providing a marker that could be detected with ABC reagents immediately after the tissue was sectioned and used to promptly assess the results. Because of the inherent reliability of the [3H]leucine method, we first charted the AR signal and then examined the parallel set of BDA material for labeling of axons and terminals in corresponding locations, rather than vice versa. Although we made no specific effort to document instances in which BDA labeling was present without corresponding AR signal, which would indicate the presence of an artifact caused by uptake by fibers passing through the injection site, such were fairly common.

4.3. Acquisition and utility of projection magnitude data A further strength of the present study was the semiquantitative assessment of projection magnitudes, as presented in Table 1. The nominal, 5-point scale served to normalize the variability in AR signal levels between rats. Based on these normalized ratings, it was possible to arrange the cases in Table 1 according to the dorsoventral position of their injection sites, yielding an internally consistent data set. As summarized in Fig. 3, this data set was consistent with the idea that the VNTB contains at least 4 separate neuronal populations, as defined by their projections. This interpretation is supported by several previous studies in the rat and cat which show that within the VNTB, there is a partial segregation of neurons projecting to different targets. Thus, in approximate dorsoventral

sequence, there are VNTB neurons which project to the IC (Adams, 1983a; Faye-Lund, 1986), the cochlea (White and Warr, 1983; Vetter and Mugnaini, 1992), the CN (FayeLund, 1986), and the LSO, reported here. Recently, a more precise description of this segregation of cell types in VNTB was obtained using immunostaining for ChAT combined with retrograde cell marking from CN, which showed that, as can be seen in Fig. 3, neurons which project to the CN are ventromedially adjacent to a cluster of larger ChAT-positive neurons which were presumed to be medial olivocochlear neurons (Sherriff and Henderson, 1994). In addition, double retrograde labeling experiments indicate that, except for some medial olivocochlear neurons, it is not characteristic for VNTB neurons to make collateral connections with more than one major target (Adams, 1983a), as appears also to be the case in the rat (Warr and Lopez, 1993). It seems clear, therefore, that the VNTB contains several spatially and connectionally distinguishable neuronal populations.

4.4. Projections to the DCN One of the most significant findings was that VNTB projects massively to the opposite DCN in the form of terminal nests in the molecular layer. In contrast, the projections to the ipsilateral DCN were sparse and, although not strictly confined to any one layer, were perhaps most prominent in the deep layer of the DCN. A similar asymmetry in magnitude and layer of termination was previously observed in the projections to DCN from VNTB using the AR method in the cat (Spangler et al., 1987). That and the present study are apparently the only ones to document the existence of an exogenous projection to the molecular layer of the DCN. All other exogenous inputs, except those from CN granule cells, terminate in the fusiform layer or deeper, including those from the cochlea (Lorente de N6, 1933; Osen, 1970), VCN stellate cells (Oertel et al., 1990) and the IC (Caicedo and Herbert, 1993). The nest-like terminal arbors in the DCN molecular layer, visualized here with BDA, have apparently not been described previously, either with axon marking techniques or with the rapid Golgi method (Ramon y Cajal, 1909; Lorente de NO, 1933, 1981). In fact, Lorente de N6 believed that the DCN molecular layer did not receive exogenous inputs, except from granule cells (Lorente de N6, 1981). The fibers we observed do not fit the description of mossy fiber afferents to the DCN because the latter terminate in granule cell regions (Mugnaini et al., 1980a, Mugnaini et al., 1980b), whereas the former appeared to be virtually repelled by granule cells (Fig. 5A). Thus it would appear that the terminals in the molecular layer represent a different, previously unknown, class of afferents to the DCN molecular layer. Recent evidence indicates that many small neurons in the rat's VNTB immunolabel for choline acetyltransferase

W.B. Warr, J.E. Beck/Hearing Research 93 (1996) 83-101

(CHAT) and that these same neurons project preponderantly to the opposite CN, including the DCN, via the ventral acoustic stria (Sherriff and Henderson, 1994). However, opposing the implications of our data are quantitative studies in the Sprague-Dawley albino rat showing that the DCN molecular layer contains significantly lower levels of ChAT than does either the fusiform or deep layer (Godfrey et al., 1987a) and that severing the ventral acoustic stria produces no greater loss of ChAT activity in the molecular layer (34%) than in the fusiform cell (41%) or deep layer (33%) (Godfrey et al., 1987c). Such findings leave open the possibility of an inhibitory role for the projection to DCN from VNTB and, in fact, many VNTB neurons immunolabel for glycine, T-aminobutyric acid (GABA), or its synthesizing enzyme, glutamic acid decarboxylase (Mugnaini and Oertel, 1985; Moore and Moore, 1987; Roberts and Ribak, 1987; Wenthold et al., 1987; Helfert et al., 1989; Adams and Mugnaini, 1990) and many of the synaptic terminals in the DCN, particularly the molecular layer, are likewise singly or doubly labeled for glycine a n d / o r GABA (Osen et al., 1990; Kolston et al., 1992). In confirmation of these data, regional microassays (Godfrey et al., 1978) and receptor binding studies (Juiz et al., 1989, 1994) of the rodent CN demonstrate that the DCN molecular layer contains by far the highest levels of GABA of any region of the CN. However, countering the idea that the contralateral VNTB might have inhibitory projections to CN is the finding that injections of [3H]glycine into the entire CN of the guinea pig retrogradely labels predominantly ipsilateral rather than contralateral periolivary neurons in the guinea pig (Benson and Potashner, 1990). 4.5. Projections to VCN

The present findings indicate that the VNTB projects to the VCN bilaterally and with more or less equal magnitude. Depending on species, previous investigators have variously reported VCN input from VNTB to be preponderantly contralateral in the cat (Elverland, 1977; Adams, 1983b; Spangler et al., 1987), preponderantly ipsilateral in the guinea pig (Shore et al., 1991), or more or less equal, depending on the specific ventromedial periolivary cell group in the tree shrew (Covey et al., 1984), indicating that the organization of projections from this region cannot be generalized across species. Our data further indicate that, although the rostral VNTB projects to almost the entire rostrocaudal extent of the VCN, these inputs increase in magnitude the more rostral the level. Although our controls implicate the MNTB as the source of a part of the input to the rostral VCN ipsilaterally, as in the guinea pig (Winter et al., 1989), our findings are in general accord with those in the cat in which a similar projection gradient was found after [3H]leucine injections in the rostral VNTB (Spangler et al., 1987). The latter findings, together with complementary

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gradients in the strength of retrograde labeling in VNTB following HRP injections in rostral versus caudal CN of the cat, led Spangler and his colleagues to hypothesize that rostral VNTB projects preferentially to anteroventral CN and that the caudal VNTB projects preferentially to the posteroventral CN, an arrangement previously demonstrated to prevail for lateral periolivary cell groups in the cat (van Noort, 1969). Our qualitative observations of BDA labeling suggest that projections from VNTB to any given region of the VCN consist of two general types of terminal axons which we broadly categorize as thicker and thinner, which form, respectively, pericellular contacts or free terminations in the neuropil. Similarly, the AVCN of the kitten was found to receive axons from the ventral acoustic stria which possessed somewhat comparable differences in diameter and type of terminal arbor (group I and group III fibers), as visualized with the rapid Golgi method (Cant and Morest, 1978). 4.6. Projections of olivocochlear neurons

Previous studies in the rat have traced collateral branches from the OCB to the medial border of the VCN and subpeduncular comer regions (Osen et al., 1984), and in the mouse approximately 65% of all medial olivocochlear axons send a collateral to either the medial border or the granule cell lamina separating DCN from VCN (Brown et al., 1991). In our material, the only granule cell region that could be somewhat reliably detected as a probable projection target of medial olivocochlear neurons was the subpeduncular granule cell region. At least two factors account for this: first, the small injection sites in this study labeled relatively few olivocochlear neurons, and second, and more importantly, the injections labeled a much larger number of VNTB neurons that pass through the medial sheet of granule cells on their way to the VCN and DCN. A third factor which may account for our not having found a more robust projection to the subpeduncular corner region is suggested by the fact that, in the mouse, OCB collaterals to the subpeduncular comer and superficial layer arise only from fibers innervating high frequency regions of the cochlea (Brown et al., 1991). If, as seems evident from Fig. 1C,D, our injections consistently missed the medial olivocochlear neurons in the dorsomedial portion of VNTB, which represents high frequencies, this fact could account for the generally low levels of signal observed in the subpeduncular comer area and the complete absence of signal in the superficial layer of granule cells overlying CAV and RAV. Considerations of the tonotopy of injection sites may also to account for apparent discrepancies in the literature relating to olivocochlear projections in the cat. Thus, while Elverland's injections of [3H]leucine in the low frequency part of VNTB produced no evidence of collateral inputs from the OCB to CN (Elverland, 1977), an injection in the high-frequency region of the VNTB

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produced clear projections to the superficial granule cell layer (Spangler et al., 1987). The paucity of OCB projections we found in the rat are not altogether surprising since cutting the entire OCB causes only slight reductions in levels of AChE in the CN (Osen et ai., 1984) and only about 15% reductions in micro-assays for ChAT in CN granule cell regions (Godfrey et al., 1987b), unlike the cat (Godfrey et al., 1990). Similarly, in the guinea pig a quantitative study using double retrograde labeling indicated that less than 10% of medial olivocochlear neurons have collaterals to the superficial granule cell layer (Winter et al., 1989). 4.7. Projections to LSO and LNTB

The projections of the VNTB to the contralateral LSO were among the more unexpected findings of the present study, although a similar study in the cat revealed a crossed projection to the region of the dorsal hilus of the LSO, but not to the body of the nucleus, and to the LNTB (Spangler et al., 1987). In addition, a projection to the contralateral LSO from the VNTB has recently been observed to occur in the chinchilla, where an inhibitory role for these fibers is implied by the fact that the VNTB cells of origin were determined to be immunoreactive for GABA (Helfert et al., 1993). Particularly surprising was the density of the terminal bands of AR signal within the LSO, which equalled or exceeded any other projection from the VNTB in the present study. BDA material revealed terminal nests consisting of tangles of fibers oriented in the plane of the isofrequency laminae. These tangles filled the depth of the tissue section and may have filled the entire rostrocaudal extent of an isofrequency lamina, in the same way that inputs to this nucleus from the CN and MNTB do (Scheibel and Scheibel, 1974; Kuwabara and Zook, 1991). Close examination of these terminal nests produced no evidence of their being pericellular. The connections between the opposite VNTB and both the LSO and LNTB are functionally coherent to the extent that both the nucleus of origin and its targets derive their ascending excitatory input from the CN of the same side, crossed in the case of the VNTB and uncrossed in the case of the LSO and LNTB. Thus, these projections from VNTB presumably bring together neuronal activity driven by sounds in the same acoustic hemifield. 4.8. Intrinsic and local projections of the VNTB

A projection both arising and terminating within the VNTB has apparently not been previously reported. The varicosities on the fibers contributing to the terminal pattem created images not unlike the masses of puncta demonstrated by immunocytochemical staining for glutamic acid decarboxylase (Moore and Moore, 1987; Roberts and Ribak, 1987) and glycine (Mugnaini and Oertel, 1985;

Wenthold et al., 1987; Helfert et al., 1989). Because the terminal field of this projection encompassed nearly the entire longitudinal extent of the VNTB, activity within the rostral part of this nucleus could presumably affect VNTB neurons at the its caudal extremity. Such an intrinsic system of connections was not found after control injections of either the MNTB or SPN. A recent study using intracellular staining in slice preparations in the mouse and gerbil, among others, describes clear axonal ramifications from certain VNTB neurons to the immediately adjacent MNTB (Kuwabara et al., 1991). Because of the proximity between these cells and their axonal targets in the MNTB, the injections in the present study would have been too large and diffuse to allow the reliable detection of this connection using autoradiography. What can be said, however, is that such a projection does not appear to exist, at least in the rat, for VNTB neurons situated some distance away from the MNTB (e.g., animal 15). 4.9. Projections to IC

The projections of the VNTB to IC in the rat appeared to be almost completely limited to the CNIC and the rostrally adjacent ECIC, as defined by Faye-Lund and Osen (1985), and this projection was virtually entirely ipsilateral, which confirms previous retrograde labeling studies (Beyerl, 1978; Faye-Lund, 1986; Aschoff and Ostwald, 1988). The terminal field of VNTB projections in both CNIC and ECIC coincided closely with regions staining positively for ACHE. Although some scattered tracks of AR signal were occasionally found in the laterally adjacent, AChE-negative portion (layer 3) of ECIC, a region also known as the 'lateral nucleus' of the IC (Oliver and Huerta, 1992; Oliver and Beckius, 1993), no evidence of a terminal pattern was found there, except when injections affected SPN. The region of AChE-positive staining in the rat's IC has previously been noted to be connectionally significant in that it was devoid of descending inputs from auditory cortex (Herbert et al., 1991). In a complementary way, what is presently known about the ascending projections to IC in the rat suggests that the AChE-positive region is the predominant target of not only the VNTB, but also other ascending auditory inputs, including those from the SPN and the cochlear nucleus (Oliver and Beckius, 1993). However, unlike the situation in the cat (Oliver and Huerta, 1992), some ascending projections appear to reach the laterally adjacent ECIC, including those from the DCN in rat (Oliver and Beckius, 1993) and mouse (Ryugo et al., 1981), as well as those from the SPN (present study). The projections we observed in the IC were surprisingly sparse, considering the substantial numbers of neurons that are retrogradely labeled in VNTB by tracer injections into the rat's IC (Beyerl, 1978; Faye-Lund, 1986; Aschoff and Ostwald, 1988). Such retrograde labeling clearly defines a horizontally oriented, longitudinal column, of large multi-

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polar neurons in the dorsal portion of the VNTB which is centered immediately beneath the MSO. One explanation for the somewhat modest AR signal we found in the IC is that our selective injections of VNTB were situated ventral to the actual cell bodies which project to IC, in order to avoid labeling the nearby MSO and SPN. Thus, the projections of these olivocollicular neurons would be expected to have less than optimal magnitudes of AR signal.

4.10. Tonotopic organization of the rat VNTB We found a pattern of VNTB projections to CN, LSO and to CNIC that is consistent with its known tonotopy. That neurons in the VNTB in the rat are tonotopically organized is indicated by their labeling with the protooncogene, c-fos, as a consequence of stimulation with tones (Friauf, 1992). These latter data reveal a lateral to medial gradient of labeling corresponding, respectively, to low- versus high-frequency sound stimulation. In addition, there is a corresponding descending projection from CNIC to VNTB which is organized in a manner consistent with this tonotopy (Caicedo and Herbert, 1993). Although this predominantly ipsilateral descending projection is most profuse in the region containing VNTB neurons projecting to the CN (Faye-Lund, 1986; Caicedo and Herbert, 1993; Thompson and Thompson, 1993), it also contacts the dendrites of medial olivocochlear neurons, as well (Vetter et al., 1993).

Acknowledgements Supported in part by research Grants 5 RO1 DC 00372 and P50 DC 00215 from the National Institute on Deafness and Other Communication Disorders, National Institutes of Health. We gratefully acknowledge the contributions of Kevin S. Spangler, who participated in the initial phases of the study, and of Dolores Lopez for her advice in using the BDA method. We also thank Laura Bruce and Bernd Fritzsch for their comments on the manuscript. I thank Diane Schmidt, Skip Kennedy and LaVon Bowman of the Media Graphics Department at BTNRH for their able computer graphics work.

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