THE JOURNAL OF COMPARATIVE NEUROLOGY 373255-270 ( 1996)

Glutamatergic Connections of the Auditory Midbrain: Selective Uptake and Axonal

Transport of D-[3H]Aspartate

RICHARD L. SAINT MARIE Department of Neuroanatomy, House Ear Institute, Los Angeles, California 90057 and

Department of Otolaryngology-HNS, University of Southern California School of Medicine, Los Angeles, California 90033

ABSTRACT D-L3H]aspartate was used to identify potential glutamatergic connections of the chinchilla

inferior colliculus (10. High-affinity uptake of D-[3Hlaspartate is considered a selective marker for glutamatergic synapses, and neurons retrogradely labeled from such injections are believed to use glutamate, or a closely related compound, as a transmitter. Injections of D-[3H]aspartate suggest that glutamatergic endings in the IC arise primarily from intrinsic connections, the opposite IC, layer 5 of temporal cortex, nucleus sagulum, and lzteral lemniscal nuclei. Neurons giving rise to the principal sensory (lemniscal) projections to the IC, i.e., those from the cochlear nuclei, superior olive, and the majority of projections from the lateral lemniscal nuclei, did not label in these experiments, indicating that their synapses do not recognize D-[3H]aspartate as a suitable substrate and may use inhibitory or other excitatory transmitters. After IC injections, fiber and diffuse labeling was found ipsilaterally in the medial geniculate body, superior colliculus, and dorsolateral pontine nuclei, contralaterally in the IC, and bilaterally in the superior olive and cochlear nuclei. Such labeling was attributed to anterograde transport of D-L3H1aspartate within the efferent collaterals of labeled IC neurons, suggesting that many of the IC’s efferent projections may also be glutamatergic. This interpretation was confirmed in separate experiments in which D-[3H]aspartate, injected in the medial geniculate body, retrogradely labeled neurons in the IC as well as in layer 6 of temporal cortex. Finally, the mesencephalic trigeminal nucleus and tract labeled in some cases and may have local glu- tamatergic connections.

Indexing terms: excitatory neurotransmitters, chinchilla, inferior colliculus, medial geniculate body,

‘i 1996 Wiley-Liss, Ine.

mesencephalic trigeminal nucleus

The inferior colliculus (IC) is an obligatory terminus for nearly all of the ascending and descending pathways of the central auditory system. These include bilateral projections from the ventral and dorsal cochlear nuclei, lateral superior olive, periolivary nuclei, and dorsal nuclei of the lateral lemniscus; and ipsilateral projections from the medial superior olive and the ventral and intermediate nuclei of the lateral lemniscus (see Aitkin, 1986; Irvine, 1986; Oliver and Shneiderman, 1991; Oliver and Huerta, 1992). Addition- ally, extensive commissural projections connect the IC’s on both sides. Ascending pathways continue as the IC projects to the medial geniculate body, which in turn projects to auditory cortex. Descending pathways project from audi- tory cortex to the IC and from the IC to the cochlear and superior olivary nuclei. Despite our considerable knowledge of the anatomy of these connections, the transmitters that they use are known in only a few cases, primarily those which use the inhibitory transmitters, y-aminobutyric acid

(GABA), and glycine (e.g., Adams and Mugnaini, 1984; Hutson, 1988; Saint Marie et al., 1989; Saint Marie and Baker, 1990; Glendenning et a1.,1992; Shneiderman et al., 1993; Oliver et al., 1994; Winer et al., 1996).

Glutamate (or a closely related compound) has been shown to be a major excitatory transmitter in the IC. Iontophoresis of glutamate or the glutamate analogs, aspar- tate and N-methyl D-aspartate (NMDA), increases activity and lowers acoustic thresholds of many IC neurons (Fain- gold et al., 1989, 1991). Postsynaptic receptors for gluta- mate have been localized in the IC pharmacologically and

Accepted May 10, 1996. This manuscript is dedicated belatedly to Alan Peters on the occasion of

his sixty-fifth birthday (J. Comp. Neurol. 355: 1-51, Address reprint requests to Dr. Richard L. Saint Marie, Neuroanatomy

Department, House Ear Institute, 2100 West Third Street, Los Angeles, CA 90057.

i 1996 WILEY-LISS, INC.

256 R.L. SAINT MARIE

with receptor binding and immunocytochemistry (e.g., Fain- gold et al., 1989, 1991; Petralia and Wenthold, 1992; Petralia et al., 1994a,b). Significant levels of glutamate and aspartate are found in the IC (Adams and Wenthold, 1979; Ottersen and Storm-Mathisen, 1984; Banay-Schwartz et al., 1989, 1992), as are enzymes for their synthesis (Kaneko et al., 1989; Najlerahim et al., 1990). Intrinsic connections (Ottersen and Storm-Mathisen, 19841, ascending projec- tions from the contralateral lateral superior olive (Glenden- ning et al., 1992), commissural projections from the oppo- site IC (Smith, 1992), and descending projections from auditory cortex (Adams and Wenthold, 1979; Feliciano and Potashner, 1995) have all been implicated as possible sources of glutamatergic synapses in the IC.

In the present study, D-L3HJaspartate was injected in the IC or medial geniculate body to identify possible glutamater- gic connections of the auditory midbrain. D-aspartate is an analog of L-glutamate (and L-aspartate) that is selectively taken up by high-affinity transporters expressed on presyn- aptic endings that use L-glutamate or a closely related dicarboxylic acid as a transmitter (Davies and Johnston, 1976). Once inside the presynaptic ending, some of the D-["HJaspartate is transported back to the parent cell body, where, because it is poorly metabolized, it accumulates and can be detected autoradiographically (Streit, 1980; Cuenod et al., 1981, 1982; Cuenod and Streit, 1983). The selectivity of D-I 3H laspartate as a transmitter-specific retrograde marker has been demonstrated in many brain pathways (e.g., Cuenod et al., 1982; Ottersen and Storm-Mathisen, 1984), including the cochlear nerve and lateral olivoco- chlear projection (Oliver et al., 1983; Ryan et al., 1987, 19901, in each case corroborating other evidence that the projections are glutamatergic.

MATERIALS AND METHODS Fourteen chinchillas (Chinchilla laniger, 400-600 g, 9

female and 3 male) were injected with 3.2-6.4 pCi of D-13HJaspartate (New England Nuclear, Boston, MA; spe- cific activity: 13.5 Ciimmol). Eleven received unilateral injections in the IC and three received unilateral injections in the medial geniculate body. The tracer was concentrated by evaporation under a constant stream of dry Nitrogen gas and reconstituted at 40 pCiIp1 (3 mM) in sterile normal saline. This concentration was chosen to increase the intensity of the transported signal and thereby reduce the long autoradiographic exposures times. Concentrations of 3-10 mM have been shown to be selectively transported by glutamatergic neurons (e.g., Oliver et al., 1983; Bernays et al., 1988).

Prior to surgery and euthanasia, animals were deeply anesthetized with sodium pentobarbital (60 mgikg, i.p.1 and diazepam (1 mgikg, i.m.1. The depth of anesthesia was monitored by cutaneous nociceptive and corneal reflexes prior to and during the procedures and anesthetics were supplemented as required. Animals also received presurgi- cal injections of atropine sulfate (0.05 mg/kg, i.m.1. All protocols were pre-approved by an institutional animal care and use committee and appropriate veterinary care was provided by an accredited animal care facility.

After a partial craniotomy, parts of the occipital cortex were aspirated to expose the IC or medial geniculate on one side. Small pressure injections of D-[3HH]aspartate in saline were made using glass micropipets (20-40 pm tip diam- eters) attached to a calibrated, pulsed, air-pressure injec-

TABLE 1. Summary of D-l:'Hlaspartate Injections in the Inferior Colliculus and Medial Geniculate Body

Tracer Case Survival number DosaEc Volume Injection Site' time (hour)

Ch3101690 Chl102490 Ch1101690 Ch 11 12990 Ch1040991 Ch 1032691 Ch 10 1089 1 Ch1031391 Ch1031191 Ch2112690 Chl112890

Ch1062591 Ch2070191 Ch1070191

6.4 pCi 6.4 pCi

6.4 pct 6.4 pCi 6.4 pCi 6.4 WCI 6.4 pCi 6.4 pCi 4.8 pCi 4.0 $21

%2 pCf

3.2 pCi 3.2 pCi 3.2 pCi

Inferior colliculus injections . 1 6 ~ l DC IdeepIandCNidorsall 16 ~1 DC tdeepl and CN (dorsal,

.OH pl DC (deep1 and CN tdorsdi

.I6 pI DC ideepl and CN IdorsalJ

.16 pl CN (laterall

. I6 111 CN (laterall

. I6 pI CN icentrdl 16 p1 C N imediali

. I6 pl DC (caudal1

.12 pl DC (central)

. I0 pl DC irostral) Medial geniculate injections .08 pl Dorsal .OR pl Dorsomedial and pretectum .08 uI Central

3 6

12 24 12 18 14 12 16 48 24

3 12 20

'DC, dorsal cortex d 1 C . CN, central nucleus d I C

tion system (Picospritzer 11, General Valve Corp.). The location of the injection sites are summarized in Table 1. After survival times of 3 to 48 hours, animals were re- anesthetized and sacrificed by transcardial, vascular perfu- sion at 37°C. A brief perfusion with 50-100 ml of 0.1 M phosphate buffer (pH 7.2) to clear the vasculature of blood preceded perfusion with 500-750 ml of buffered 1% parafor- maldehyde and 3% glutaraldehyde. The brains were dis- sected after 1 hour and stored overnight in the buffered fixative at 4°C. Most brains were Vibratome-sectioned at 60 pm the next day. One IC case was stored in buffered fixative with 30% sucrose for 7 days, and then frozen in dry ice, and cut at 40 pm with a freezing microtome. Brain sections were mounted on glass slides, defatted, air dried, and coated with Kodak NTB-3 emulsion. Coated slides were exposed in the dark a t 4°C for 1 week to 32 months, depending on the strength of the signal. The emulsion was developed with Kodak D-19 developer, and the sections counterstained with Cresyl Violet, dehydrated, and coverslipped. Sections were viewed with brightfield or fiber-optic Darklite (MVI) illumination and photographed with a Nikon Microphot SA or Optiphot 11. Low power drawings of autoradiographically developed sections were made at ~ 4 0 with a Nikon Opti- phot I1 equipped with a drawing tube.

RESULTS D-r3H]aspartate injection sites in IC

The eleven cases with IC injections varied in several parameters, including the volume of tracer injected, sur- vival time, and the position of the injection sites (Table 1). In the core of the injection sites there was a dense accumu- lation of silver grains that did not permit neuropilar, somatic, or glial label to be distinguished. In the corona of the injection sites, on the other hand, there was a selective accumulation of radiolabel over neuronal profiles, including their dendrites, and over axons. A less dense, diffuse radiolabel was also apparent in the corona, which decreased with distance from the injection sites.

Intrinsic connections Many labeled neuronal somata and dendrites were found

in the corona of the IC injection sites. Several cases had injections placed centrally in the IC. The resulting injection

GLUTAMATERGIC CONNECTIONS OF AUDITORY MIDBRAIN 257

Fig. 1. Distribution of radiolabel in one case with a large injection of D-[?H]aspartate in the inferior colliculus (IC). Injection site (black core) was at the boundary of the deep dorsal cortex and superficial central nucleus, and included parts of both. Three kinds of radiolabel were plotted: diffuse 1 stippling), axonal (wavy lines), and somatic (black circles). The IC was divided into a dorsal cortex (DC) and lateral (L), dorsomedial (DM), commissural (CM), rostra1 (R), and central nuclei;

the central nucleus was further divided into medial (m), central (el, ventral (v), ventrolateral (vl), and lateral (1) parts after Morest and Oliver (1984). BIC, brachium of IC; CG, central gray; DNLL, dorsal nucleus of the lateral lemniscus; INLL, intermediate nucleus of the lateral lemniscus; PBG, parabigeminal nucleus; Sag, nucleus sagulum; SC, superior colliculus. (Case 1112990; 1 week exposure.) See text for details. Scale bar = 2 mm.

sites (Table 1) included the deep layers of the dorsal cortex and the dorsal part of the central nucleus. The injection site and distribution of radiolabel within the midbrain for one of these cases is illustrated in Figure 1. Labeled neuronal profiles were abundant within 200-300 pm of the injection sites in all directions. Additionally, diffuse label and labeled neurons and axons extended dorsomedially and rostrally

toward the IC commissure for up to 1 mm from the injection sites (e.g., Fig. 1). Many of the labeled neurons in this area were rounded or stellate, others were fusiform in shape (Fig. 2A). They ranged in size from 12 to 20 pm (ave. diam.). The predominant orientation of the labeled den- drites and fibers was ventrolateral to dorsomedial. Ventral to the injection sites, fiber, cellular, and diffuse label

258 R.L. SAINT MARIE

Figure 2

GLUTAMATERGIC CONNECTIONS OF AUDITORY MIDBRAIN 259

extended obliquely, up to 600 pm from the injection sites (e.g., Fig. l), sometimes reaching the ventralmost part of the central nucleus. The oblique orientation was reminis- cent of the fibrodendritic laminae described in this region of the IC in cats (Morest and Oliver, 1984; Oliver and Morest, 1984; Oliver and Shneiderman, 1991). Labeled fibers were also oriented obliquely,though some ran orthogonally. Many labeled somata and dendrites in the central nucleus showed no particular orientation and may have been stellate neu- rons (Fig. 2B). Others, particularly in the central and medial parts of the central nucleus, were orientated ventro- lateral to dorsomedial along the presumed fibrodendritic laminae and appeared to be disc-shaped neurons. The medial part of the central nucleus, which was labeled by more medial injections, contained the greatest proportion of labeled neuronal somata and dendrites with this orienta- tion. Labeled somata ranged in size from 10 to 15 pm (average diameter) in the central, ventral, and medial parts of the central nucleus, but a few neurons as large as 18-25 pm were also found.

IC neurons also labeled at a distance from the injection sites in these experiments (e.g., Fig. 1). After injections that included the central part of the central nucleus, clusters of labeled neurons were often observed in the ventral and lateral parts of the central nucleus, and in areas correspond- ing to the lateral (Fig. 2C), ventrolateral, dorsomedial, and rostral nuclei (Fig. 2E) described in the cat IC (Morest and Oliver, 1984; Oliver and Morest, 1984). Fiber and diffuse label were also elevated in these areas. Typically, regions of low label separated the latter areas from each other and from the injection site corona. Labeled neurons in the lateral part tended to be oriented orthogonal to the orienta- tion of found in the central and medial parts of the central nucleus, which was also reminiscent of the organization described in the IC of cats (Morest and Oliver, 1984; Oliver and Morest, 1984). Additionally, there appeared to be labeled projections from the central part to the medial part of the central nucleus and a heavy projection from the lateral nucleus to lateral parts of the central nucleus. These latter could not be known with certainty, however, because the injection sites in these cases spread partly into the presumed projection area, and local connections could have contributed some or all of the observed retrograde label. Except from areas immediately contiguous to the injection sites, extensive intracollicular projections to the dorsal cortex were not observed.

Other midbrain connections In the IC contralateral to the injections, diffuse label and

labeled fibers and neurons were always observed at a

Fig. 2. Brightfield (A and F) and darkfield (B-E, G , and H) autoradiographs of labeled midbrain structures. A: Commissural nucleus of the IC contains many radiolabeled neurons with large round, small round, or small spindle-shaped somata. B: Radiolabeled neurons in the central part of the IC central nucleus have predominantly small round somata and dendrites oriented obliquely. C: Radiolabeled neurons in the IC lateral nucleus are more stellate in appearance. D: In the contralateral IC, a solitary labeled cell and its presumed dendrites resemble those of a “disk-shaped’’ neuron. E: Fiber, diffuse, and somatic label extends into the rostral nucleus of the IC. F: A dense diffuse radioactivity and many labeled neurons (arrows) fill the nucleus sagulum after injection of the IC dorsal cortex. G: Neurons of the MeV are intensely labeled after a ventral medial injection of the IC. Cases: A, C, and D (1112990); B (1101690); E (3101690); F (1031191); and G (1031391). Scale bar = 125 pm in A-E, 150 pm in F, 200 pm in G.

position that mirrored that of the injection site (e.g., Fig. 1). The contralateral label, though weaker in intensity and numbers of neurons, also included areas that corresponded to the regions within the corona of the injection site, ipsilaterally. The weaker label could be compensated for by increasing the autoradiographic exposure times for the contralateral IC, but the numbers of labeled neurons contralaterally still remained many fewer than those ob- served ipsilaterally. Intranuclear connections that labeled ipsilaterally after injections of the central part of the central nucleus (e.g., those from the ventral and lateral parts of the central nucleus and from the lateral, ventrolat- eral, dorsomedial, rostral pole, and commissural nuclei of the IC) were often mirrored contralaterally, though less intensely and in far fewer numbers. One notable difference was that a greater proportion of labeled neurons in the central, medial, and lateral parts of the contralateral cen- tral nucleus were fusiform shaped, oriented parallel to presumed local fibrodendritic laminae, and were probably disc-shaped neurons (Fig. 2D). The course of the contralat- eral projection was via the IC commissure, which contained many labeled decussating fibers (Fig. 1). No labeled fibers were found in the commissure of Probst.

Other midbrain projections that transported the D-i3H]as- partate from the IC injections included those of the nucleus sagulum, intercollicular tegmentum, cuneiform and subcol- licular areas, central gray, and the mesencephalic trigemi- nal nucleus (MeV). Projections from the nucleus sagulum, intercollicular tegmentum, and central gray were bilateral, though the ipsilateral projection was always much heavier, both in numbers of neurons and intensity of label. Labeled neurons in the nucleus sagulum were observed only after the dorsal cortex of the IC was injected (Fig. 2F). A dense accumulation of labeled fibers was evident in the nucleus sagulum after all IC injections and these continued ros- trally to join the brachium of the IC (Fig. 1). The intercol- licular tegmentum, which is wedged between the bodies of the inferior and superior colliculi (Morest and Oliver, 1984), contained much diffuse label as well as many labeled fibers and some neurons after injections of the central nucleus, but not the dorsal cortex. Neurons and fibers of the adjacent central gray also labeled bilaterally, especially after injections of the central nucleus and even when well removed from the apparent injection site (Fig. 1). After injections of the deep dorsal cortex, many labeled fibers were found projecting mediolaterally through layers I11 and IV of the superior colliculus, bilaterally (Fig. 1). A few extended vertically into layer I1 and appeared to terminate there. Contralaterally, labeled fibers joined the brachium of the IC. No labeled neurons were found in the superior colliculi on either side. Some neurons also labeled in the ipsilateral cuneiform and subcollicular areas after injec- tions of the central nucleus. After the most medial injec- tions, cell bodies and fibers of the MeV and its tract were sometimes labeled (Fig. 2G). Thick labeled fibers extended caudally to the motor nucleus of the trigeminal, which contained diffuse and fine-fiber labeling. Labeling of the MeV and its tract was most likely due to the ventral and medial diffusion of the tracer into the periaqueductal gray and the MeV itself, and not to an auditory projection.

Hindbrain connections After large injections that involved the deep dorsal cortex

and the superficial central nucleus, labeled fibers accumu- lated ventrolaterally in the injected IC and converged

260 R.L. SAINT MARIE

Fig. 3. Distribution of fiber (wavy lines) and somatic (black circles) radiolabel in and around the lateral lemniscus (LL; and its dorsal, DNLL; intermediate, INLL; and ventral, VNLL, nuclei) after an injection of the ipsilateral dorsal cortex and central nucleus of the IC. Sections proceed from rostral (extreme right) to caudal (extreme left).

toward the lateral lemniscus (e.g., Fig. 1). The fibers penetrated the dorsal nucleus of the lateral lemniscal (DNLL), but no labeled neurons were found there (Fig. 3). More ventrally, labeled fibers were found in the rostral, caudal, lateral, and medial limbs of the lateral lemniscus, but were most concentrated laterally and rostrally. The labeled fibers completely encapsulated the intermediate (INLL) and ventral (VNLL) nuclei of the lateral lemniscus. Medially, labeled fibers also extended through the paralem- niscus. Labeled neurons were evident in INLL and VNLL after as little as 3 hours survival. Some were scattered in these nuclei, but most were found in clusters near the boundary of INLL and VNLL (Fig. 4A) and along the medial border of VNLL (Fig. 4B). The majority of neurons in the core of INLL and VNLL were unlabeled. Near the boundary of INLL and VNLL, labeled fibers sometimes formed a ladder-like arrangement of horizontal bands that penetrated the nuclei and mingled with labeled horizontally oriented neurons and dendrites (Fig. 4A). Near the ventral extreme of the lemniscus, many fibers in the dorsolateral cortical spinal tract were also labeled (Figs. 3, 4 0 . Some fiber and diffuse label extended into the dorsolateral pon- tine nuclei. A few labeled fibers were found contralaterally in the lateral lemniscus, paralemniscus, and dorsolateral corticospinal tract and pontine nuclei. A few labeled neu- rons were sometimes found in the contralateral INLL.

After 6 or more hours of survival, labeled fibers formed a capsule around the ipsilateral superior olivary complex (Fig. 5). Ventrally, labeled fibers passed within the trap- ezoid body and formed a dense plexus immediately ventral to the medial superior olive and a second more extensive plexus more medially in the ventral nucleus of the trapezoid body (Fig. 4D). A less extensive band of labeled fibers projected dorsally in the dorsolateral, dorsal, and dorsome- dial periolivary nuclei (superior paraolivary nucleus of rats) (Fig. 5). After 12 or more hours some labeled fibers ex-

CT, corticospinal tract; MCP, middle cerebellar peduncle; PL, paralem- niscus; PN, pontine nuclei; Sag, nucleus sagulum; SCP, superior cerebellar peduncle. (Case: 1112990; 4 month exposure). See text for details. Scale bar = 2 mm.

tended across the midline of the trapezoid body and formed similar, though much less dense, fiber plexuses in the contra- lateral superior olivary complex. Labeled fibers extended dorsolaterally in the ipsilateral trapezoid body and entered the cochlear nucleus medially as multiple fascicles at the levels of the posteroventral and dorsal cochlear nuclei (Figs. 4E, 5 ) . Diffuse label and sparse fiber label were evident in the external granular layer of the ventral cochlear nucleus and in the fusiform and deep layers of the dorsal cochlear nucleus (Fig. 4F). By 24 hours the contralateral cochlear nucleus was similarly labeled (Fig. 5). Injections of the caudal dorsal cortex of the IC produced the most intense fiber and diffuse label in the superior olivary complex, but injections confined to the central nucleus of the IC also produced good labeling there. Other differences in the intensity or topography of labeling in the superior olivary complex or cochlear nuclei were not consistently observed. Notably, no labeled neurons were ever found in the superior olivary complex or cochlear nuclei after survival times up to 24 hours and autoradiographic exposure times as long as 1 year. The absence of labeled neurons in these hindbrain structures could not be attributed to insufficient transport time, since 12 hour survival times were adequate to produce prominent fiber and diffuse labeling in the cochlear nuclei and superior olive and to retrogradely label neurons in the more distant temporal cortex (see below).

Forebrain connections Labeled fibers in the injected IC were concentrated in the

external layers of the dorsal and caudal cortices and in the lateral nucleus. These converged and extended rostrally into the brachium of the IC (e.g., Fig. 1). A few neurons were labeled ipsilaterally in the brachial nucleus. Labeled brachial fibers were joined by a separate concentration of labeled fibers that extended in the dorsal pyramidal tract, the adjacent rostral limb of the lateral lemniscus, and the

GLUTAMATERGIC CONNECTIONS OF AUDITORY MIDBRAIN 261

Fig. 4 Darkfield autoradiographs of labeled hindbrain structures. A: Diffuse, fiber, and somatic radiolabel in the intermediate nucleus of the lateral lemniscus forms a ladder-like arrangement, which extends horizontally between the vertically oriented lemniscal fibers. B: In the ventral nucleus of the lateral lemniscus, a cluster of radiolabeled neurons appears medially (upper left), but the core of the nucleus (bottom half) is unlabeled. C: Radiolabled fibers at the ventral extreme of the lateral lemniscus (LL) are continuous with similarly labeled fibers in the lateral corticospinal tract (CT). Some fiber and diffuse label

is also evident in the lateral pontine nucleus (PN). D: Dense fiber and diffuse label forms two patches in the ventral nucleus of the trapezoid body, but no labeled somata are found here. E: Fascicles of radiolabeled fibers extend dorsally between the anteroventral cochlear nucleus (AVCN) (right) and the restiform body (left). F: The latter fibers eventually give rise to fiber and diffuse label in inframolecular layers of dorsal cochlear nucleus (DCN). Cases: A (3101690); Band C (1101690); D-F (1 112990). Scale bar = 200 pm in A-E, 320 pm in F.

262 R.L. SAINT MARIE

Fig. 5. Distribution of radiolabeled fibers in the superior olivary and cochlear nuclei after injection of the dorsal cortex and central nucleus of the IC. Labeled fibers in the superior olive are concentrated in periolivary regions bilaterally, especially in the ipsilateral ventral nucleus of the trapezoid body (VNTB), with sparse label in the principal nuclei, the lateral superior olive (LSO), medial superior olive (MSO), and medial nucleus of the trapezoid body (MNTB). Some fiber label continues dorsolaterally in the trapezoid body, passes medial to the

lateral surface of the tegmentum. These fibers passed around the parabigeminal nucleus as they extended dor- sally and rostrally. Some joined the much more numerous brachial fibers as they entered the medial geniculate body (MGB), while others continued rostrally in the cerebral peduncle (Fig. 6A). Labeled fibers extended between the peduncle and brachium throughout most of the rostrali caudal extent of the MGB. Within the MGB there was a dense concentration of diffuse and fiber label in the bra- chium after survivals as short as 3 hours. Extensive fiber and diffuse label in the ventolateral and dorsal nuclei of the MGB (after Morest, 1964) resulted from injections of the IC dorsal cortex. Injections of the IC central nucleus, on the other hand, usually resulted in heavy fiber and diffuse label in regions analogous to the ventral, ovoidal, and medial

AVCN, and terminates in the inframolecular layers of the DCN, bilaterally. Only sparse label is evident in AVCN and posteroventral cochlear nucleus (PVCN). 5, spinal trigeminal nucleus; 5T, spinal trigeminal tract; 7, facial nucleus; 7N, facial nerve; CT, corticospinal tract; ICP, inferior cerebellar peduncle; V, vestibular nerve root. (Case: 1112990; 12 month exposure). See text for additional details. Scale bar = 2 mm.

nuclei described in the cat MGB (Morest, 1964). Labeled neurons were only rarely observed in the MGB after IC injections.

Cerebral cortical projections were traced in three cases, which were selected because of their strong fiber label in the cerebral peduncle at the level of the MGB. Survivals ranged from 12 to 24 hours and each had a good injection of the IC dorsal cortex. The projections are illustrated for one of the cases in Figure 7. Beyond the MGB, labeled fibers contin- ued in the dorsal-most part of the cerebral peduncle and could be traced into the internal capsule, where they passed through the dorsal aspect of the putamen and into the temporal cortex. Lateral to the putamen, the fibers first extended dorsally, then flexed back ventrally and laterally to enter the cortex. Large layer 5 pyramidal neurons were

GLUTAMATERGIC CONNECTIONS OF AUDITORY MIDBRAIN 263

Fig. 6. Darkfield autoradiographs of labeled forebrain structures. A: Dense accumulations of fiber and diffuse label are evident in parts of the ventral nucleus (v) of the medial geniculate body (MGB), with more moderate levels present in the medial (m) and dorsal (d) nuclei. A dense concentration of labeled fibers can be seen in the dorsolateral part of the

strongly labeled in the temporal cortex (also Fig. 6B). Label continued to the surface of the cortex and much of this could be attributed to the apical dendrites of the labeled layer 5 neurons. In some cases the terminal tufts of labeled apical dendrites could be seen bifurcating in layer 1 (Fig. 6C).

Contralaterally, many of the labeled fibers crossing cau- dally in the commissure of the IC formed an external capsule around the IC, being confined primarily to the lateral nucleus and the superficial (molecular) layer of the dorsal and caudal cortices (Fig. 1). These fibers converged rostally in the brachium of the IC with other labeled fibers crossing through the caudal superior colliculus. Fiber and diffuse label in the contralateral MGB mirrored that in the MGB on the injected side, but was very much less intense. Labeled fibers decussating rostrally in the superior collicu- lus streamed ventrally, medial to the MGB, and joined other labeled fibers in the dorsal part of the cerebral peduncle. These fibers were many fewer in number than those ipsilateral to the injection and were not followed any farther. They were presumably the axons of layer 5 pyrami-

corticospinal tract (CT) at this level. B: A cluster of layer 5 pyramidal neurons (arrows) and their apical and basal dendrites are labeled in temporal cortex. C: The labeled apical dendrites continue to layer 1 where they end in characteristic terminal tufts (arrows). Cases: A-C (1112990). Scale bar = 600 pm in A, 120 pm in B, 150 bm in C.

dal neurons in the temporal cortex, contralateral to the injections.

D-[3Hlaspartate injections in MGB One interpretation of the fiber and diffuse label found in

the MGB and in the cochlear nuclei, superior olive, and dorsal pontine nuclei after D-l3H]aspartate injections in the IC is that the efferent collaterals of labeled intrinsic IC neurons also become labeled over time by a combination of retrograde and anterograde transport mechanisms. It is known that many IC neurons have local connections, including those with efferent projections (Oliver et al., 1991). If such neurons are glutamatergic then they should be able to accumulate D-["HJaspartate from their efferent targets as well as from their local connections in the IC. To test this hypothesis, D-13Hlaspartate was injected in the MGB in three additional cases. One case had a small injection in the dorsal part of the MGB, which labeled neurons bilaterally and caudally in the dorsal cortex of the IC. Diffuse and fiber label was found in the caudal parts of

264 R.L. SAINT MARIE

Fig. 7. Distribution radiolabel in temporal cortex. Labeled fibers (wavy lines) pass through the internal capsule, the dorsal part of the putamen tPu), and the deep layers (5 and 6 ) of the cerebral cortex. The cells of origin are layer 5 pyramidal neurons (black circles), whose labeled apical dendrites extend through the superficial cortical layers to the surface of the cortex. Ca, caudate nucleus; LV, lateral ventricle. (Case 1112990,8 month exposure). See text for additional details. Scale bar = 2 mm.

the brachium and the sagulum. The survival time in this case was only 3 hours and no more distant connections were labeled. A second case had an injection dorsomedially in the MGB and included the pretectal area. This case produced strong retrograde labeling of layer 6 neurons in the tempo- ral and occipital cortices, but no labeling was found in the IC. A third case had an injection in the core of the MGB, near the border of the dorsal and ventral parts of the MGB, just lateral to the fibers of the brachium (Fig. 8A). This case produced strong labeling of neurons in the ipsilateral IC, near the border of the dorsal cortex and central nucleus (Fig. 8B). Labeled neurons were also found scattered throughout the central nucleus, in the lateral nucleus, and in the rostra1 IC. Fiber and diffuse label filled the brachium, ipsilaterally, and continued within the lateral nucleus and into the sagulum. Fiber labeling was scattered throughout

the ipsilateral IC and in the commissure. Labeled cells and fibers, though fewer in number, were also found in the contralateral IC, near the border of the dorsal cortex and central nucleus. A few labeled neurons were found ipsilater- ally in the sagulum, the cuneiform nucleus, and the periaq- ueductal gray. Rostrally, this injection produced strong retrograde labeling of small, layer 6, pyramidal neurons in the temporal cortex (Fig. 8C). The labeled apical dendrites of these neurons extended to the superficial layers of the cortex, and dense bands of diffuse label, presumably the result of labeled axon collaterals, were seen in layers 6 and 4 (Fig. 8D).

DISCUSSION Methodology

D-L3H]aspartate has been widely used as a retrograde marker of glutamatergic projections since its competitive uptake by high-affinity L-glutamate and L-aspartate synap- tic transporters was first described (Balcar and Johnston, 1972; Davies and Johnston, 1976; see also Danbolt, 1994; Storm-Mathisen et al., 1995; Fonnum and Hassel, 1995). Its advantage as a retrograde tracer is that, unlike the L-isomers of the dicarboxylic amino acids, glutamate and aspartate, D-L3Hlaspartate is slowly metabolized. It sur- vives in neurons long enough to be transported to the cell body (and along axon collaterals, see below), where it accumulates and can be detected autoradiographically (Streit, 1980; Cuenod et al., 1981, 1982; Cuenod and Streit, 1983). Retrograde transport of D-L3Hlaspartate has been shown to selectively label brain pathways where there is considerable other evidence that glutamate is the transmit- ter. Among these are the primary sensory afferents of the visual, auditory, and somatic sensory systems (including the MeV in the present study), as well as colliculothalamic. thalamocortical, striatal-pallidal, olivocerebellar, hippocam. pal mossy fiber, and many cerebral corticofugal projections, including corticocortical, corticostriatal, corticoamygdaloid, corticothalamic, corticopontine, corticocollicular, corticospi . nal, and corticorubral connections (reviewed in Cuenod et al., 1982; Ottersen and Storm-Mathisen, 1984; see also Bernays et al., 1988; Beart et al., 1990; Carnes et al., 1990; Amaral and Insausti, 1992; Johnson and Burkhalter, 1992, 1994; Ray et al., 1992; Pirot et al., 1994; White et al., 1994).

The present results are consistent with the interpreta- tion that D-L3Hlaspartate is selectively taken up by a subpopulation of presynaptic endings in the IC and trans- ported retrogradely to their parent cell bodies, both within and beyond the injected IC. For example, neurons in the adjacent and contralateral dorsal nuclei of the lateral lemniscus, which project to the IC but are thought to use the inhibitory transmitter, GABA (e.g., Adams and Mug- naini, 1984; Hutson, 1988; Shneiderman et al., 1993), did not transport D-L3H1aspartate in the present study. These and other projections not labeled in the present study are discussed in detail below.

The present results are also consistent with observations that, once inside a neuron, D-l3Hlaspartate can be trans. ported anterogradely from axonal branch points to label collateral terminal fields of the parent neuron (e.g., Baugh man and Gilbert, 1980, 1981; Beaudet et al., 1981; Cuenod et al., 1981). Baughman and Gilbert found that when layei 6 neurons in visual cortex were retrogradely labeled witk, D-l 3H]aspartate after injections of the lateral geniculatc nucleus, there was also a diffuse label present in cortica

GLUTAMATERGIC CONNECTIONS OF AUDITORY MIDBRAIN 265

Fig. 8. Results of D-I:iHlaspartate injection in the MGB (Case 1070191 1. A: Brightfield autoradiograph showing the size and position of the injection, just lateral to the brachial fibers (br) and near the junction of the dorsal and ventral MGB subdivisions, LGB, lateral geniculate body, m, medial subdivision of the MGB. d, dorsal subdivi- sion of the MGB; v, ventral subdivision of the MGB. B: Darkfield autoradiograph showing diffuse and fiber label and retrogradely labeled neurons (arrows) near the border of the dorsal and ventral subdivisions

of the ipsilateral IC. C: Retrogradely labeled pyramidal neurons in layer 6 of the temporal cerebral cortex. Apical dendrites are prominently labeled. D: At lower magnification, the apical dendrites can be seen to extend into the supragranular layers of the cortex. Two prominent bands of diffuse label are seen in layers 4 and 6 of the cortex and these can be attributed to the labeled axon collaterals of the layer 6 neurons. Scale bar = 750 pm in A, 200 pm in B and C, 375 pm in D.

266 R.L. SAINT MARIE

layer 4, indicative of the local interlaminar connections of the labeled layer 6 cells. Conversely, following cortical injections, they found diffuse but not cellular label in the lateral geniculate nucleus. They attributed the latter to anterograde transport within the efferent collaterals of layer 6 neurons, which took up the D-L3H1aspartate by way of their local axon collaterals. A similar diffuse labeling in layer 4 of temporal cortex was observed in the present study after retrograde labeling of layer 6 neurons with projections to the medial geniculate body. Significantly, most if not all of the efferent targets of the IC contained fiber and diffuse label after injections of D-[”]aspartate in the IC. This included efferent projections to the MGB, contralateral IC, superior olive, cochlear nuclei, and dorsolateral pontine nucleus. The inference is that glutamate may be the principal efferent transmitter of the IC. In the case of the colliculocollicular and colliculogeniculate projections, this was confirmed in the present study by retrogradely labeling IC neurons after injections of D-L3Hlaspartate in the contra- lateral IC or the MGB.

Labeling of the MeV and its tract in the present study is also consistent with this interpretation. Recently it has been shown that some MeV neurons in the rat have fine locally ramifying processes, which resemble axons and which have terminal-like boutons (Luo et al., 1991; Ra- apana and Arvidsson, 1993). These processes ramify near the somata and in the neighboring periaqueductal gray, areas that were included in the injection sites of the most medially placed injections of the present study. Uptake of D-[“H]aspartate by synapses of the local axons could ex- plain the somatic labeling of the MeV neurons, and trans- port by collateral axons could explain the labeling of the MeV tract fibers and their terminations in the motor nucleus of the trigeminal. This labeling suggests that these somatosensory neurons may also be glutamatergic. The numbers of labeled MeV neurons in the present study, however, suggest that MeV neurons with locally ramifying axons may be quite prevalent.

Glutamatergic connections of the IC Many kinds of neurons in the IC accumulated exogenous

D-L3HJaspartate in the present study and are probably glutamatergic. Neurons in all of the principal subdivisions of the IC were labeled. These included small to large neurons, presumed disc-shaped and stellate neurons, neu- rons with local axonal plexuses, those with longer intra- nuclear projections that interconnect the different subdivi- sions of the IC, and commissural neurons that connect the two IC’s. The principal efferent projections of the IC also appeared to label (discussed above). This is consistent with findings that many IC neurons contain elevated concentra- tions of glutamate and the enzymes for glutamate and/or aspartate synthesis (Ottersen and Storm-Mathisen, 1984; Kaneko et al., 1989; Najlerahim et al., 1990).

The large number of labeled neurons immediately sur- rounding the injection sites in the dorsal cortex and central nucleus of the IC in the present study were labeled presum- ably through their local axonal connections. Injections of the central part of the central nucleus (and deep dorsal cortex) produced distinct clusters of labeled neurons in the ventral, ventrolateral, and lateral parts of the central nucleus and in the neighboring lateral, dorsomedial, com- missural, and rostra1 nuclei of the IC. This suggests that many of the connections between subdivisions of the IC may be glutamatergic. Another consistent feature of these

experiments was the large number of labeled neurons in the opposite IC, supporting the proposition of Smith (1992) that commissural IC projections provide monosynaptic excitation by way of glutamatergic synapses. Somatic up- take of D-13HJaspartate by neurons in the injection site, an alternative explanation for the most immediate labeling, has not been previously reported (e.g., Baughman and Gilbert, 1980, 1981; Cuenod et al., 1881). Oliver et al. (1983) used tracer concentrations identical to those em- ployed here and found no somatic labeling of principal neurons in the ventral cochlear nucleus, though radiola- beled glia, axons, and terminal plexuses were plentiful in their injection sites. Moreover, injections of MGB in the present study failed to produce somatic labeling of neurons in or around the injection sites, as would be expected from indiscriminate somatic uptake. The lack of somatic labeling in and around the injection sites by principal neurons of the ventral cochlear nucleus and MGB may indicate the ab- sence of glutamatergic neurons there or, if present, they may not possess local synaptic plexuses by which to accumu- late the transmitter analogue.

Many of the efferent projections of the IC may also be glutamatergic, including those to the MGB, opposite IC (described above), superior olivary complex, cochlear nu- clei, and the dorsolateral pontine nucleus. Once inside neurons, D-L3H]aspartate can be transported anterogradely to collateral target fields (e.g., Baughman and Gilbert, 1980, 1981; Beaudet et al., 1981; Cuenod et al., 1981). This may account for the considerable diffuse label observed in the MGB after IC injections. Since there were no labeled neurons in the MGB, its most likely source was the anterogradely labeled collateral fields of labeled neurons in the injected IC. The MGB is the principal target of IC neurons, both disc shaped and stellate (Oliver, 1984b). Both neuronal types appeared to label in the present experiments and, in addition to their efferent projections, both types have extensive local collaterals (e.g., Oliver et al., 19911, through which they could have accumulated D-L3H]aspar- tate. The synaptic morphology of the colliculogeniculate projection is one that has been associated with excitatory transmission (i.e., asymmetric, round vesicle; Jones and Rockel, 19711, and recently Hu et al. (1994) demonstrated that IC projections provide a fast excitatory pathway to the MGB, which is mediated by glutamate acting on both NMDA and non-NMDA receptors. In the present study, the preferential distribution of radiolabel in regions analogous to the ventral, ovoidal, and medial subdivisions of the MGB after IC central nucleus injections, and in regions analo- gous to the dorsal and ventrolateral subdivisions of the MGB after IC dorsal cortex injections was consistent with the topography of the colliculogeniculate projection de- scribed in cats (e.g., Aitkin, 1986; Oliver and Shneiderman, 1991; Oliver and Huerta, 1992). Finally, injections of D-L3Hlaspartate in the MGB in the present study confirmed the presence of a glutamatergic colliculothalamic projec- tion. This projection presumably parallels the smaller GABAergic projection recently described in cats (Winer et al., 1996).

Fiber and diffuse label were routinely observed in the dorsolateral pons after 3-hour survivals, in the superior olivary complex after 6 hours, and in the cochlear nucleus after 12 hours. Since no labeled cells were observed in any of these regions, the origin of this label must have been from projections originating elsewhere. For example, the bilateral label in the superior olivary complex, with its

GLUTAMATERGIC CONNECTIONS OF AUDITORY MIDBRAIN 267

preponderance in the ipsilateral ventral nucleus of the trapezoid body, was characteristic of the descending col- liculo-olivary projection (recently treated by Caicedo and Herbert, 1993; Thompson and Thompson, 1993; Vetter et al., 1993). The IC also has robust descending projections to the cochlear nuclei, and the distribution of labeled fibers in the lateral limb of the ipsilateral lateral lemniscus and in the trapezoid body bilaterally was consistent with the trajectory of this pathway (reviewed by Saldana, 1993). As in the present study, the bulk of this projection terminates in the dorsal cochlear nucleus and granule cell area of the ventral cochlear nucleus, bilaterally. Degeneration studies showed that IC projections provide excitatory-like synapses (round vesicle, asymmetric contact) with the dendrites of fusiform cells and granule cells in the dorsal cochlear nucleus (Kane, 1977). Alternatively, fiber and diffuse label in the superior olive and cochlear nuclei may have origi- nated from collaterals of labeled auditory corticocollicular neurons. Feliciano et al. (1995a) describe projections from auditory cortex to the superior olive and cochlear nucleus (see also, Weedman et al., 1994, 1995). Lesions of descend- ing projections (including presumably those from the IC and auditory cortex) have failed to show deficits of D-[3HJas- partate uptake and release in the cochlear nuclei (Staatz- Benson and Potashner, 1988), but the proportion of gluta- matergic endings relative to those of the cochlear nerve and intrinsic granule cell population (Oliver et al., 1983) may have been too small to be detected in that study. The dorsolateral pontine nuclei are innervated by the auditory cortex, intercollicular tegmentum, and extracentral parts of the IC (e.g., Hashikawa, 1983; Huffman and Henson, 1990; Spangler and Warr, 1991). Labeled fibers found dorsally in the cerebral peduncle at midbrain and pontine levels were indicative of a corticopontine projection. The intercollicular zone and extracentral parts of the IC also contained labeled neurons and may have contributed labeled endings in the pons.

Several afferent projections to the IC accumulated D-[”]aspartate in the present study and may be glutama- tergic. These included major projections from temporal cortex and the opposite IC (discussed above), as well as projections from the ventral and intermediate nuclei of the lateral lemniscus, nucleus sagulum, periaqueductal gray, intercollicular tegmentum, and cuneiform and subcollicu- lar areas. The glutamatergic nature of the projection from auditory cortex is supported by a body of evidence. Lesions of auditory cortex have been shown to deplete glutamate levels in extracentral regions of the IC (Adams and Wen- thold, 1979), which are the primary targets of corticocollicu- lar projections (Andersen et al., 19801, and to depress D-[:3H]aspartate uptake and release in the IC (Feliciano and Potashner, 1995). Mitani et al. (1983) reported that electri- cal stimulation of auditory cortex monosynaptically excited neurons in the dorsal cortex of the IC, and corticocollicular synapses in the cortices and central nucleus of the IC have an “excitatory” morphology characterized by round vesicles and asymmetric contacts (Rockel and Jones, 1973a,b; Feli- ciano et al., 1995b). These corticocollicular projections, and the corticothalamic projections retrogradely labeled in the present study, resemble other corticofugal projections in their ability to selectively accumulate exogenous D-f3HJas- partate (discussed above), which further supports the hy- pothesis that most if not all cortical pyramidal neurons are glutamatergic.

Supporting evidence for the other IC afferents is sparse. While the bulk of the projection from the ventral nucleus of the lateral lemniscus is probably inhibitory (Hutson, 1988; Saint Marie and Baker, 1990; Glendenning et al., 1992; Saint Marie, 1993), up to 10% of its neurons in cats are not immunoreactive for the inhibitory transmitters, GABA and glycine (Saint Marie, unpublished), and may use another, possibly excitatory, transmitter. These may account for the small proportion of neurons that labeled in the present study. Conversely, the great majority of neurons in the intermediate nucleus of the cat lateral lemniscus are not immunoreactive for GABA or glycine (Saint Marie, 1993) and are presumed to be excitatory. Yet, only a fraction of INLL neurons transported D-L3HJaspartate in the present study, suggesting that INLL may contain multiple transmit- ter types. The periventricular gray contains moderate numbers of glutamate-immunoreactive neurons (Ottersen and Storm-Mathisen, 1984), and as we find here some of its neurons can be selectively labeled with D-i3H laspartate (Wiklund and Biittner-Enever, unpublished; cited in Cue- nod et al., 1982). D-13HIaspartate labeling of neurons in the nucleus sagulum from IC injections in the rat has been noted previously (Wiklund, unpublished; cited in Cuenod et al., 1982; Beitz, 1990).

Others have attempted the experiments reported here but without similar success. These are known from anec- dotal comments (Oliver et al., 1983; Glendenning et al., 1992; Wiklund, unpublished; cited in Cuenod et al., 1982) and from one brief report (Schwarz and Schwarz, 1992). Discrepancies between those studies and the present one may be methodological. In the present study survival times greater than 12 hours were required to demonstrate the most distant projections. In some cases signal weakness required autoradiographic exposures up to 12 months. D-L3HJaspartate was used at a concentration that saturated high-affinity synaptic transporters (Davies and Johnston, 1976), and animals were perfused with buffered 3% glutar- aldehyde to maximize the retention of amino acids, includ- ing the D-l3H]aspartate (Storm-Mathisen and Ottersen, 1990a,b). Most findings reported here were reproduced in all eleven cases with IC injections. Any variations could be attributed to the site and size of the injections or to the rate of transport (for the shortest survival times).

Projections that did not transport D-PHIaspartate

The selectivity of D-L3Hlaspartate as a transmitter spe- cific marker was further evidenced by its failure to label auditory projections known not to be glutamatergic. A previous study using HRP, a non-specific retrograde marker, in chinchillas had confirmed the presence of bilateral projections to the IC from the ventral and dorsal cochlear nuclei, lateral superior olive, periolivary nuclei, and dorsal nuclei of the lateral lemniscus and ipsilateral projections from the medial superior olive and ventral and intermedi- ate nuclei of the lateral lemniscus (Saint Marie and Baker, 1990). Some of these projections, i.e., those from periolivary nuclei and the dorsal and ventral nuclei of the lateral lemniscus, are thought to use inhibitory transmitters (e.g., Adams and Mugnaini, 1984; Hutson, 1988; Glendenning et a]., 1992; Saint Marie et al., 1989; Saint Marie and Baker, 1990; Saint Marie, 1993; Shneiderman et al., 1993). Other projections are thought to be excitatory. Projections from the caudal cochlear nuclei have been shown to provide monosynaptic excitation of IC neurons (Semple and Aitkin,

268 R.L. SAINT MARIE

1980), and synaptic endings in the IC originating from dorsal and ventral cochlear nuclei have a morphology (round vesicle, asymmetric contact) typically associated with excitation (Oliver, 1984a, 1985, 1987). Some evidence suggests that neurons in the dorsal cochlear nucleus may be glutamatergic or aspartatergic (Najlerahim et al., 1990), but lesions of cochlear nuclear projections failed to affect glutamate levels or calcium-dependent release of glutamate in the IC (Adams and Wenthold, 1979; Suneja et al., 1995), suggesting that other excitatory transmitters may be used by these projections. The latter is supported by the present findings and those of Oliver et al. (1983) and Glendenning et al. (1992), all of which failed to show D-["]aspartate labeling of cochlear nucleus projections to the IC.

The most surprising outcome of the present study was the failure to label any of the principal excitatory afferents from lower auditory structures. In addition to those from the cochlear nuclei, projections from the ipsilateral medial superior olive, ipsilateral INLL, and contralateral lateral superior olive are thought to be excitatory. This supposition is not based on direct evidence, but rather on the general response characteristics of IC neurons and the fact that these structures are neither GABA nor glycine immunore- active and do not transport ["IGABA or [3Hlglycine from the IC (Hutson, 1988; Saint Marie et al., 1989; Saint Marie and Baker, 1990; Glendenning et al., 1992; Saint Marie, 1993 ). Ottersen and Storm-Mathisen (1984) reported sparse glutamate immunoreactivity in neurons of'the mouse supe- rior olivary complex and lateral lemniscal nuclei, but Glen- denning et al. (1992) reported that the projection from the contralateral lateral superior olive to the IC in cats was glutamate immunoreactive and may provide glutamatergic excitation to the IC. That conclusion is not supported by the present findings in chinchillas. This discrepancy is probably related to methodological rather than species differences, for, as in the present study, those authors also failed to demonstrate D-L3H]aspartate transport by lateral superior olivary projections to the IC (Glendenninget al., 1992). The difficulty of distinguishing immunocytochemically between metabolic pools of glutamate, which are present in all neurons, and transmitter pools has been noted previously (e.g., Ottersen and Storm-Mathisen, 1984) and may con- found the interpretation of glutamate-immunoreactive pro- jections to the IC. The selectivity of D-L3H1aspartate uptake may also have its limitations. So-called "false negatives" have occasionally been reported with D- ["Jaspartate uptake and transport where there is other evidence that a projection may be glutamatergic (e.g., Cuenod et al., 1982; Bernays et al., 1988).

Acetylcholine is another excitatory transmitter found in the IC (reviewed in Morley et al., 1985; Aitkin, 1989; Faingold et al., 1991) but is unlikely to be the principal excitatory transmitter of the ascending pathways. Adams and Wenthold (1979) showed no deficit of cholinergic enzymes in the IC after lesioning projections from the cat cochlear nucleus. Moreover, choline acetyltransferase (an enzyme marker for cholinergic neurons) is not present in the principal neurons of the cochlear or lateral lemniscal nuclei (Kimura et al., 1981). Cholinergic neurons are found throughout the superior olive (Kimura et al., 1981; Osen et al., 1984; Vetter et al., 1991) but are believed to be part of the descending olivocochlear projection (e.g., Warr, 1992).

The most straightforward interpretation for the failure in the present study to label the principal excitatory afferents to the IC from the lower auditory brainstem is

that these pathways are not glutamatergic. In all likelihood, whatever transmitters they do use are probably substances whose transmitter roles have yet to be discovered. An alternative explanation is that there exists a subpopulation of presynaptic glutamate transporters that do not recognize D-["HJaspartate as a suitable substrate, but no such trans- porter has yet been described.

CONCLUSIONS It is concluded that glutamate, or a closely related

compound, is probably the principal excitatory transmitter of many intrinsic and efferent projections of the IC, and of descending cerebral corticocollicular, corticothalamic, and corticobulbar projections. I t is significant that D-L3H]aspar- tate was accumulated by corticocollicular, commissural collicular, and colliculogeniculate projections, which are thought from stimulation and ablation studies to be gluta- matergic. I t is also significant that the principal projections of the dorsal and ventral nuclei of the lateral lemniscus, ipsilateral lateral superior olive, and periolivary nuclei, which are thought to use other (inhibitory) transmitters, did not label. Neither did projections from the cochlear nuclei, which are excitatory, but which have been shown from ablation studies not to be glutamatergic. This selectiv- ity lends credence to the method and suggests that other projections labeled in the present study, e.g., colliculopon- tine, colliculoolivary, and colliculocochlear nuclear projec- tions, will also be shown to be glutamatergic. Finally, the principal sensory pathways to the IC, i.e., those from the cochlear and superior olivary nuclei, did not transport D-L3H]aspartate. This suggests that there may be a chemi- cal dichotomy in the excitation of the IC, with intrinsic, commissural, and corticocollicular connections using a glutamatergic transmitter, but with the main sensory (lemniscal) pathways using non-glutamatergic excitatory transmitter(s).

ACKNOWLEDGMENTS This research was supported by a Public Health Service

rant, RO1 DC00726, from the National Institute on Deaf- ness and Other Communication Disorders of NIH. The author thanks Dr. D.K. Morest for generously providing facilities for these experiments, Dr. J.K. Moore and Dr. S.J. Potashner for their critical reviews of the manuscript, and Dr. Lin Luo and Lisa Seman-Tobin for providing valuable technical assistance.

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