230

Brain Research, 562 (199!) 230-242 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03~50 A DONIS 0~K1689939] 17080U

BRES 17080

Auditory projections from the cochlear nucleus to pontine and mesencephalic reticular nuclei in the rat Karl Kandler and Horst Herbert Department of Animal Physiology, University of Tiibingen, Tiibingen (E R. G. ) (Accepted 28 May 1991)

Key words: Sensorimotor interface; Audio-motor reflex; Startle response; Preyer's reflex; Auditory system; Reticular formation; Fluoro-Gold; Phaseoulus vulgaris leucoagglutinin

We investigated projections from the cochlear nucleus in the rat using the anterograde tracer Phaseolus vulgaris-leucoagglutinin. We focused on nuclei in the brainstem which are not considered to be part of the classical auditory pathway. In addition to labeling in auditory nuclei, we found presumed terminal fibers in 4 pontine and mesencephalic areas: (1) the pontine nucleus (PN), which receives bilateral projections from the antero- and posteroventral cochlear nuclei: (2) the ventrolateral tegmental nucleus (VLTg), which receives a contralateral projection from the rostral portion of the anteroventral cochlear nucleus: (3) the caudal pontine reticular nucleus (PnC), which receives bilateral input originating predominantly in the dorsal cochlear nucleus: and (4) the lateral paragigantocellular nucleus (LPGi). which receives projections from all subdivisions of the cochlear nuclei. In the VLTg and PnC, anterogradely labeled varicose axons were often found in close apposition to the primary dendrites and somata of large reticular neurons. Injections of the retrograde fluorescent tracer Fluoro-Gold into the VLTg demonstrated that the neurons of origin are mainly located contralaterally in the rostral anteroventral cochlear nucleus and in the cochlear root nucleus. The relevance of these auditory projections for short-latency audio-motor behaviors and acoustically elicited autonomic responses is discussed. INTRODUCTION

The cochlear nucleus (CN) is the first central relay in the ascending auditory pathway of mammals. The auditory nerve fibers synapse on CN neurons, which in turn project to auditory nuclei in the brainstem and midbrain (i.e. nuclei of the superior olivary complex, nuclei of the lateral lemniscus, and the inferior colliculus (review in ref. 31). In addition to CN efferents terminating in nuclei of the classical auditory pathway, projections to brainstem nuclei have occasionally been described which are not considered a part of the auditory pathway proper. These include nuclei of the reticular formation (e.g, ref. 58) and cranial motor nuclei 9"51. It is possible, therefore. that fibers leaving the auditory pathway at the level of the CN mediate short latency audio-motor reflexes. In the cat, the CN projects to tensor tympani motoneurons, and thus is part of a reflex arc which has only two central synapses 33. Other short latency acoustic reflexes are the Preyer's reflex 22 and the acoustic startle reflex 42. Both can be elicited by sudden, loud acoustic stimuli. Because of the short latencies of these responses to acoustic stimuli 13"2°, it has been proposed that the pathways responsible for these reflexes involve only a few

synaptic relay stations. Caeser et al. ~3 suggested three in the CNS for the head muscle components of the startle reflex, and Davis et al. 2° suggested 4 central relay stations for the body movements of the startle reflex, Although the startle reflex circuit is short, simple associative as welt as non-associative learning can occur (reviewed in ref. 19). In order to study the plasticity of this reflex with neurophysiologicat and neuropharmaeological tools, one must know the neuronal substrate. For the proposed three-synapse pathway 13. the involvement of the CN and the cranial motoneurons is mandatory. Since second order nuclei of the auditory pathway do not project to the motor nuclei that supply facial muscles (e.g. facial and trigeminal motor nuclei), a premotor structure located outside the main auditory pathway must receive auditory input from the CN. So far. no study has focused on efferents originating in the CN and terminating in areas which also contain premotor neurons 3., 35.52.57

The aim of the present study was to map the distribution and analyse the morphology of CN fibers which terminate in nuclei outside the main auditory pathway and are known to contain premotor neurons. We injected the anterograde tracer Phaseotus vulgaris-leuco-

Correspondence: K. Kandler. Universitat Tiibingen, Tierpbysiologie, Auf der Morgenstelle 28. D-7400 Tilbingen. F.R.G. Fax: (49) 7071 294634.

231 a g g l u t i n i n ( P H A - L 24) i n t o t h e C N a n d f o u n d t h a t 4 s u c h nuclei r e c e i v e a p r o m i n e n t C N p r o j e c t i o n . O n e o f t h e s e target areas, the ventrolateral tegmental nucleus (VLTg), was s u b s e q u e n t l y i n j e c t e d w i t h t h e r e t r o g r a d e fluoresc e n t t r a c e r F l u o r o - G o l d ( F G ) to d e m o n s t r a t e t h e locat i o n a n d m o r p h o l o g y o f t h e n e u r o n s o f o r i g i n in t h e C N . P r e l i m i n a r y r e s u l t s h a v e b e e n p u b l i s h e d 37.

hydrogen peroxide. Finally, the sections were mounted on gelatinecoated slides, air dried, dehydrated in alcohol, cleared in xylene, and coverslipped with Entellan. Data analysis. Sections were viewed using brightfield illumination and Nomarski optics. The distribution of PHA-L labeled fibers in the brainstem was mapped with a camera lucida drawing tube. Cytoarchitectural boundaries were defined by superimposing projections of the corresponding thionin-stained sections with the drawings, using brain outlines and blood vessels as reference points.

Retrograde tracing experiments MATERIALS AND METHODS

Anterograde tracing experiments Thirty-one female Spraquc-Dawley rats weighing 180-240 g were anesthetized with 7% chloral hydrate (0.5 mill00 g) and fixed in a stereotaxic frame. The injection pipette was lowered through the brain in a slightly oblique direction from dorsorostral to ventrocaudal in order to avoid injury to the transverse sinus. Tracer deposits were guided into the CN by recording evoked potentials to acoustic stimuli (clicks, 0. l ms, 40-50 dB SPL) delivered through hollow ear bars, Recordings were done with the same glass micropipettcs (tip diameter of 20-30 ~m), from which iontophoretic injections of 2.5% PHA-L (Vector) in 50 mM Tris-buffered saline (pH 7.6) were made, using a pulsed positive current of +5 /~A with a 50% duty cycle for 15-20 rain. In order to label many efferent fibers from the CN, we placed multiple PHA-L injections into 2-5 different sites of the CN. In 7 animals, the PHA-L injections were restricted to only one subnucleus of thc CN, either to the posterovcntral CN (PVCN; n = 5) or the anteroventral CN (AVCN, n - 2). In the remaining cases, the injection sites included both subnuclei (AVCN and PVCN, n 3) or covered parts of all 3 subnuclei (dorsal CN, as well as AVCN and PVCN, n = 5). Injections with substantial involvement of adjacent structures in the injection site were excluded from further analysis. After 8-12 days, animals were anesthetized and perfuscd through the aorta with 0.9% saline, followed by the fixative according to the two-step pH change protocol of Berod et al.V. After perfusion with 180 ml of ice-cold 4% paraformaldehyde in 0.1 M sodium acetate buffer (pH 6.5) followed by 300 ml of ice-cold 4% paraformaldehyde in 0.1 M borate buffer (pH 11), the brains were removed and postfixed overnight at 4 °C in the borate buffer fixative containing 5% sucrose. The brains were then blocked and soakcd for two days in 30% sucrose in 0.1 M phosphatc buffer (pH 7.4) at 4 °C. These blocks wcrc cut transversely from the lower medulla to the posterior thalamus on a freezing microtome at 40/~m and divided into two series. One series was further processed by immunoperoxidase staining and the other one was stained with thionine. lmrnunoperoxidase staining. Sections were immunohistochemically processed by the peroxidasc-antiperoxidase (PAP) method to identify PHA-L-labeled fibers. First, we rinsed the sections in several changes of TBS and then we incubated them in a blocking solution containing 10% normal swine scrum, 2% bovine serum albumine (BSA), and 0.3% Triton X-100 in TBS. After 1 h, the sections wcre transferred into the primary antibody solution of rabbit antiPHA-L (DAKO) diluted 1:3000 in a carrier containing 1% normal swine serum, 1% BSA, and 0.3% Triton X-t00 in TBS, and incubated for 2 days at 4 °C. The sections were then rinsed for 30 min in several changes of TBS, transfcrrcd into unlabeled swine antirabbit lgG antiserum (Dako) diluted l:50 in carrier, and incubated tot 1.5 h at room temperature. Then they were extensively rinsed in TBS and incubated in the rabbit-PAP complex (Dako) diluted 1:200 in carrier. After 1.5 h at room temperature, the sections were again rinsed in several changes of TBS. The final visualization of the anterogradely transported PHA-L was done by reacting the sections with nickel-enhanced diaminobenzidine 2~'. Incubation was for 15 rain in 50 mM TBS at pH 8.0 containing 0.6% nickel-ammonium sulfate, 0.02% 3,3-diamidinobenzidine (DAB) and 0.01%

In 7 rats, we injected the retrogradely transported fluorescent dye Fluoro-Gold (FG: Fluorochrome Inc.) into the VLTg. A 2% solution of FG in 0.1 M cacodylate buffer at pH 7.5 was iontophoretically injected through glass micropipettes with tip diameters of 40-60 ,urn (+5/~A for 10-15 min, 5 s on/off). After 6 days, the rats were deeply anesthetized and perfused through the aorta with 0.9% saline followed by 500 ml of ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The brains were then removed, postfixed overnight in the fixative containing 10% sucrose, cut on a freezing microtome in the transverse plane at 50/~m, and divided into two series. One series was Nissl-stained with thionin, the second series was mounted on gelatine-coated slides, air dried, and coverslipped with DePex. Data analysis. The latter sections were viewed with fluorescent illumination using a UV filter set for FG and the distribution of retrogradely labeled neurons was mapped with the aid of an X/Yplotter coupled to the microscope stage. Cytoarchitectural boundaries were defined by superimposing the adjacent thionin-stained sections with the plots.

RESULTS

Anterograde tracing experiments I n j e c t i o n s o f P H A - L r e s u l t e d in a r e a c t i o n p r o d u c t in the CN with a homogeneous

black center and brown

surroundings with black labeled neuropil and somata (Fig. 1). W e a s s u m e t h a t P H A - L

is o n l y i n c o r p o r a t e d

i n t o , a n d t r a n s p o r t e d f r o m , l a b e l e d n e u r o n s in t h e cent e r a n d its i m m e d i a t e s u r r o u n d i n g s , a n d t h u s r e f e r to t h e s e r e g i o n s as ' t h e i n j e c t i o n site'. In m o s t cases a n a l ysed, t h e i n j e c t i o n site was c l e a r l y r e s t r i c t e d t o t h e C N with o n l y a m i n o r s p r e a d o f t r a c e r , w h i c h r e s u l t e d in a diffuse

brown

trigeminal

staining

in a d j a c e n t

sensory nucleus,

(e.g.

the

the vestibular nerve,

regions

the

paraflocculus, the inferior or middle cerebellar penduncle). H o w e v e r , in t h e s e cases, we a l m o s t n e v e r o b s e r v e d l a b e l e d fibers e m e r g i n g f r o m t h e s e s t r u c t u r e s . O c c a s i o n ally, a b r o w n r e a c t i o n p r o d u c t c o u l d b e f o u n d a l o n g t h e p i p e t t e t r a c k in t h e c e r e b e l l u m . A g a i n , v e r y few fibers, if any, e m e r g e d

f r o m t h e s e sites. A f t e r i n j e c t i o n s o f

P H A - L i n t o t h e C N , we o b s e r v e d l a b e l e d fibers l e a v i n g the CN through the dorsal, intermediate,

and ventral

a c o u s t i c striae. L a b e l e d a x o n s w e r e c o n s i d e r e d to r e p r e s e n t ' t e r m i n a l l a b e l i n g ' w h e n t h e y e x h i b i t e d a x o n a l arborization, had a curved and irregular course, and had e n p a s s a n t a n d t e r m i n a l swellings o n t h e f i b e r b r a n c h e s . In c o n t r a s t , t r a v e r s i n g fibers g e n e r a l l y h a d s t r a i g h t traj e c t o r i e s a n d few, if a n y , a x o n a l swellings. In a d d i t i o n to labe'ling in s e c o n d a r y a u d i t o r y n u c l e i ,

232 presumed terminal labeling was consistently apparent in several brainstem nuclei which are not considered a part of the main auditory pathway. These are the pontine nuclei, the VLTg, the caudal pontine reticular nucleus, and the lateral paragigantocellular nucleus (terminology according to Paxinos and WatsonS°). In the few cases with larger injections sites slightly extending into regions surrounding the CN, we also observed isolated fibers in the vestibular nuclei, the parabrachial nucleus, the locus coeruleus and the cerebellum. We attribute this minor anterograde labeling to incorporation of tracer substance in structures immediately adjacent to the CN (i.e. vestibular fibers, sensory trigeminal fibertracts, middle or inferior cerebellar peduncle). Because of the inconsistent appearance of this labeling and the paucity of fibers, they will not be considered further. Pontine nuclei (PN). Injections of P H A - L into either the A V C N or the P V C N labeled presumed terminal fibers bilaterally in the PN with a contralaterat predominance (Fig. 2 A ' ) . Ascending auditory fibers could be traced leaving the ventral lateral lemniscus and entering the PN from a dorsocaudal direction. The fibers ran rostrally for about 500/~m before they began to branch. The terminal field was restricted to a vertically oriented band in the lateral third of the PN. a region which corresponds to the medial portion of the pontine nucleus' lateral subnucleus 47, This labeling extended up to 400/~m rostrocaudally in the PN. Due to the numerous axonal and terminal boutons (Fig. 3D,E), it is likely that the labeled axons make synaptic contact with PN neurons. Ventrolateral tegmental nucleus (VLTg). We found P H A - L labeled fibers contralaterally in the VLTg (Fig. 2A.B) in 3 animals with P H A - L injections into the rostral part of the A V C N (Fig. 1). The VLTg is located ventrolateral to the oral pontine reticular nucleus (PnO) and just medial to the ventral nucleus of the lateral lemniscus (VLL; Fig. 3A. see also plate 51 in ref. 50). The auditory fibers entered the VLq~g either caudally from the rostral periolivary region (RPO) or laterally from the lateral lemniscus. Furthest rostrally in the VLTg, the fibers were located medial to and above the rubrospinal tract, while more caudally, labeled fibers were found at sites just ventrolateral to the VLL. At the most caudal level, P H A - L labeled fibers were located just dorsomedial to the rostral periolivary nucleus (RPO). The axons located within the VLqg could easily be distinguished from fibers within the R P O by their morphology. The

Fig. 1. Photomicrograph of a transverse scction through the left AVCN illustrating a PHA-L ir~jection sile (experiment NC 31 ) Bar = 500 ~m.

former were characterized by their large diameter, sparse branching and thick varicosities (Fig. 3B), whereas in thc RPO. presumed terminals were fine and highly arborized. The large axonal and terminal varicosities in the VLTg mostly appeared in close proximity to somata of large neurons (Fig. 3B,C). presumably making axo-somatic contacts. Moreover. the distribution of anterogradely labeled terminal fibers in the VLTg matched thc area where large VLTg neurons with diameters of approximately 30-40 # m are located (compare thioninstained section in Fig. 3A with Fig. 2A~. Caudal pontine rencular nucleus (PnC). Following P H A - L injections into various CN subnuclei, two types of anterogradely labeled fibers could be distinguished in the PnC by their diameters and trajectories (Fig. 4). One class of axons was fairly thick, with diameters of about 2 ~m. and ran straight from the ipsi- to the contralateral side (arrowheads in Fig. 4B). Fibers in this class rarely gave off collaterals and had almost no en passant boutons. suggesting no or only few synaptic contacts be-

Fig. 2. Line drawings of coronal sections through the midbrain and the pons in two experiments (NC 31 and NC 25) illustrating the distribution of anterogradely labeled fibers (A-D, rostral to caudal). The animals received multiple injections of PHA-I. into the AVCN (NC 31. A-D) or the PVCN/DCN (NC 25. A'-D'). The center of the injection site is drawn in black, the marginal zone is dotted. Thin lines dcpic~ anterogradely labeled fibers. Bar = 1 rnm.

233

CasNeC 31

CasNeA, C ~

A

r

~''"

'

VLL

L ~ Gr [

PVON

L N T B ~

D

~ )

~

VNTB

PvcN iD

LPGi

234

Fig. 3. Photomicrographs of transverse sections through the ventrolateral midbrain of animal NC 31 ( A - C ) , which received a P H A , L injection into the left AVCN. and animal NC 25 {D,E), which received a PHA-L injection into the left PVCN/DCN (dorsal is up, lateral is right). A: thionin-stained section through the ventrolateral part of the mesencephatic brainstem Note the large somata in the VLTg just medial to the VLL. The box indicates the area enlarged in B. Bar = 400 urn. B: PHA-L-labeled AVCN fibers in the VLTg contralateral to the injection site. The section shown is immediately rostral to the thionin-stained section in A. Note the thick fiber fragments and their close proximity to the large VLTg neurons (arrow). Bar = 100 gin. C: high power photomicrograph with Nomarski optics of a terminal ending in the VLTg presumably contacting a large background stained neuron. Bar = 20/~m. D: presumed terminal labeling in the lateral part of the PN. The arrow points to the axon which is enlarged in E. Bar = 200 #m. E: high power photomicrograph of a presumed axonal terminal in the PN. Bar = 50 um.

235

Fig. 4. Photomicrographs of coronal sections through the PnC of animal NC 25 which received PHA-L injections into the PVCN and DCN (dorsal is up. lateral is right). A: thionin-stained section through the pontine brainstem illustrating the location of the PnC characterized by large reticular neurons. Bar = 500 pm. B: photomicrograph through the PnC illustrating PHA-L-labeled fibers contralateral to the injection site. Arrowheads point to a thick fiber with few varicosities running in the dorsal acoustic stria. Arrows indicate thin, beaded axons coursing ventrally and dorsally, and are assumed to contact PnC neurons. Bar = 200 pm. C: high power photomicrograph with Nomarski optics illustrating a PHA-L-labeled varicose fiber juxtaposed to the soma and proximal dendrite of a background stained PnC neuron. Bar = 20

tween these axons and PnC neurons. These fibers were found in all animals with injections into the CN. Due to their location and their course in the brainstem, we consider these fibers to belong to the dorsal acoustic stria. Fine, intensely beaded axons (diameters smaller than 0.5 pm) were only seen when the injection site included parts of the dorsal CN (DCN; Fig. 2, case NC 25). These fibers entered the reticular formation together with the thick axons in the dorsal acoustic stria. In contrast to the latter, these axons did not run straight through the PnC, but frequently coursed ventrally and dorsally (note arrows in Fig. 4B), showing numerous axonal as well as terminal varicosities. These boutons were frequently found closely opposed to the somata and proximal dendrites of large PnC neurons (Fig. 4C) suggesting synaptic contact between auditory fibers and these large PnC neurons. Lateral paragigantocellular nucleus (LPGij. PHA-L injections into various subnuclei of the CN always re-

sulted

in labeling

of presumed

terminal

fibers

in the

LPGi, a reticular nucleus situated ventrally in the medullary brainstem (Figs. 2C’,D,D’ and 5A). Labeled fibers were located just ventromedial to the facial nucleus (MOM), stretching along its rostrocaudal extent. Furthest rostrally, the LPGi-labeling was continuous with the fiber labeling in the caudal periolivary region. An exact transition zone could not be distinguished. Caudally, the labeling was restriced to a narrow, cell-poor region close to the ventral surface of the medulla (Fig. 5B), which extended up to the anterior part of the inferior olive. Labeled axons in the LPGi formed a dense plexus of fine fibers with many terminal and en passant varicosities (Fig. 5C), suggesting that they make numerous synaptic contacts with hitherto unknown postsynaptic targets. Labeling in the main auditory pathway. Injections of PHA-L into the CN resulted in anterogradely labeled fibers in acoustic striae and in auditory nuclei whose overall distributions were the same as those found in earlier

236

B

Fig. 5. Photomicrographs of coronal sections through the ventromedial medulla of animal NC 28. which received PHA-L injections into the AVCN and PVCN (dorsal is up, lateral is right). A: thionin-stained sections showing the location of the ventral LPGi lateral to the pyramidal tract (py) and medial to the facial motor nucleus. B: PHA-L-labeled beaded fibers in the LPGi in the section adjacent to the thionmstained section in A. The box indicates the area enlarged in C. C: note the highly arborized and varicose fibers, suggesting numerous svnaptic contacts of the auditory fibers in the LPGi. Bars in A and B - 500 urn. C = 100 um.

studies (e.g. refs. 17,23,28,31), and hence will only briefly be summarized here. Terminal fibers were found in the contralateral CN, bilaterally in the superior olivary complex (SOC), in the nuclei of the lateral lemniscus, and in the inferior coUiculi (IC; Fig. 2). In the $OC, we found labeled fibers ipsilaterally in the lateral superior olive, bilaterally in the medial superior olive, contralaterally in the medial nucleus of the trapezoid body, and bilaterally in the ventral and lateral nuclei of the trapezoid body. In the periolivary region, terminal fibers were present mainly contralaterally in the dorsal and rostral parts, and bilaterally in the caudal periolivary region. In the ventral nucleus of the lateral lemniscus (VLL), intense labeling was found contralaterally, but a few fibers were also seen in the ipsilateral V L L , as well as contralaterally in the intermediate and dorsal nucleus of the lateral lemniscus. Labeling in the IC was predominantly in the contralateral central nucleus. A projection to the ipsilateral IC was only apparent in cases with large tracer deposits, and a projection to the external and dor-

sal cortex appeared only after injections which included the DCN. Control experiment. We placed a P H A - L injection into one animal's ventromedial paraflocculus. In,this case,~ labeled fibers were found in various parts of the cerebellum including the contralateral paraflocculus, lobuli 4-6, the ventral spinocerebellar tract, and the inferior and middle cerebellar penduncle. However, no labeled fibers were found in the PN, PnCi VLTg, nor LPGi, nor in any nucleus of the auditory pathway. Retrograde tracing experiments

Projections from auditory nuclei to the PN, PnC and the LPGi have already been studied with retrograde transport techniques (see Discussion for references). To identify the location and the type of CN neurons that project to the VLTg, we injected this nucleus with the retrograde tracer FG. Three animals received injections which were clearly restricted to ~the VLTg without a spread of tracer into adjacent auditory structures (e.g.

237

B

l

~f"

DeN

Ave N

55\\

Fig. 6. Fluorescence photomicrograph of a Fluoro-Gold injection site in the right VLTg (dorsal is up, lateral is right). B: line drawings of the resulting pattern of retrogradely labeled neurons in the contralateral cochlear nucleus. Each dot represents one retrogradely labeled neuron (B-D: dorsal is up, lateral is left). Bars in A = 500/~m, B = l mm. C,D: fluorescence photomicrographs of a transverse section through the contralateral rostral AVCN, illustrating the distribution and morphology of retrogradely labeled neurons. Arrows in C and D point to the same neuron. Bars in C = 200/~m, D _+ 20/~m. Dorsal is to the top, lateral to the left.

VLL, SOC). In these cases, no retrogradely labeled neurons were found in the central nucleus of the IC, the SOC, the NLL, nor in the ipsilateral CN. By contrast, in animals in which the injection site partly covered the VLL and/or the R P O , large numbers of retrogradely labeled cells were present in the above m e n t i o n e d auditory nuclei. These brains were excluded from further analysis. We present the results of an experiment in which the injections site was located in the VLTg just medial to the

VLL and ventral to the rubrospinal tract without any spread of tracer into the adjacent VLL (Fig. 6A). Many retrogradely labeled neurons were found in the contralateral CN (Fig. 6B,C), with the largest n u m b e r present in the AVCN (Fig. 6B). More caudally, labeled neurons were located mainly in the dorsal portion of the AVCN. The somata of the retrogradely labeled neurons were oval with an average diameter of 16 x 10/zm (n = 15). They usually had one thick dendritic trunk or a few short dendrites extending from one side of the somata

238 (Fig. 6D). Some heavily labeled neurons were also found in the cochlear r o o t n u c l e u s 27"46"49 (see 8n in Fig. 6B). These cells were large with diameters of about 30 #m and were mostly circular in shape. Furthermore, a few weakly labeled neurons were found in the DCN. In addition to the neurons in the contralateral CN, we found retrogradely labeled neurons in one other auditory area, the external cortex of the IC. Besides auditory input to the VLTg, we found strong projections from second order somatosensory nuclei including the principal sensory trigeminal nucleus, the spinal trigeminal nucleus, the dorsal column nuclei, and the vestibular nuclei. DISCUSSION Injections of P H A - L into the cochlear nucleus of rats yielded labeled fibers and presumed terminals in the nuclei of the auditory pathway and in four nuclei of the pons andmidbrain: the pontine nuclei (PN), the ventrolateral tegmental nuclei (VLTg), the caudal pontine reticular nuclei (PnC), a n d the lateral paragigantoeeltular nuclei (LPGi).

Technical considerations Recently it was shown that P H A , L might be taken up by fibers of passage and t r a n s ~ r t e d both anterogradety as well as retrogradelyS3, Hence, it is possible that fibers in the vicinity of the CN (e.g. inferior or middle cerebellar peduncle, vestibular nerve, spinal trigeminal tract) might have incorporated and transported P H A , L , leading t o false positive results in our experiments. Some labeled fibers were, in fact, observed in fiber tracts close to the CN, but this labeling was very sparse compared to labeling in the main auditory pathway or the PN, VLTg, PnC or LPGi. Furthermore, we did not find any correlation between the number of labeled axons in fiber tracts adjacent to the CN and the amount of labeling in the PN, VLTg, PnC or LPGi. Also, a control injection into the cerebellum did not yield labeled fibers in the PN, VLTg, PnC and LPGi. Therefore, we are quite confident that the labeled axons in these 4 target areas originate in the CN. Projections to potential premotor nuclei Three of the 4 regions receiving CN efferents (VLTg, PnC, and PN) contain neurons which project to either cranial 3°'52"57 or spinal motoneurons 35'43 and thus could be relays in reflex pathways which mediate acoustically elicited short latency motor responses.

Ventrolateral tegmental nucleus Injections of PHA-L into the rostral AVCN resulted

in anterogradely labeled large diameter fibers loosely distributed in the contralateral VLTg (Figs. 2B and 3B. C I. This pattern, together with the finding that the varicose fiber fragments are mostly found in close proximity to large somata (Fig. 3B,C), suggests that these AVCN fibers might preferentially contact a subpopulation of large neurons scattered in the VLTg. FG injections into the VLTg labeled CN neurons predominantly in the AVCN and in the cochlear root n u c l e u s 27"46"49, suggesting that at least some of the latter neurons do not project only to the trapezoid body 46. The location of labeled neurons in the AVCN (Fig. 6B) corresponds roughly to cell regions I and III of the rat AVCN 27. The somato-dendritic morphology of most of the retrogradely labeled neurons in the AVCN (Fig. 6C) is similar to that of bushy cells described in the cat m. A sparse projection from the contralaterat AVCN to a mesencephalic area whose location corresponds to the VLTg was previously described for the cat 29'58. In the bat, the CN projects bilaterally to the nucleus of the central acoustic tract (NCAT)14, which shows similarities in location and cell morphology to the VLTg in rats. The pattern of afferent neurons projecting to the oral pontine reticular nucleus (PnO), including the medial part of the VLTg, was also described by lrvine and Jackson 32 for the cat and by Shammah-Lagnado et al. s4 for the rat. In contrast to our results, neither study reported a projection from the CN to the PnO. However. our injection sites were more ventrolaterally located in the reticular formation than theirs (i.e. in the VLTg rather than the PnO), suggesting a somewhat different afferent pattern to the PnO and VLTg. This observation further supports Mehler's 45 early suggestion that the VLTg is separate from the PnO. Functional implications. Based on latency measurements, it was suggested that the acoustic startle pathway which mediates the short latency contractions of head muscles consists of 3 central synaptic relays ~3. Since no direct projection from second order auditory nuclei onto motoneurons supplying facial muscles is known, the postulated sensory-motor interface must be located outside the ascending auditory pathway. The VLTg is a structurc which recmves primary auditory input from the CN (present study), and in turn projects to the trigeminal and facial motor nuclei ~'35"52"s7 Furthermore. electrical stimulation studies of mesencephalic reticular areas revealed that the VLTg requires the lowest current thresholds to elicit short latency EMGs in facial muscles, and that at the same electrode positions, the highest amplitudes of startle-related auditory evoked potentials arc found 3~. These data point to the VLTg as a sensorymotor interface mediating the head components of thc startle reflex.

239 Since neurons in the ventrolateral PnO (corresponding to VLTg) project to the ventral horn of the spinal cord 35, they also could mediate the whole body movements of the startle response. Startle-like body movements can be elicited by electrical stimulation of the nuclei of the lateral lemniscus and adjacent reticular structures including the VLTg TM. Lesion 2° and pharmacological studies 56 further support this view. Although Davis and co-workers 15.19.20 consider the ventral nucleus of the lateral lemniscus, and not the closely located VLTg, as a relay of the startle pathway, their data are not in contrast to ours, as their sites of lesions, electrical stimulations, and pharmacological injections usually included the VLTg. Currently it is unclear whether the large VLTg neurons directly activate spinal motoneurons in the startle reflex, or whether VLTg neurons excite neurons in the PnC, which in turn mediate the reflex down to the spinal cord 2°'41. Given the facts that lesions of the ventral nucleus of the lateral lemniscus and VLTg abolish not only the acoustic, but also the tactile startle responseS5, and that the VLTg receives strong somatosensory input from primary somatosensory nuclei, it is quite likely that the latter nucleus also plays a role in mediating the tactile startle response.

Caudal pontiac reticular nucleus In the PnC, we found two types of anterogradely labeled fibers: thick unbranched axons and fine, arborized ones with numerous varicosities, often located in close proximity to the somata and primary dendrites of large PnC neurons. Using electrophysiological and anatomical methods, Lingenhtihl and Friauf 43 showed that these neurons do receive direct auditory input from the CN. They further demonstrated that the acoustically responsive PnC neurons project to both the facial nucleus and the spinal cord. The fact that electrical stimulation in the PnC can elicit monosynaptic excitatory postsynaptic potentials (EPSPs) in spinal motoneurons 25 identifies the spinal projection as an excitatory pathway. Functional implications. The acoustic input to large PnC neurons and their projection to cranial and spinal motoneurons makes them possible mediators for short latency audio-motor behaviors such as the Preyer's reflex or the acoustic startle response. Lesion and electrical stimulation studies 2°'4j support this assumption. The present data suggest that both the VLTg and the PnC are parts of 3-synapse audio-motor pathways in the brain. At present, however, it is unclear whether the two nuclei mediate the same bet'.aviors, or whether they belong to different functional circuits. As the locations of these presumed sensory-motor interfaces are known, physiological studies on a single cell level could investi-

gate learning and plasticity associated with the Preyer's or the acoustic startle reflex such as habituation, sensitization, and prepulse inhibition 19

Pontine nuclei Our results demonstrate that the VCN provides auditory input to the PN, predominantly contralaterally to the lateral aspect of the PN, as defined by Mihailoff et al. 47. Our results, however, do not match the findings of Faye-Lund 21, who injected W G A - H R P into the dorsolateral portion of the rat PN and reported that the only positive finding in auditory nuclei was a few labeled cells in the periolivary region. Her injection sites, though, were more lateral in the PN (see her Fig. 7B), and do not match with the termination area of auditory fibers from the CN in our study (Figs. 2A" and 3D of the present study). Aside from projections from the VCN and the periolivary region el, auditory input to the lateral PN also derives from the inferior colliculus (e.g. refs. 12,21) and the auditory cortex ~'~ The findings that acoustically responsive neurons in the dorsolateral PN of the cat have latencies of 16-17 ms 2 brings into question the existence of a direct projection of CN fibers to the lateral PN. However, one should take into account that the projection from the CN to the PN is quite sparse and that the authors could have missed auditory responsive PN neurons which are excited by CN fibers. Species differences could also be responsible for the controversial findings. Functional implications. Neurons in the lateral PN project to the midvermal area of the cerebellar cortex 39 which is considered an audio-visual integration area 55. Involvement of the pontocerebellar system in pre-programming, initiation, and ongoing control of limb movement was discussed (e.g. ref. 6). Interestingly, the cerebellar vermis is also essential for the long-term habituation of the acoustic startle response 44. The pontocerebellar system, however, seems inadequate for directly relaying motor responses with latencies as short as those in the Preyer's or startle reflex (about 6 ms for pinna muscle responseS3), due to the much longer pathway. Although recent studies show that neurons in the PN also project to the facial and trigeminal motor nuclei 52, direct input by CN fibers onto these premotor neurons remains to be shown. Additional anatomical and electrophysiological data are necessary to determine the possible role of this audio-motor loop for the startle reflex and its plasticity.

Lateral paragigantocellular nucleus Following injections of PHA-L into the AVCN and PVCN, we consistently found anterogradely labeled fibers bilaterally in the ventromedial medullary reticular

240 formation. This area corresponds to the ventral p o r t i o n of the L P G i 3"48 and is characterized by small and medium-sized fusiform cells located in a lattice of neuropil on the ventral surface of the brainstem 48 (see also Fig. 5A). Bilateral auditory input from the P V C N to the L P G i was previously described for the rat by A n d r e z i k et al. 4, a finding which could not be confirmed by Boeckstaele et al. 8. In the cat, projections from the CN to the L P G i were also d e m o n s t r a t e d 36"48'59, but discrepan-

a possible pathway for the acoustically elicited orienting or defense response, both of which are characterized by arousal and cardio-respiratory changes 34. It is tempting to further speculate that analgesia produced by intense acoustic stimuli (e.g. startle stimuli tS) might also be mediated via the C N - L P G i r o u t e

cies exist over the location of the neurons of origin in the CN and w h e t h e r this pathway is uni- or bilateral. Neurons in the L P G i also project back to the CN and even into the cochlea 1"36"6°. W a r r 6° therefore considered the L P G i as the caudal extent of the posterior periotivary region, and thus a part of the olivocochlear bundle ( O C B ) . O u r data suggest that auditory fibers originating in the CN terminate not only on caudal neurons of the O C B , located adjacent to the rostral L P G i , but also on serotonergic neurons not belonging to the olivocochlear system. D o u b l e - l a b e l i n g experiments in our l a b o r a t o r y (unpublished observations) revealed a dense network of P H A - L l a b e l e d V C N fibers closely o p p o s e d to dendrites of serotonergic L P G i neurons. A d d i t i o n a l auditory input to the L P G i originates in the IC 48, and primary somatosensory input derives from the dorsal column nuclei in the cat 36, suggesting an integration of sensory informa-

The present studv used the a n t e r o g r a d e transport of P H A - L to d e m o n s t r a t e that. in addition to nuclei o f the classical auditory pathway, 4 structures in the b r a i n s t e m receive considerable auditory input from the cochlear nucleus. T h r e e of the regions, the pontine nucleus, the ventrolateral t e g m e n t a l nucleus, and the caudal pontine reticular nucleus, contain p r e m o t o r neurons. These nuclei may, therefore, act as links in pathways which mediate short latency a u d i o - m o t o r behaviors, Current data favor the ventrolateral tegmental nucleus and the caudal pontme reticular nucleus as s e n s o r y - m o t o r interfaces in the startle pathway, mediating either the head components or the whole body m o v e m e n t of the reflex, respectively. The fourth structure receiving auditory input from the CN is the L P G i . We p r o p o s e that this nucleus may be involved in mediating acoustically elicited autonomic responses such as the orienting or defense response.

tion of different modalities by the L P G i . Functional implications. In addition to the role of the L P G i in autonomic regulation and analgesia (review in ref. 16), this nucleus provides a major input to the locus coeruleus (LC) 5. The LC, in turn, projects to n u m e r o u s areas along the neuraxis and influences sensory processing at all central levels, including that of the auditory nuclei 4°'41. The auditory input to the L P G i could provide

Acknowledgements. The authors thank Helga Ziltus for technical assistance, Andrea Piepenstock for access to histological material. and Krista Nadakavukaren for correcting the English. The comments of Drs. Eck Friauf and Jo Ostwatd on the manuscript were most beneficial and are also greatly appreciated. Finally, we wish to thank Dr. Kent Morest for his valuable criticism whichimproved the paper considerably. This work was supported by the Deutsche Forschungsgemeinschaft SFB 307.

ABBREVIATIONS Aq AVCN CN DAS DLL DCN EC EPSP FG Gi GiA GrC HRP IAS IC icp IFP ILL IO LNTB

aqueduct anteroventral cochlear nucleus cochlear nucleus dorsal acoustic stria dorsal nucleus of the lateral lemniscus dorsal cochlear nucleus external cortex of the inferior colliculus excitatory postsynaptic potential Fluoro-Gold gigantocellular reticular nucleus gigantocellular reticular nucleus, part alpha granular layer of the cochlear nucleus horseradish peroxidase intermediate acoustic stria inferior colliculus inferior cerebellar peduncle interfascicular nucleus peduncle intermediate nucleus of the lateral lemniscus inferior olive lateral nucleus of the trapezoid body

Conclusions

LPGi LSO mcp MNTB Mo5 Mo7 MSO NLL PAG PBg PHA-L PN PnC PnO Pr5 PVCN PY RPO RtTg RVL s5 SC

lateral paragigantocellular nucleus lateral superior olive middle cerebetlar peduncle medial nucleus of the trapezoid body motor trigeminal nucleus motor facial nucleus medial superior olive nuclei of the lateral lemniscus penaqueductal gray parabigeminal nucleus

Phaseolus vulgaris-leucoagglutinin pontine nucleus caudal pontine reticular nucleus oral pontine reticular nucleus principal sensory trigeminal nucleus posteroventral cochlear nucleus pyramidal trae~ rostral periolivary region rubrospinal tract reticulotegmental nucleus ot the ports rostroventrolateral reticular nucleus sensory root of the trigeminai nerve superior colliculus

241 SOC Sp50 SPO tb VAS VCN

superior olivary complex spinal trigeminal nucleus, oralis superior paraolivary nucleus trapezoid body ventral acoustic stria ventral cochlear nucleus

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