direct input from cochlear root neurons to pontine reticulospinal...

Direct Input from Cochlear RootNeurons to Pontine Reticulospinal

Neurons in Albino Rat

FERNANDO R. NODAL1* AND DOLORES E. LOPEZ2

1University Laboratory of Physiology, Oxford OX1 3PT, United Kingdom2Instituto de Neurociencias de Castilla y Leon, Universidad de Salamanca,

37007 Salamanca, Spain

ABSTRACTThe cochlear root neurons (CRNs) are thought to mediate the auditory startle reflex

(ASR) in the rat, which is widely used as a behavioral model for the investigation of thesensorimotor integration. CRNs project, among other targets, to the nucleus reticularispontis caudalis (PnC), a major component of the ASR circuit, but little is known about theorganization of this projection. Thus, we injected biotinylated dextran amine (BDA) in CRNsto study their projections with light and electron microscopy. Also, we performed double-labeling experiments, injecting BDA in the CRNs and subunit B of the cholera toxin orFluorogold in the spinal cord to verify that CRNs project onto reticulospinal neurons.Electron microscopy of the labeled CRNs axons and terminals showed that even their mostcentral and thinnest processes are myelinated. Most of the terminals are axodendritic, withmultiple asymmetric synapses, and contain round vesicles (50 nm diameter). Double-labelingexperiments demonstrated that CRN terminals are apposed to retrogradely labeled reticu-lospinal neurons in the contralateral nucleus reticularis PnC and bilaterally in the lateralparagigantocellular nucleus. Analyses of serial sections revealed that multiple CRNs synapseon single reticulospinal neurons in PnC, suggesting a convergence of auditory information.The morphometric features of these neurons classify them as giant neurons. This studyconfirms that CRNs project directly onto reticulospinal neurons and presents other anatom-ical features of the CRNs that contribute to a better understanding of the circuitry of the ASRin the rat. J. Comp. Neurol. 460:80–93, 2003. © 2003 Wiley-Liss, Inc.

Indexing terms: acoustic startle reflex; reticular formation; biotinylated dextran amine;

fluorogold; subunitB cholera toxin; nucleus reticularis pontis caudalis

The acoustic startle response (ASR) is a defensive be-havior made up of a series of rapid and phasic contractionsof the skeletal muscles throughout the body and elicitedby sudden and loud sounds (Landis and Hunt, 1939; Hoff-man and Ison, 1980). In the rat, the ease with which thisbehavior (Davis et al., 1982) and its modifications (Pilzand Schnitzler, 1996) — prepulse inhibition, sensitization,habituation, and fear potentiation — can be quantifiedmake it a useful behavioral animal model for studyingsensorimotor integration (Mansbach et al., 1992; Koch,1999).

Although several different neural circuitries for theASR have been proposed (reviewed in Yeomans andFrankland, 1996), all the authors agree that the nucleusreticularis pontis caudalis (PnC) is a main component ofthe ASR (reviewed in Koch, 1999). Among the differentneuronal types within the PnC (Valverde, 1962), the giant

neurons appear to mediate this response. In fact, lesions ofthe PnC show a correlation between the number of sur-viving giant neurons and the amplitude of the ASR (Koch

Grant sponsor: Basque Government; Grant number: BFI 95.119; Grantsponsor: European Union; Grant number: QLGI-CT-1999-51291; Grantsponsor: Junta de Castilla y Leon; Grant number: JCyL/FSE: SA 084/01;Grant sponsor: Fondo de Investigacion Sanitaria del Ministerio de Sanidady Consumo; Grant number: PI021697.

*Correspondence to: Fernando R. Nodal, University Laboratory of Phys-iology, Park Road, OX1 3PT Oxford, UK.E-mail:[email protected]

Received 22 October 2002; Revised 20 December 2002; Accepted 26December 2002

DOI 10.1002/cne.10656Published online the week of March 31, 2003 in Wiley InterScience

(www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 460:80–93 (2003)

© 2003 WILEY-LISS, INC.

et al., 1992). Also, intracellular staining and recordings ofgiant neurons in PnC revealed that they receive very shortlatency auditory inputs (2.2 ms) and project to the spinalcord (Lingenhohl and Friauf, 1994).

The way in which acoustic inputs reach the PnC, andspecifically giant neurons, is the most controversial pointabout the neural circuitry of the ASR. In the rat, Lee et al.(1996) have demonstrated that the inputs from the co-chlear root neurons (CRNs) are essential to elicit the ASR.However, other studies have implicated the dorsal co-chlear nucleus (Meloni et al., 1998), ventral cochlear nu-cleus (Scott et al., 1999), and the superior olivary complex(Wagner et al., 2000) in the complete expression of the ASR.

CRNs, only described in some rodent (Lopez et al., 1993)and marsupial species (Aitkin, 1995), constitute the audi-tory nerve nucleus (ANN) (Harrison and Warr, 1962).CRNs respond to auditory stimuli with short latencies(Sinex et al., 2001). They project principally to the con-tralateral PnC, but also project to other brainstem nuclei,directly or secondarily related to motor control (Lopez etal., 1999). Thus, CRNs appear to be a specialized neuronaltype that mediates alert and escape behaviors driven bysound, including the ASR itself.

Although much data support the involvement of CRNsin the ASR, it still remains unclear if CRNs project di-rectly onto reticulospinal neurons and what type of syn-apses they form. This study aims to address these issuesusing double tract-tracing experiments and by ultrastruc-tural observations of the CRN terminals. Some of theseresults have been reported previously (Nodal et al., 1997).

MATERIALS AND METHODS

Animals and experimental design

Ten adult female Wistar rats (Charles River, Barcelona,Spain), weighing 300 g, were used in the present study(Table 1). The experiments were conducted according tothe Guidelines of the European Communities Council Di-rective (86/609/EEC) and the current Spanish legislationfor the use and care of laboratory animals (BOE 67/8509-12, 1998). All animals received an injection of biotinylateddextran amine (BDA) (BDA 10,000, #D-1956; MolecularProbes, Eugene, OR, USA) into the left cochlear nerveroot. Five of them received an additional injection of eitherSubunit B of Cholera Toxin (CT-B) (#103; List BiologicalLaboratories, Campbell, CA, USA) (n � 3) or FluoroGold�(FG) (Fluorochrome, Denver, CO, USA) (n � 2) into the

cervical spinal cord. The animals were housed in groups ofthree before surgery and individually after it, with waterand laboratory chow continuously available.

Injection of neuronal tracers

The injection of the neuronal tracers was performedunder deep anesthesia using a mixture of Xylacine (7mg/kg) and Ketamine (40 mg/kg) administered intramus-cularly. When necessary, one-fifth of the initial dose wasgiven during surgery.

After placing the animal in a stereotaxic frame usingthe head-positioning procedure described by Paxinos andWatson (1998), the skull was exposed by a midline incisionscalp and a craniotomy was made over the injection coor-dinates. The coordinates for the left cochlear nerve were–0.9 mm anteroposterior, 1.3 mm mediolateral, and �0.1mm vertical from the interaural point (see Lopez et al.,1999, for details). BDA (10% in saline) was iontophoreti-cally delivered through a glass micropipette (20 �m inter-nal diameter) with 3–5 �A of positive current for a periodof 20 minutes with a half duty period of 7 seconds. Afterthe scalp was sutured, the animals were allowed to re-cover for 7 days. Three animals with single injections inthe cochlear nerve root were processed for light micros-copy (97040, 97085, and 97116) and two for electron mi-croscopy (98003 and 98016).

Immediately following the cochlear nerve injection, fiveanimals received spinal cord injections. An incision wasmade in the midline of the neck. The trachea and esoph-agus were isolated and protected by a silicon tube so thatthey could be retracted laterally without occluding them.The body of the third or fourth vertebra was exposed,drilled, and the spinal meninges were retracted. EitherCT-B (1% in saline) or FG (4% in saline) was pressure-injected into the spinal cord using a Hamilton syringe (1�l) driven by a micromanipulator (#5000; Kopf, Tujunga,CA, USA). To minimize the lesion, a glass micropipette (40�m internal diameter) was glued to the needle of theHamilton syringe. Between 0.3 and 0.6 �l of tracer wasinjected. Following all injections the pipettes were left inplace for a period of 15 minutes before withdrawal toreduce reflux up the track.

Tissue preparation for light microscopeobservation

At the end of the postinjection survival time, the ani-mals were deeply anesthetized with sodium pentobarbital

TABLE 1. Animals Used in This Study1

Case Tracer Method of Injection Injection site Sectioning Processing

97040 BDA Iontophoresis ANN Coronal LM97085 BDA Iontophoresis ANN Parasagittal LM97116 BDA Iontophoresis ANN Horizontal LM98003 BDA Iontophoresis ANN Coronal EM98016 BDA Iontophoresis ANN Parasagittal EM96043 BDA

CT-BIontophoresisPressure

ANNSpinal cord

Coronal LM

96048 BDACT-B

IontophoresisPressure

ANNSpinal cord

Coronal LM

97027 BDACT-B


ANNSpinal cord

Coronal LM

97086 BDAFG


ANNSpinal cord

Coronal LM

97108 BDAFG


ANNSpinal cord

Coronal LM

1Cases in bold are used to illustrate this study. ANN, auditory nerve nucleus; BDA, biotinylated dextran amine; EM, electron microscopy; FG, FluoroGold; CT-B, subunit B ofchlolera toxin; LM, light microscopy.

81CRN PROJECT TO RETICULOSPINAL NEURONS

(60 mg/kg) and then perfused transcardially with 100 mlof fresh Ringer’s calcium-free buffer (NaCl, 145.45 mM;KCl, 3.35 mM; NaHCO3, 2.38 mM) pH 6.9 at 38°C, fol-lowed by 1,000 ml of fresh solution of 1% paraformalde-hyde and 1% glutaraldehyde in phosphate buffer (PB 0.06M, pH 7.4) at room temperature. The brains were removedand cryoprotected by immersion in 30% sucrose in PBuntil they sank. Then, the brains were cut at 40 �m usinga freezing sliding microtome in the appropriate plane (seeTable 1). The serial sections, collected in PB, were dividedinto 10 series.

Visualization of the BDA was made by incubating thefree-floating sections with avidin-biotin-peroxidase com-plex (ABC, #PK 4000; Vector Laboratories, Burlingame,CA, USA). Diaminobenzidine tetrahydrochloride (DAB)and nickel ammonium sulfate (Hancock, 1982) were thechromogens used for the peroxidase reaction. Briefly, thesections were washed in Tris 0.05 M, pH 8, incubated for90 minutes at room temperature in ABC, and, after sev-eral washes, reacted for 5–15 minutes in a solution con-taining H2O2 (9.14 mM), DAB (0.4 mM), and nickel am-monium sulfate (2.53 mM) to produce a black reactionproduct. The reaction was stopped by two washes withTris 0.05 M, pH 8.

In the double-injection experiments, FG or CT-B werevisualized immunochemically after BDA was revealed; thesections were incubated with either rabbit anti-FG (1/2,000 #AB-153; Chemicon, Temecula, CA, USA) or goatanti-CT-B (1/5,000 #703; List Biological) for 72 hours at4°C. Then they were incubated with biotinylated second-ary antibodies, either goat antirabbit (1/40 #PK-4001; Vec-tor Laboratories) or rabbit antigoat (1/40 #PK-4002; Vec-tor Labs.) for 90 minutes. Finally, the sections werereacted with ABC as described above, using only DAB asthe chromogen to produce a brown reaction product.

For each brain, all sections were mounted on slides andthree alternate series were counterstained with cresylviolet; the others were dehydrated and coverslipped withEntellan� Neu (Merck, Darmstadt, Germany).

Tissue preparation for electron microscopeobservation

The animals were perfused transcardially with 100 mlof Ringer’s calcium-free buffer and 1,000 ml of freshlyprepared fixative (1% paraformaldehyde and 1.25% glu-taraldehyde in PB 0.06 M, pH 7.4). The brains were re-moved and 60 �m thick sections were cut using a Vi-bratome. The sections were collected in cold PB (0.1 M, pH7.4) and incubated overnight at 4°C with gentle agitationin ABC Elite (Vector Laboratories; #PK-6100) in PB (0.1M, pH 7.4). After several washes with PB, sections werereacted with H2O2 (9.14 mM) and DAB (0.4 mM) in PB at4°C for 15–30 minutes. Then the sections were transferredto cacodylate buffer (0.1 M, pH 5.5) and osmicated for 2hours in 1% osmium tetroxide (EMscope, Ashford, UK).After several washes in distilled water and one in 50%ethanol, the sections were incubated overnight in a solu-tion of 1% of uranyl acetate and 70% of ethanol. The nextday the sections were dehydrated in a series of alcohols(90% and 100%), and then infiltrated with EMbed 812resin (all components from Electron Microscopy Science,Fort Washington, PA) and polymerized at 70°C betweenacetate sheets.

Sections for further study were selected under lightmicroscopy. These sections were chosen because terminal

axonal fields could be seen in the contralateral PnC. Se-lected areas of these sections were mounted on EMbed 816blocks for semithin (0.5 �m) and serial ultrathin section-ing (silver-gold interference �70 nm).

Cytoarchitectonic criteria

The terminology and abbreviations for the different nu-clei are from the atlas of the rat brain by Paxinos andWatson (1998). The subdivisions of the reticular formationare based on the descriptions of Newman (1985) and Lopezet al. (1999). We recognize four main divisions in thepontine magnocellular reticular formation. From caudalto rostral these are: the ventral pontine reticular nucleus(PnV); the nucleus reticularis pontis caudalis (PnC); theventrolateral tegmental nucleus (Vltg); and the nucleusreticularis pontis oralis (PnO). For the subdivisions in thespinal cord we used the Rexed subdivisions adapted to therat (Molander et al., 1989).

Data analysis

The sections were studied using a Leitz DMRB (Leica,Madrid, Spain) light microscope equipped with a drawingtube and digital camera and a Zeiss (EM 900) electrontransmission microscope. Negatives from electron micros-copy photographs and camera lucida drawings were digi-tized by an Epson (perfection 1240) scanner and correctedfor brightness and contrast. Occasional spurious back-ground was deleted. The morphometric analysis of thelabeled reticulospinal neurons was made with ScionImage(Beta 4.02, NIH Image) software.

The CRN axons were reconstructed using camera lucidadrawings of serial sections, drawn with a �40 objective.Drawings of adjacent sections were aligned by the contourof the sections and blood vessels.

To count the reticulospinal neurons in the PnC we em-ployed a modification of the physical fractionator. Thesections covering the PnC were divided into nine groupsand a pair of sections in each group was randomly andsystematically sampled. In one section of each pair (thereference section), we counted the labeled somata presentthroughout the nucleus and eliminated those also presentin the adjacent section (the mirror section). Then wecounted the labeled somata in the mirror section (now thereference section) minus the somata also present in theadjacent reference section (now the mirror section). Thisway we obtained two samples equally distributed throughthe PnC (Gundersen, 1986). The total number of labeledneurons was estimated by multiplying the sum of all theneurons counted in the different pairs by the fraction ofsections that they represent for the entire PnC. We calcu-lated the error using the formula of Cruz-Orive (1990).

RESULTS

Projection of cochlear root neurons

Injection sites in the cochlear nerve root. All injec-tion sites were restricted to the cochlear nerve root and didnot spread to the ventral division of the cochlear nuclei orthe trapezoid body (Fig. 1). In all cases the injectionslabeled primary auditory afferents that terminated in theipsilateral cochlear nuclei (Fig. 1D). In half of these casesprimary vestibular afferents were labeled. The vestibularafferents terminate in the ipsilateral vestibular nuclei andthus do not contribute any source of error for the analyses

82 F.R. NODAL AND D.E. LOPEZ

Fig. 1. Injection sites (IS) in the cochlear nerve root in cases 96043(A,B) and 97085 (C–E). A: photomicrograph of a coronal section at thelevel of the injection site (IS) in case 96043. A band of labeled termi-nals can be seen in the ventral division of the cochlear nuclei (PVCN)and thick-labeled fibers can be seen in the trapezoid body (arrows).Scale bar � 500 �m. B: The frame shows the location of the retro-gradely labeled cochlear root neurons (CRNs) drawn with a cameralucida. Scale bar � 25 �m. C: Photomicrograph from a Nissl stained

parasagittal section that shows the injection site (IS) in case 97085.Labeled CRNs are located in the deepest part of the nerve. Scale bar �500 �m. D: Camera lucida drawing of a more medial sagittal sectionshowing the terminals and axons in ipsilateral cochlear nuclei and thefibers traveling in the trapezoid body. Scale bar � 500 �m. E: Pho-tomicrograph of the trapezoid body from section in D where only fibersof thick diameter are visible. Scale bar � 250 �m.


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of this study. These data indicate that both cochlear andvestibular primary afferents take up the tracer as theytravel through the injection site as fibers of passage. La-beled axons arising from cochlear neurons were only ob-served in the ventral acoustic stria, the trapezoid body(tz). Most of these labeled fibers had a diameter of around5–6 �m. In case 96043, with a very small injection site(Fig. 1A), the serial reconstruction of these thick axonsrevealed their origin from three labeled neurons that layin the deepest part of the cochlear nerve root (Fig. 1B).These neurons were characterized by round-oval somataand dendrites oriented perpendicular to the primary au-ditory afferents. Based on their morphology and locationwe classified them as CRNs. The number of labeled CRNsaxons varied from case 96043 with three thick axons inthe trapezoid body to case 97085 with 19 axons (Fig.1D,E).

Labeled cells outside the cochlear nerve root were rarelyobserved and were located mainly in the ventral divisionsof the cochlear nuclei and in a few occasions in the supe-rior olivary complex, usually in the ventral nucleus of thetrapezoid body or in the periolivary nuclei. The axons ofthese retrogradely labeled neurons were thin, so they werereadily distinguishable from the thick CRN axons.

Targets and terminal fields of the CRNs. The tra-jectory and main targets of the CRNs described here (Fig.2) are in agreement with previous studies by us (Lopez etal., 1993: Lopez et al., 1999). CRNs, as a population,project bilaterally to the lateral paragigantocellular nu-cleus (LPGi) and the nucleus reticularis pontis caudalis(PnC). They also project ipsilaterally to the facial motornucleus (7) and contralaterally to the ventrolateral teg-mental nucleus (VlTg), the nucleus reticularis pontis ora-lis (PnO), the horizontal cell area of the lateral lemniscus,the intercollicular tegmentum, and the deep layers of thesuperior colliculus (SC).

Although all of the nuclei innervated by CRNs are sensustricto out of the auditory pathway, CRNs axons travel inthe tz and the most rostral part of the contralateral laterallemniscus (Fig. 2). All of the CRN axons give off collateralsto the contralateral PnC, VlTg, and PnO, but only a pro-portion of them innervate the rest of the nuclei.

In the ipsilateral tz, the labeled CRN axons have a largediameter (�6 �m) (Fig. 1) that is maintained for most oftheir length and only present a substantial reduction (lessthan 3 �m) in their most distal parts, as they becometerminal fields. Under the light microscope, the thicknessof the axons and the narrowing at the branching pointsthat resemble Ranvier nodules (Fig. 3) suggest that theyare myelinated. CRNs axons exhibited a consistent pat-tern of branching among animals. During their trajectory,each parent axon branches several times, giving off fromone to three collaterals at each branching point to inner-vate the corresponding target nuclei (Fig. 2). They thenramify profusely to form terminal fields where en passantand terminal synaptic boutons were seen (Fig. 3).

The reconstruction of serial sections of CRN axons re-vealed a lack of any topographic projection in the targetnuclei. The terminal fields of different neurons overlappedin each target nucleus. Furthermore, individual neuronsreceived terminals from multiple CRN axons. Several col-laterals from each CRN axon innervated each of the dif-ferent nuclei in the pontine reticular formation. The mor-phology and arrangement of the axonal varicosities (1–6�m diameter) were similar in all the target nuclei. Thedensity of the terminal innervation varied for each targetnucleus. This density is determined by the area of eachnucleus innervated by the CRN and by the cellular densityof each nucleus. The most extensive terminal field wasfound in the contralateral PnC, while the greatest termi-nal density was present in the horizontal cell area of thelateral lemniscus (Figs. 2, 3).

Fig. 3. Distal-labeled CRN axons. A: Photomicrograph of a para-sagittal section at the level of the contralateral horizontal cell area inthe lateral lemniscus. One thick parent axon running in the laterallemniscus can be seen in the left bottom part showing a tight branchpoint (arrow). Numerous terminal and en passant boutons are present

(doubleheaded arrows). B: Photomicrograph of a parasagittal sectionat the level of the contralateral PnC. Several axons of different diam-eters can be seen branching (arrows) and terminal and en passantboutons are present (doubleheaded arrows). Scale bars � 50 �m.


Fig. 4. Ultrastructure of CRN terminals in PnC. A: photomontageshowing two labeled terminals (T1,T2) that synapse (white arrow-heads) on the same dendrite. The postsynaptic membrane contains anelectron-dense thickening at these synapses. In terminal 1 (T1), part

of the axon is surrounded by myelin sheaths (arrowheads). Scalebar � 1 �m. B,C: Photographs of the terminal 2 (T2) taken fromdifferent ultrathin-sections. bv, blood vessel. Scale bar � 0.5 �m.

Light microscopic observations indicated that most ax-onal varicosities, en passant or terminal, occurred verynear the neuronal somata and very few were closely ap-posed with them.

Electron microscopy of the labeled axons and terminalsfrom the CRN (Figs. 4, 5) in the contralateral PnC con-firmed some of the features observed with light micros-copy. Myelin sheaths were observed surrounding labeledaxons and, on occasion, they were interrupted at the endof the axon where a terminal bouton was present (Fig. 4).Most of the boutons are round, but oval boutons wereoccasionally observed (Fig. 5). We observed only one axo-somatic contact established by an en passant bouton; therest of the synaptic contacts studied were axodendriticterminals.

Most labeled terminals contacted primary dendrites, ascan be inferred by the diameter of the postsynaptic pro-files and by the presence of mitochondria in them (Fig. 4).Normally, each terminal included several synaptic areas.Synapses were about 250 nm in length, exhibited anelectron-dense material in the postsynaptic membraneand were classified as asymmetric. Although the CRNpresynaptic terminals were very densely stained, makingit difficult to observe their morphology, round vesicles(diameter 50 nm) were observed within them (Fig. 5).

Distribution and morphology of pontinereticulospinal neurons

Injection sites in the spinal cord. All the injectionsites in the spinal cord were located at cervical levels, inC3 or C4. The most restricted injection site (case 96043)included the medial and ventral funiculus of the righthemicord, with some spread into the right and left ventralhorns. The remaining cases had more extensive injectionsites: in some the medial funiculus was affected bilaterallyand in others the gray matter was widely affected. Thebiggest injection site corresponded to case 97086, whichincluded almost all of the right hemicord and spared onlypart of the lateral funiculus on that side (Fig. 6).

The size of the injection sites depended on the amount oftracer injected, the depth at which the injection was made,and the type of tracer employed in each case. Althoughequal parameters were used to deliver the tracers, FGinjection sites were bigger than those produced by CT-B incoronal sections. However, the diffusion of both tracerswas similar in the rostrocaudal extension. In all cases theinjection site was restricted to the cervical spinal cord,typically centered at the C3 level covering the whole seg-ment and some spread of the tracer could be observed intothe adjacent segments C2 and C4.

In the FG cases, an area of gliosis was observed at thecenter of the injection site. However, this did not obviouslyaffect the behavior or the motor control of those animals.The morphology of the retrogradely labeled neurons in thereticular formation was similar with both tracers em-ployed, although FG provided a more extensive filling ofthe neurons than CT-B.

Fig. 5. Ultrastructure of CRN terminals in PnC. A: Photomicro-graph of a labeled terminal (T1) in PnC; round vesicles can be ob-served in it. The adjacent nonlabeled terminal (*) contains pleomor-phic vesicles. B: Photomontage showing an elongated terminal (T2)with several synaptic contacts (white arrows). Scale bars � 0.5 �m.


Fig. 6. Distribution of reticulospinal neurons and CRN terminalfields in case 97086. The injection site of FluoroGold in the spinal cordis shown in the top left drawing. In the series of camera lucidadrawings (a–i), the distance between serial sections is 400 �m. Re-ticulospinal neurons are represented by dots. Stars indicate the loca-tion of reticulospinal neurons with appositions from CRN axons on

them. The gray areas show the terminal fields from the CRN. Injec-tion site in the cochlear nerve is located in section c. Gi, gigantocel-lular reticular nucleus; GiA, gigantocellular reticular nucleus alpha;IRt, intermediate reticular nucleus; PL, paralemniscal area; RtTg,reticulotegmental nucleus of pons; SubCD, subcoeruleus dorsal part;SubCV, subcoeruleus ventral part. Scale bar � 1 mm.

The number and proportion of somata in PnC variedfrom one animal to another in relation to the location andextension of the injection site. In cases 96043 and 97027,whose injection sites were restricted to the right side ofthe spinal cord, we observed labeled somata bilaterally,but with a clear ipsilateral predominance as the ratio ofthe number of neurons in right PnC versus left PnC was5.24 and 7.03, respectively. In the other cases, in whichthe injection spread at least into the left ventral funiculus,this ratio was around 1 (0.77, 0.98, and 1.01; Table 2).

Distribution of labeled neurons. Case 96086 re-ceived the largest injection into the spinal cord and wefound labeled neurons in the vestibular nuclei, magnocel-lular reticular formation, deep layers of the superior col-liculus, red nucleus, substantia nigra, and motor cortex. Itis beyond the scope of this report to make an exhaustivedescription of these descending pathways and their cells oforigin; thus, we are only describing the morphology of thelabeled cells present in those nuclei innervated by CRNs(Fig. 6).

Labeled cells in LPGi present an elongated soma ori-ented parallel to the dorsal surface of the pyramidal tract.Most of the LPGi cells are reticulospinal, as is shown incase 96086. The majority of these cells project bilaterallyto the spinal cord, as they were labeled on both sides evenif the injection was restricted to one hemicord (Case96043). LPGi neurons normally present 3–5 dendritictrunks. Two of them arise from opposite poles and areoriented in the same direction of the largest diameter ofthe soma. The other primary dendrites run perpendicularto the main axis of the soma, arising ventrally or dorsally.Usually, the primary dendrites ramify in the vicinity ofthe soma.

The somata of labeled neurons in PnC and PnO range indiameter from 20–50 �m and are classified as medium-sized (20–40 �m) and giant (�40 �m) neurons. Most ofthe labeled neurons projected ipsilaterally to the spinalcord, but some of them also projected contralaterally. Inthe coronal plane, the reticulospinal neurons in PnC andPnO had a multipolar morphology, with a polygonal somaand three to six primary dendrites that lacked any pre-ferred direction. The dendrites often extended far fromtheir soma and usually intermingled with other dendritictrees arising from neurons in the same vicinity and to-gether these formed a complex lattice.

In single sections through PnC, labeled neurons weremore numerous in the central part of the nucleus than inits periphery (Fig. 6), but no difference in the number ofneurons was observed in the rostrocaudal extension of thenucleus when we compared different sections. The giantreticular neurons were located predominantly in the cen-tral part of PnC while the medium-sized ones were morehomogeneously distributed in the nucleus.

Terminals of the CRNs onto reticulospinalneurons

In all the cases with double injections into the cochlearroot nerve and the spinal cord, we observed a spatialcoincidence between the terminal fields in the LPGi, PnC,VlTg, and PnO that arise from CRNs and the location ofthe retrogradely labeled reticulospinal neurons in thesenuclei (Fig. 6). On the other hand, the SC-labeled neuronswere located in the most lateral part of layer VI, whereasthe terminal fields of the CRNs were located more medi-ally within the same lamina.

Of the nuclei in which CRN targets overlapped thereticulospinal neurons, we only observed labeled termi-nals apposed to retrogradely neurons in the LPGi and thePnC, and these were present bilaterally (Fig. 6). In theLPGi these neurons were approximately equally distrib-uted on both sides. On the other hand, CRNs predomi-nantly synapse on contralateral PnC neurons.

In case 96086, in which both injection sites are thebiggest in each category, we found 60 retrogradely la-beled neurons in the contralateral PnC with terminalappositions from the CRN on them. The dimensions ofthe major and minor diameters of these PnC neuronswere (mean � SEM) 49.19 �1.12 �m and 32.21 �0.79�m, respectively, and all of them were therefore classi-fied as giant neurons.

One-half of neurons that were found in close proxim-ity to labeled CRN axons had more than one, usually2– 4 appositions on them (Fig. 7). These appositionswere mainly located on the somata and on the veryproximal dendrites but this observation may have beenbiased by the fact that neither FG nor CT-B labeled thewhole cell.

DISCUSSION

The present study investigates the projection of theCRN to reticulospinal neurons and the ultrastructure ofthe CRN terminals. Restricted injections of BDA in thecochlear nerve were analyzed using EM and revealed thatCRN axons are myelinated and that they establish enpassant and terminal contacts in the PnC. These termi-nals exhibit round vesicles and asymmetric synapses, sug-gesting that they are excitatory. Double-labeling experi-ments demonstrated that CRNs project bilaterally to thePnC and the LPGi where they synapse with reticulospinalneurons. Most of these reticulospinal cells in the PnC aregiant neurons. Thus, the projection from CRN onto theseneurons could constitute one of the shortest possible cir-cuitry for the auditory startle reflex (Lee et al., 1996;Yeomans and Frankland, 1996; Koch and Schnitzler,1997; Koch, 1999).

TABLE 2. Labeled Neurons in PnC1

Case Tracer Total Ipsilateral PnC Contralateral PnC Ipsi/Contra

96043 CT-B 656 551 84% 105 16% 5.2496048 CT-B 1020 505 49.6% 515 50.4% 0.9897027 CT-B 458 401 87.5% 57 12.4% 7.0397086 FG 2094 916 43.7% 1178 56.3% 0.7797108 FG 1790 902 50.4% 888 49.6% 1.01

1The number of neurons was estimated by the physical fractionator. In the cases where both sides of the spinal cord were affected by the injection site the most contained traceris considered as ipsilateral. FG, FluoroGold; OM, CT-B, subunit B of chlolera toxin.


An essential point in any tract-tracing experiment is todelineate the effective injection site in order to quantifythe extent of the labeled structures. This becomes moredifficult in double tract-tracing experiments, in which theobjective is to study the convergence of the labeled struc-tures from both neuronal tracers used.

Because CRNs axons course through the tz (Lopez et al.,1999, and present study), we define the effective injectionsite in the cochlear nerve root in terms of labeled-thick axons

running in the tz irrespective of the location or extent of theinjection site. The number of labeled CRN axons in ourexperiments (range 3–19) constitutes �10–50% of the wholepopulation of one side, estimated at 40–50 neurons (Mer-chan et al., 1988). The targets of the CRN are consistentamong the cases examined in this study and with those ofprevious studies (Lee et al., 1996; Lopez et al., 1999) and weexpect that the unlabeled neurons would have had the samepattern of projections and targets.

Fig. 7. Photomicrographs (leftcolumn, bar 25 �m) and camera lu-cida drawings (right column, scalebar � 30 �m) of coronal sectionsfrom case 97086 showing severalretrogradely labeled PnC neurons(in brown) with appositions fromanterogradely labeled CRN axons(in black) on them (arrows). Neu-rons in A, C, and D were located inthe PnC contralateral to the injec-tion site in the cochlear root nerve.Neuron in B is located in the ipsi-lateral PnC.


The employment of different sectioning planes and the3D reconstruction of the CRN axons in this study providesa more comprehensive picture of their spatial arrange-ment than in previous studies based only in the coronalplane. Our results show that terminal fields of individualaxons clearly cross the boundaries of PnC, Vltg, PnO, andparalemniscal area. The reconstruction of individual ax-ons shows that the same collaterals usually provide ter-minals in several nuclei. The terminals in PnO have adouble source. Some terminal branches arise continuouslyfrom a main collateral axon that enters the PnC ventrallyand then runs rostrally to reach PnO, whereas others havetheir origin from collaterals that emerge from the parentaxons at different positions in the rostral part of the lat-eral lemniscus and then traverse in a caudal direction toend in the paralemniscal regions and PnO. In a panoramicview, the CRN terminal fields in the reticular formationcan be interpreted as a unique field that extends along allthese reticular nuclei. This interpretation agrees withsome of the descriptions of the reticular formation (New-man, 1985; Valverde, 1961, 1962) in which distinct cyto-architectonic boundaries cannot be delineated.

The reconstruction of individual axons also shows thatnot all the axons innervate the same targets. For a totalnumber of 19 axons traced in case 97085, all of them werefound to have a contralateral projection to the LPGi, PnC,VlTg, PnO, the horizontal cell area of the LL and thetectum. Ipsilateral projections are more restricted, as onlythree of these axons innervated the ipsilateral PnC, seventhe ipsilateral LPGi and two of the latter also innervatedthe facial nucleus. In case 96043 only one of three labeledCRNs projected to the ipsilateral PnC, even though thethree CRN somas lay close together in the cochlear nerveroot. We believe this difference cannot be attributed to anincomplete labeling of the axonal trees, because more cen-tral and thinner collaterals were traced for the same axon.Moreover, retrograde injections in PnC (Lingenhohl andFriauf, 1994, and pers. obs.) label CRNs bilaterally, al-though the number is always greater on the contralateralside. More experiments are needed to determine if theseneuronal types have distinct functions or if they receive adifferential innervation by the primary afferents.

Although the acoustic startle reflex in the rat is a widelyused behavioral model, relatively little attention has beenfocused on its neural circuitry (Yeomans and Frankland,1996). The PnC and the LPGi, in which CRNs project ontoreticulospinal neurons, are related to different aspects ofthe ASR. The projection to LPGi could explain in part therapid autonomic response observed in the ASR (Baundrieet al., 1997). Early lesion (Davis et al., 1982) and electro-physiological (Wu et al., 1988) studies have shown thepivotal role of PnC in the motor control of the ASR. Fur-thermore, most of the circuitries responsible for modifica-tions of the ASR converge on PnC neurons (Koch, 1999;Fendt and Fanselow, 1999; Fendt at al., 2001). In the rat,among the different neuronal types described in the PnC,the giant neurons appear to mediate this reflex as they arereticulospinal and receive auditory information (Lingen-hohl and Friauf, 1992). In addition, there is a correlationbetween the number of giant cells in PnC and the ampli-tude of the ASR (Koch et al., 1992).

The data from the labeling experiments may underesti-mate the total number of CRN terminals on reticulospinalneurons if only a portion of the reticulospinal neuronswere labeled or if their dendrites were partially labeled.

Lingenhohl and Friauf (1994) demonstrated that giantreticulospinal neurons that receive acoustic informationproject to a large extension of the spinal cord by collateralsemerging along their descending trajectory. To maximizethe number of labeled reticulospinal neurons, we exploitedthe fact that FG and CT-B can be taken up by fibers ofpassage (Dado et al., 1990; Chen and Aston-Jones, 1995).Thus, the injections in the spinal cord were made using aventral approach to ensure that the reticulospinal tract(Siegel et al., 1983) was affected. In addition, the pressureinjections were made at the cervical level to label as manyreticulospinal axons as possible, because at this level theaxons either project to the injection site or they runthrough it to innervate more distal segments of the spinalcord (Matsuyama et al., 1997, 1999). Therefore, it is notpossible to determine what musculature is affected by theactivity of the labeled reticulospinal neurons because theycould take up the tracer by their terminals or as fibers ofpassage.

In PnC and PnO we observed overlap between the ter-minal fields from CRN and the distribution of the reticu-lospinal neurons in these nuclei. It is impossible to deter-mine the number of boutons that innervate the labeledneurons because FluoroGold and Subunit B of the CholeraToxin produce incomplete retrograde filling and only allowus to visualize the terminals located on the soma or on themost proximal parts of the dendritic tree. Some of theboutons observed in the proximity of the retrogradely la-beled neurons could participate in this innervation. Thereconstruction of the axons that synapse on the labeledneurons shows that they arise from different CRNs, indi-cating a convergence onto single neurons.

The ASR comprises the participation of numerous mus-cles along the whole body from the head to the hind limbs.Although lesions of the CRN have demonstrated theirparticipation in the ASR (Lee et al., 1996), little attentionhas been paid to the possible implications on other behav-iors like head orientation towards a new stimulus. Per-haps this is due to the fact that the latency of the head-orienting response is much longer, 40–100 ms (Thompsonand Masterton, 1978) than for the ASR (7–10 ms). But theCRN can contribute to it by providing fast auditory infor-mation to deeper layers of the SC, paralemniscal area,facial nucleus, and PnC (for a review of brainstem controlof head orienting, see Isa and Sasaki, 2002). In the deeplayers of the SC an auditory space map has been shown(King and Hutchings 1987) and also in this nucleus arethe tectoreticular and tectospinal neurons that control theneck musculature (Corneil et al., 2002). After injections incervical spinal cord, several labeled neurons were locatedin the SC, although no contact on them from CRN wereobserved. The CRN input to the PnO has a more impor-tant role in orienting movements than the CRN projectionto the SC, since the projection to the PnO is much biggerthan to the SC. Based on comparative connections andhistology, the lateral part of PnO could be homologous tothe paralemniscal area described in the cat that partici-pates in the control of the pinna movements (Henkel,1981). The CRN also project to motoneurons, innervatingpinna muscles, in the facial nucleus (Friauf and Herbert,1985), which could similarly contribute to generatingpinna movements.

An additional circuit by which the PnO might influenceorienting movements exists through its projections withinthe reticular formation, i.e., from PnO and VlTg to the


PnC; although it is unknown if the same PnC neurons areinnervated by both the CRN and PnO and/or VlTg. Fromour injections in the spinal cord it is not possible deter-mine which musculature is controlled by the labeled re-ticulospinal neurons. However, the most reliably labeledneurons should be those that project to the spinal level ofthe injection site, so we cannot exclude the possibility thatsome of the labeled neurons in PnC received terminalsfrom CRN participate in the control of the neck muscula-ture.

Yeomans and Frankland (1996) proposed the existenceof two parallel circuits that mediate the ASR: “monosyn-aptic” and “disynaptic” that converge in PnC. In the mono-synaptic circuit the acoustic information would reach PnCdirectly from CRN neurons. In the disynaptic pathway,that information reaches PnC through Vltg. Although le-sions of Vltg do not abolish the ASR (Lee et al., 1996), thisnucleus may amplify the signal from the CRN. The CRNsproject to the Vltg and this nucleus projects to the PnC(Herbert et al., 1997). Thus, the PnC is innervated bothdirectly and indirectly by the CRNs. Intracellular record-ings of giant neurons in PnC show that they have a lowmembrane resistance and long time constant (Wagner andMack 1998), indicating that they respond weakly andslowly to their synaptic inputs. This apparently contradic-tory property for mediating a very short latency behaviorlike the ASR may be offset by increasing the number ofinputs. Thus, the convergence of different CRN projectionsdirectly on a single PnC neuron and indirectly throughipsilateral Vltg and/or contralateral PnC neurons

(Hempel et al., 1993) may be critical for producing theASR. Also, this loop can provide a time delay to integrateother auditory signals from the DCN (Meloni and Davis,1998), VCN (Scott et al., 1999), and the superior olivarycomplex (Wagner et al., 2000).

The obligatory role of the CRN in the ASR (Lee et al.,1996) requires a high speed of conduction of the actionpotentials by CRN. The large diameter of the labeled CRNaxons examined with light microscopy (Lopez et al., 1993,1999) suggests that they are myelinated. Their ultrastruc-ture reveals myelin sheaths surrounding them until theybecome terminal boutons, providing a high-speed conduc-tion. The round shape of the vesicles in the labeled termi-nals and the asymmetry of the synapses observed in ourmaterial are consistent with an excitatory neurotransmit-ter (Uchizono, 1965). The reduction of the amplitude of theASR after infusion of glutamate antagonists in PnC(Krase et al., 1993) indicates that some acoustic inputs areglutamatergic.

The location of the CRNs in the auditory nerve allowsthem to receive a copy of all the incoming auditory infor-mation. In fact, their discharge pattern resembles thoserecorded for auditory nerve (Sinex et al., 2001). Extracel-lular recordings from CRNs (Sinex et al., 2001) show theyhave a short first-spike latency (2.2 ms) and they aretuned to high frequencies, but above 50 dB SPL, which iswell below the threshold for the ASR, they respond to awide range of frequencies (0.4–55 kHz) and their firingrates seem to reach a maximum. Consequently, if only adirect CRN-PnC projection was sufficient to elicit theASR, this reflex should be produced by any sound above 50dB SPL. Thus, other mechanisms, such as the intrinsicmembrane properties of giant neurons, reverberant cir-cuitries (CRN-Vltg-PnC), and/or inputs from other audi-tory nuclei may provide an effective control of the ASR.

In summary, the myelinization of the CRN axons andthe ultrastructural data of their terminal boutons areconsistent with their role in the ASR. In the rat, the CRNprojection to giant reticulospinal neurons in PnC can con-stitute a rapid route for mediation of the ASR (Fig. 8).Moreover, their projection to the Vltg also implicates arole of the CRN in the full expression of the ASR and notonly in its initiation.

ACKNOWLEDGMENTS

The authors thank Ignacio Plaza for technical assis-tance and Dr. Andrew King for reviewing an early versionand for the use of his laboratory.

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