functional neuroanatomy of auditory pathways in the sound-producing fishpollimyrus

Functional Neuroanatomy of AuditoryPathways in the Sound-Producing Fish

Pollimyrus

JAMES KOZLOSKI1* AND JOHN D. CRAWFORD1,2

1Graduate Group in Neuroscience, University of Pennsylvania, Philadelphia,Pennsylvania 19104

2Department of Psychology, University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACTWe have described the acoustic pathway from the ear to the diencephalon in a

sound-producing fish (Pollimyrus) based on simultaneous neurophysiological recordings fromsingle neurons and injections of biotin pathway tracers at the recording sites. Fundamentaltransformations of auditory information from highly phase-locked and entrained responses inprimary eighth nerve afferents and first-order medullary neurons to more weakly phase-locked responses in the auditory midbrain were revealed by physiological recordings.Anatomical pathway tracing uncovered a bilateral array of both first- and second-ordermedullary nuclei and a perilemniscal nucleus. Interconnections within the medullaryauditory areas were extensive. Medullary nuclei projected to the auditory midbrain by meansof the lateral lemniscus. Midbrain auditory areas projected to both ipsilateral and contralat-eral optic tecta and to an array of three nuclei in the auditory thalamus. The significance ofthese findings to the elucidation of mechanisms for the analysis of communication sounds andspatial hearing in this vertebrate animal is discussed. J. Comp. Neurol. 401:227–252, 1998.r 1998 Wiley-Liss, Inc.

Indexing terms: inferior colliculus; superior olive; octavolateralis; auditory communication;

electric fish; mormyrid

Despite the critical position of fishes in vertebratephylogeny and the importance of hearing and acousticcommunication in almost all vertebrates, an integrativeneuroethology of auditory communication in fish has beenrelatively slow to develop. The neuroethological approachhas been extremely instructive in elucidating fundamentalbehavioral and neurocomputational mechanisms in theelectrosensory systems of fishes (Heiligenberg, 1991;Kawasaki, 1997), and in the analysis of the auditorysystems of other vertebrates such as anurans (e.g.,Capranica, 1965; Feng et al., 1990; Simmons et al., 1996;Narins, 1995), birds (Doupe and Konishi, 1991; Konishi,1993), and mammals (Suga, 1990) that rely on sounds fortheir natural behavior. Many fish species use sounds insocial behavior (Myrberg, 1997), and many have evolvedaccessory structures for coupling sound pressure to mecha-nosensory hair cells of the inner ear (Schellart and Popper,1992). In this paper, we present results from a functionalneuroanatomical study of a fish that produces sounds andthat has accessory structures for pressure transduction.These results form an essential new component of anintegrated behavioral and physiological analysis of audi-tory communication in this species.

To date, research in fish auditory systems has succeededin applying behavioral, physiological, and anatomical ap-proaches to the problems of vertebrate audition. However,much of this research has focused on species that are notknown to use sounds in their natural behavior (e.g.,Furukawa and Ishii, 1967; Echteler, 1984, 1985a,b; Coombsand Fay, 1989; Fay, 1995; Wubbels and Schellart, 1997) oron fishes that lack specializations for pressure transduc-tion and that apparently detect low-frequency vibrationswith otolithic endorgans (Fine, 1981; Yan and Popper,1993; Lu et al., 1996; Lugli et al., 1996 Bass et al., 1997).Our goal in the present study is to advance the neuroetho-logical study of auditory communication in fishes by

Grant sponsor: National Institute on Deafness & Other CommunicationDisorders; Grant number: NIH DC01252; Grant sponsor: PennsylvaniaLions Hearing Research Foundation; Grant sponsor: University of Pennsyl-vania Research Foundation; Grant sponsor: NIMH; Grant number: PBNF31 MH11270–01A1.

*Correspondence to: Dr. James Kozloski, Department of Psychology,University of Pennsylvania, 3815 Walnut Street, Philadelphia, PA 19104.E-mail: [email protected]

Received 16 February 1998; Revised 3 June 1998; Accepted 19 June 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 401:227–252 (1998)

r 1998 WILEY-LISS, INC.

providing an analysis of the auditory pathways of a speciesthat has specializations for pressure transduction, usessounds for reproduction, and continues to yield importantinformation on the behavioral and neurophysiologicalmechanisms of auditory communication.

The African mormyrid fish, Pollimyrus adspersus, pro-duces one of the most elaborate sonic displays knownamong fishes. Males use sounds to call and to courtfemales. The sounds are species specific, and each indi-vidual male has his own acoustic signature (Crawford,1997b; Crawford et al., 1997a). Within the ear, eachsaccule is coupled directly to a gas-filled tympanic bubbleon each side of the head. Neurophysiological studies haveshown that auditory neurons in the medulla and midbrainare highly sensitive to acoustic stimuli like those producedby the fish (Crawford, 1993; Kozloski and Crawford, 1996)and have revealed neurons in the midbrain with complexreceptive fields that show selectivity for temporal featurescharacteristic of courtship sounds (Crawford, 1997a). Tounderstand the fate of acoustic information transduced atthe ear and the neural computations that yield the com-plex responses seen in higher auditory areas, it is essentialto have a clear picture of the auditory anatomy of thisspecies.

There have been relatively few functional neuroanatomi-cal studies of auditory systems of fishes of any kind.Elegant contributions have been made toward mappingthe central auditory pathways of several fish species(reviewed in McCormick, 1992, 1998), but few investiga-tors have combined the study of neurophysiological re-sponses to sound with neuroanatomical pathway tracing(Echteler, 1984). The auditory brainstems of those fishesthat have been investigated show complex anatomy, includ-ing both first and second-order nuclei in the medulla, andcenters of potential multimodal integration. The addedimplementation of neurophysiological recording from neu-rons in each of these areas is critical to any kind offunctional interpretation of these systems.

We used double-barrel electrodes, designed for recordingand dye injection, to introduce neuroanatomical pathway

tracers at the locations of physiologically characterizedauditory neurons within the brainstem of Pollimyrus. Asfar as we know, this article represents the first integrationof single cell auditory physiology and anatomy in a speciesof fish that possesses specializations for acoustic pressurereception and that uses sounds for communication. Weshow that Pollimyrus has a complex of seven distinct first-and second-order auditory processing areas in the rostralmedulla, a single perilemniscal cell group, and a highlyorganized array of outputs from the midbrain auditoryarea into the optic tecta and thalamus. These findingssupport and extend previous anatomical efforts to eluci-date the course of auditory information in the brainstem ofmormyrid fishes (Maler et al., 1973a,b; Szabo and Libou-ban, 1979; Bell, 1981a,b; McCormick, 1982; Haugede-Carre, 1983), other osteoglossomorphs (Braford et al.,1993), and fishes in general (reviewed in McCormick,1998). The neurophysiological results accompanying ourexperimental neuroanatomy demonstrate that there is amajor transformation in the representation of acousticinformation as it ascends this pathway from the ear to themesencephalon. Preliminary presentations of this workhave been made previously (Kozloski and Crawford, 1995,1996, 1997, 1998).

MATERIALS AND METHODS

General procedure for dye injection

We first localized auditory nuclei by recording physiologi-cal responses to sounds as electrodes were advanced. Wethen either performed single unit extracellular physiologywith a metal-filled glass electrode followed by extracellularinjection of tracer through a second barrel of the sameelectrode or performed intracellular physiology and dyeinjection with sharp electrodes. Biocytin and neurobiotinwere used as tracers in these studies. Single auditoryneurons were isolated and then characterized by usingacoustic tone bursts and click trains as stimuli. Twelveextracellular injections were made into the dorsomedial

Abbreviations

ALLn anterior lateral line nerveAnt anterior octaval nucleusELL electrosensory lateral line lobeC1 cerebellum, lobe 1CP central posterior nucleusCrCb crista cerebellarisdll decussation of the lateral lemniscusDLZ dorsolateral zone of ELLdmc dorsal mesencephalic commissuredSO dorsal secondary octaral nucleusdSO(d) dorsal secondary octaval nucleus, dendritic fielddzD dorsomedial zone of the descending octaval nucleuseff auditory efferent neuronsEGa anterior eminentia granularisELa anterior exterolateral toral nucleusELp posterior exterolateral toral nucleusH habenular nucleusizD intermediate zone of the descending octaval nucleusIG isthmic granular nucleusIRC isthmic reticular commissureIRN isthmic reticular nucleusiSO intermediate secondary octaval nucleusizD intermediate zone of the descending octaval nucleusL lateral toral nucleusll lateral lemniscusM medial octaval nucleusMC Mauthner cell

MD mediodorsal toral nucleus (n. centralis & n. ventrolateralis)MGM mesencephalic granular massmV motor nucleus of V (trigeminal)MV medioventral toral nucleusnELL nucleus of electrosensory lateral line lobenpc nucleus of the posterior commissurenV nucleus of the descending tract of V (trigeminal)OT optic tectumpc posterior commissurePE preeminential toral nucleusPEV ventral preeminential toral nucleusPG preglomerular complexPLLN posterior lateral line nervePT pretectal areapvTH posterioventral thalamic nucleusRF reticular formationTDT toro-diencephalic tractTL torus longitudinalistp toro-preeminential tractTS torus semicircularisV descending tract of V (trigeminal)VA valvulaVLZ ventrolateral zone of ELLVP ventral posterior toral nucleusvped valvular pedunclevSO ventral secondary octaval nucleus

228 J. KOZLOSKI AND J.D. CRAWFORD

division (auditory) of the midbrain torus semicircularisafter single auditory neurons were characterized.

Midbrain injections were followed by reciprocal injec-tions into previously uncharacterized auditory regions ofthe medulla and some intracellular recordings and dye fillsof medullary neurons and primary eighth nerve (nVIII)afferents. Medullary injections were localized to the de-scending octaval nucleus. In addition to injections intoP. adspersus, five additional injections into either themidbrain or medulla were made into three related speciesof mormyrid (P. isidori, Gnathonemus petersii, and Brieno-myrus niger). These allowed for confirmation of previousresults by investigators as well as providing materialfor comparative analysis within the diverse familyMormyridae.

Preparation

Experiments were done on adult P. adspersus importedfrom Nigeria by commercial dealers. Our preparation forneurophysiological recording has been described previ-ously (Crawford, 1997a). The fish were immobilized byintramuscular injection of gallamine triethiodide (Flaxedil,0.4 µg/g body weight), placed on a Plexiglas platform in theexperimental tank, and respirated with clean oxygenatedwater. Local anesthesia (lidocaine) was administered to asmall region of the skull and overlying tissues beforesurgically cutting a hole in the skull (1-mm diameter) formicroelectrode penetrations. The fish were submergedduring recordings and injections, with the center of thehead positioned 25 mm below the water surface. A shallowplastic cannula affixed to the skull and filled with Fluoroin-ert (3M, St. Paul, MN) prevented water from flooding thebraincase. Experiments were carried out in a sound attenu-ating chamber (IAC 400 series).

Experimental tank

The experimental tank was a cylindrical, water-filled,Plexiglas container with an underwater speaker (Univer-sity UW 30) positioned at the bottom (Fay and Ream,1986). Dual pneumatic vibration isolating tables (TMC)served to isolate the tank from building vibrations and tomechanically isolate the fish from the speaker; the onlydirect coupling of the sound source to the rigidly clampedpreparation was through the water column (see Crawford,1993, for details).

Stimulus generation and calibration

Stimulus generation and spike data acquisition werebased on a DTK PC (386 series with math coprocessor) anda MicroStar Labs 2400/5 DSP based Data AcquisitionProcessor (DAP) board (Crawford, 1997a). Tones and clicktrains were synthesized digitally (50 kHz; 20 µsec/point)during the experiments and output by means of a digital toanalog (D/A) converter. The signals were then bandpassfiltered (100 Hz to 5 kHz, Krohn-Hite analog filter) andattenuated before being amplified with a Crown poweramplifier and output to the speaker. Signals were cali-brated for sound pressure by using a B&K 8103 miniaturehydrophone and B&K 2106 charge amplifier.

Neurophysiological recording

We made extracellular recordings with metal-filled glassmicropipettes (Indium electrodes; Dowben and Rose, 1953)and intracellular recordings with 2 M KCl-filled glassmicropipettes. Single-barrel glass micropipettes were

pulled on a Sutter Instruments (model P97) horizontalpipette puller. A Grass P-511 amplifier was used forextracellular recordings and a World Precision Instru-ments (WPI, New Haven CT) Intra 767 electrometer forintracellular recordings. Extracellular recordings wereused in the initial localization of auditory areas andcollection of some physiological data. These recordingswere followed by penetrations with either double-barrelelectrodes for combined recording and extracellular dyeinjections or sharp micropipettes for intracellular record-ing and dye injection.

Electrodes were advanced into the brain under remotecontrol from outside the sound-attenuating room by usinga Burleigh microdrive while both tones and clicks werepresented as search stimuli. We also presented a vibratingbead (30 Hz) to stimulate the lateral line system, and anelectric stimulus to stimulate the electrosensory pathwaywhile recording auditory neurons. None of the auditoryneurons were activated by these other modalities, al-though in a few cases a vibrating bead placed very close tothe ear elicited a response; this response was expected dueto pressure stimulation of the saccule (Coombs, 1994).

Double-barrel electrodes and extracellulardye injections

Extracellular injections were made by using double-barrelelectrodes (recording 1 tracer) to ensure that injectionsoccurred within 15 µm of neurophysiologically character-ized auditory units. Double-barrel glass (WPI 2B150F)was pulled on a Kopf (model 700D) vertical puller. Thepipette tips were broken back to a diameter of approxi-mately 8 µm per barrel. One barrel was prepared as ametal recording electrode as described above, and theother was backfilled with the tracer solution.

We used two related biotin tracers in our experiments:biocytin (Sigma B4261 [Sigma, St. Louis, MO]; 5% in 2 MKCl) and neurobiotin (SP 1120, Vector Laboratories, Burl-ingame, CA; 10% in 2 M KCl); each is readily taken up byneurons after low current iontophoretic injections (1–2 µApositive DC for 10–20 minutes) and are transported antero-gradely and retrogradely to produce Golgi-like staining ofthe entire injected pathway (survival time, 1–12 hours).Dye spread from the injection site within the extracellularmatrix of each injected nucleus but did not spread beyondthe nuclear boundary. Our interpretation of the relativelylocalized projection patterns resulting from our injectionsis that the uptake of tracer was restricted to auditoryneurons damaged near the electrode tip where currentdensity was greatest during iontophoresis.

Intracellular recording and dye injection

Glass micropipettes (WPI AS100F aluminosilicate glass)were pulled on the Sutter horizontal pipette puller. Afterbackfilling with tracer solution, we beveled each electrodeto an impedance of approximately 60 MV by using a SutterBV-10 beveler. After physiological characterization, intra-cellular injections were made by applying a 2 nA (peak topeak) AC current (2 Hz) with a 0.9 nA DC offset. Injectionsof 30 minutes were followed by a 45- to 60-minute survivaltime.

Transynaptic spread

Under some circumstances, biocytin and neurobiotinmay traverse synapses; cells labeled by means of transyn-aptic spread often show weaker staining than those la-

AUDITORY PATHWAYS OF ELECTRIC FISH 229

beled directly (Bass et al., 1994). In some studies, transyn-aptic spread of tracer has been associated with synapsesthat contain gap junctions (e.g., Vaney, 1993). For thisreason, we have interpreted instances of dual cell bodylabeling after single-cell injections (i.e., dye-coupling) orweaker staining of distinct neuronal populations afterextracellular injections as potential instances of transynap-tic labeling mediated by electrotonic coupling betweeninjection sites and transsynaptically labeled neurons.

Tissue processing and microscopy

After injections, the fish were anaesthetized by anintramuscular injection of MS 222 (tricaine methanesulfo-nate, 1:2000), and perfused intracardially with salinefollowed by fixative (1.25% paraformaldehyde, 1.25% glu-taraldehyde in phosphate buffer, pH 7.1). All of the aboveanimal protocols comply with The Principles of AnimalCare published by the U.S. National Institutes of Health,and all were approved by the Institutional Animal Careand Use Committee of the University of Pennsylvania,Philadelphia.

Brains were post-fixed, embedded, and fixed within thegelatin block overnight, followed by soaking in 10%, 20%,and 30% sucrose solutions over the course of 24 hours.Brains were then frozen and sectioned at 60 µm on asliding microtome. The tissue was then processed accord-ing to widely used protocols for visualizing biotinyla-ted tracer molecules (Horikawa and Armstrong, 1988;Kawasaki and Guo, 1996; Wong, 1997).

In addition to the experimental material, we also stud-ied normal transverse sections of noninjected brains cut at15 µm and prepared with Bodian and cresyl violet stainingtechniques to resolve fiber tracts and Nissl bodies. Thesebrains were infiltrated with paraplast and cut on a rotarymicrotome. They were used for preparing an atlas (Fig. 1),which serves as a reference for all other figures. Allmaterials were studied in both brightfield and darkfieldwith an Olympus BH 50 microscope equipped with a SonyDKC digital camera and Pentium computer. Microscopicimages were digitized to create black and white digitalphotographs of approximately 0.24 pixels/µm of tissue at23 magnification, 1.2 pixels/µm at 103, 2.4 pixels/µm at203, 4.8 pixels/µm at 403, and 12.1 pixels/µm at 1003.The micrographs presented here were created from thesedigital images by using Adobe Photoshop 4.0. Brightness,contrast, and color balance were adjusted by using stan-dard Photoshop techniques. Some of the figures werecomposed as montages of images made at higher magnifi-cation to improve resolution; in these instances, bright-ness and contrast were adjusted separately within each ofthe montage components. Figures were printed with aKodak 8650PS dye sublimation printer.

Measurements and model construction

In addition to our microscopic analysis, we also usedthree-dimensional reconstructions of medullary and mid-brain nuclei (Fig. 13) from one exceptional preparation todescribe better the complex geometry of nuclei in thisregion of the auditory pathway. By using an Olympusdrawing tube, a Wacom draw pad attached to a Macintoshcomputer, and NIH Image software, we were able to collectthree-dimensional coordinates for each cell profile ob-served at 23 magnification. Coordinates were then used toconstruct a series of geometric approximations of nuclearcross-sectional areas. These areas allowed us to estimate

the approximate volume of each cell group. The coordi-nates obtained for each profile were also entered into aphoto rendering program, POV Ray, for construction of athree-dimensional model. Each profile was represented asa sphere (150-µm diameter), and all spheres were thenrendered as a ‘‘blob’’ with threshold set at 120 and strengthat 100 in POV Ray. This method effectively reduced thediameter of spheres that had no close neighbor andgenerated smooth surfaces between adjacent spheres.Profiles were assigned to separate groupings based on cellmorphology and their relative positions within the brainand were rendered in separate hues.

RESULTS

Overview

Extracellular injections of neurobiotin and biocytin atphysiologically characterized auditory loci revealed a con-stellation of cell groups and terminal fields within themedulla, midbrain, and thalamus. With few exceptions,the labeled cells corresponded well with cell groups thathad previously been identified in other mormyrid speciesthrough application of horseradish peroxidase to the saccu-lar branch of nVIII (Bell, 1981a) or to the mediodorsal toralnucleus of the midbrain (Bell, 1981b; Haugede-Carre,1983). Considerable effort has recently been devoted to thecomparative anatomical analysis of octavolateral systemsof fishes (McCormick, 1992, 1998, and personal communi-cation), and this important synthetic work has led toreinterpretation of some of the earlier anatomical findingsand to the recognition of general organizational principlesamong mormyrids and other fishes. In the presentation ofour experimental results here, we have tried to apply thenomenclature that stemmed from these recent compara-tive studies, and in particular the work of McCormick(1992, 1998). In describing the locations of cell bodieslabeled after extracellular injection into distal nuclei, wehave often described groups of neurons that constitutedonly a portion of a nucleus that has been defined previ-ously on the basis of comparative analysis of normalhistological material (e.g., anterior octaval nucleus andmedial octaval nucleus: horizontal hatching, Fig. 1C,E–G).

Seven distinct cell groups within the rostral medulla,including three secondary octaval populations (SO;Fig. 1B–G) that did not appear to receive direct input fromnVIII, were identified. The dorsomedial zone of the descend-ing octaval nucleus (dzD) (Fig. 1B–E) was the mostsubstantial of the first-order processing regions, consistingof a dense midline cluster of auditory neurons that hasbeen shown in other mormyrid species to receive inputfrom saccular afferents (Bell, 1981a). The axons of dzDneurons formed a major lemniscal projection into nucleuscentralis (MD; Fig. 1H–J) in the midbrain, as well as localprojections within the medulla. The major targets ofmidbrain auditory neurons were the isthmic granularnucleus (IG), optic tectum (OT), thalamic nuclei, anddescending projections to the perilemniscal isthmic reticu-lar nucleus (IRN; Fig. 1H–L).

Medulla

Sacculus and primary afferents. The primary affer-ents innervating the sensory epithelium of the sacculus(Fig. 2A) were revealed after recordings of auditory re-sponses and extracellular injection of neurobiotin intodzD. These fibers coursed medially from the ear to enter


Fig. 1. Atlas: (A) Horizontal section with dorsal view of brain; linesin A indicate the level of each in a series of caudal (B) to rostral (L)transverse sections (15 µm, cresyl violet) through the brainstem ofPollimyrus adspersus. B–F: Medulla, including the eighth nerve(VIII). G–K: Mesencephalon. J–L: Diencephalon. Regions identified asauditory by dye injection in this study are cross hatched. In Figures

2–11, line-drawing insets represent approximate levels of the trans-verse sections illustrated here, with a letter corresponding to theirlevels in this atlas. For abbreviations, see list. Scale bar in A refersonly to A, whereas the scale bar in B refers to high magnification(top)and low magnification (bottom) images in B–L.


Figure 1 (Continued)

the brain near the rostral margin of the electrosensorylateral line lobe (ELL; Figs. 1B–D, 2B,D; see also Bell,1981a). The diameter of the saccular nerve coursingthrough the brain was approximately 275 µm. The axonsof saccular afferents were 7–10 µm in diameter at thelateral margin of the brain and decreased to ,5 µm indiameter as they projected medially. Afferents traversedapproximately 800 µm from the edge of the brain rostrallyto their first target, the medial tip of the intermediate zoneof the descending octaval nucleus (izD), and an additional300 µm to reach dzD, where fibers appeared to makebilateral projections within this midline cell group (Fig. 2B).The descending octaval nucleus of teleosts is large andincludes several zones (McCormick, 1998). In mormyrids,the izD is separated from the dzD by a large fiber tract(dll). These two areas are considered to be part of the samenucleus based on cytoarchitecture, comparisons with otherteleosts, and the bidirectional bridge of axons linking themacross the dll (Fig. 4A,B). Somata and terminals werelabeled in izD, and also in secondary octaval cell groups(see below) after extracellular injections of neurobiotininto dzD.

Electrophysiological recordings from primary afferentsrevealed sustained responses that were highly synchro-nized to tones in the 100 to 1,000 Hz frequency band(Fig. 2C). The afferent illustrated here gave a synchro-nized response at 307 Hz during intra-axonal recordings.The afferent was filled with neurobiotin and reconstructedfrom camera lucida drawings of 60-µm transverse sectionsthrough a rostrocaudal extent of 540 µm; 120 µm of thisextent was occupied by terminal branching in the izD anddzD (Fig. 2B).

Dorsomedial zone of the dzD. The dzD is a relativelylarge midline division of the descending octaval nucleus(Fig. 1B–E) and was strongly labeled after extracellularinjections into the auditory midbrain (cf. also Bell, 1981b).The brain volume occupied by labeled dzD somata repre-sented approximately 45% of the total brain volume occu-pied by all other medullary cell bodies labeled after MDinjections. The number of cell profiles labeled in dzDindicated that this area contained the greatest number ofmidbrain projecting neurons in the auditory medulla. Bell(1981a,b) referred to this cell group as the anterior nucleusin the mormyrids he studied (Gnathonemus petersii andBrienomyrus sp.), but McCormick (1992) has argued thatthis cell group should be considered a dorsomedial zone ofthe descending octaval nucleus (dzD). According to thisview, the descending octaval nucleus is a multimodaloctavolateral cell group and has now been identified inseveral teleost groups (McCormick and Braford, 1993;McCormick and Hernandez, 1996; McCormick, 1998). ThedzD tends to be relatively enlarged in species such asmormyrids, cyprinids, and clupeids, which have periph-eral specializations (i.e., otophysic connections) for soundpressure detection (McCormick, 1992). The dzD is a fusedmidline structure in Pollimyrus and other mormyrids(Bell, 1981b), whereas in other teleost fishes (e.g., goldfish,McCormick and Hernandez, 1996), the cell groups thoughtto be homologous to the mormyrid dzD are distinctlypaired and positioned more laterally.

The portion of dzD labeled after MD injections inPollimyrus had a rostrocaudal extent of approximately 720µm, beginning approximately 240 µm anterior to wherethe saccular nerve entered the medulla. The labeled dzDcells were delimited both ventrally and laterally by decus-

sating lemniscal tracts (dll) formed by electrosensoryaxons projecting to the midbrain from ELL (Bell andSzabo, 1986). Both the dzD and the adjacent medialoctaval nucleus (M) have a dense lamina of crest cellsalong their dorsal edge. The filled dzD somata wereprimarily multipolar in our transverse sections with one tothree processes per cell observed overall (Figs. 3A,B, 5A).The labeled axons of dzD neurons could be traced into thelateral lemniscus in some cases. In addition, many pro-cesses originating in dzD neurons showed arborizationswithin dzD itself (Fig. 3A,B).

After injections into dzD or izD, large numbers of labeledaxons were observed originating from the caudal portionsof these zones and traversing the ascending lemniscaltract to form a bilateral tract (Fig. 4A,B). Fibers withinthis short tract appeared to terminate within caudal dzDand within izD (Fig. 4B, arrows), suggesting that these twocell groups form reciprocal connections. Primary afferentfibers (nVIII) were also seen entering izD, and some mayterminate here then project through the short tract toterminate in caudal dzD (Fig. 2B,D).

According to Bell (1981a), izD receives strong primaryafferent input from the mechanosensory lateral line or-gans. The anatomical connection between izD and dzD,thus, suggests integration of mechanosensory lateral lineand auditory information from the sacculus in these areas.After injections into the auditory midbrain, only a fewfibers were labeled in the tracts connecting dzD and izD.This finding suggests that important local medullarycircuits may be confined to the caudal regions of dzD andizD.

Multi-unit recordings were also used to measure thefrequency response characteristics of driven auditory activ-ity in the dzD region (Fig. 4C). The population activityrevealed that response amplitude was greatest to tones inthe 200 to 900 Hz band, with two clear peaks in themulti-unit response function corresponding to spectralpeaks in the courtship sounds made by male Pollimyrusadspersus (Crawford, 1997b; Crawford et al., 1997a).

Crest cell layer of dzD. In addition to multipolarneurons in dzD, we also observed a limited number oflabeled crest cells in the dorsal layer of dzD after MDinjections (Fig. 3A,B, large arrows). The crest cells hadboth dorsal and ventral dendrites. The dorsal dendritespenetrated the molecular layer overlying dzD, the cristacerebellaris, and the ventral dendrites entered the moreventral parts of dzD (Fig. 3A,B). In some cases, we sawcomplete retrograde labeling of pyramidal crest cells(Fig. 3A, arrow); more often, however, crest cells appearedfaintly labeled, suggesting the possibility of transynapticlabeling from deeper, polygonal dzD neurons by means ofgap junctions. Both the apical and basal dendrites of crestcells appeared smooth and did not show spines common insimilar neurons in other systems (Montgomery et al.,1995). The somata of labeled crest cells were similar in sizeto the somata labeled in the principle cell region of dzD(cf. Bell, 1981b).

We have isolated a limited number of crest cells withintracellular micropipettes. The auditory physiology ofthese neurons (Fig. 3C) was quite similar to that illus-trated for the primary afferents (Fig. 2C), which projectedinto dzD. Both were sustained and synchronized to thestimulus. However, there appears to be a quantitativeincrease in the strength and bandwidth of entrainment to


periodic stimuli in the dzD crest cell population (Kozloskiand Crawford, 1997; unpublished observations). The crestcell response depicted in Figure 3C gave one spike perstimulus cycle (i.e., 606 spikes/second) during a 606 Hztone; other neurons in this area that were isolated but notfilled also produced a single spike per cycle at rates as highas 956 Hz. These firing rates suggest a very short timeconstant for crest cells in dzD which is similar to neuronsin other systems with cerebellar-like organization(cf. Montgomery et al., 1995).

Medial octaval nucleus. A distinct group of cell bod-ies, dorsolateral to the edge of dzD, was also labeled aftermidbrain MD injections but were not labeled after injec-tions into dzD and other sites in the medulla. This group of

cells was part of the medial octaval nucleus (M; Figs. 1C,3A-2). Labeled cells in M extended 120-µm rostrocaudallywithin the same sections as labeled portions of dzD, andthis group of neurons had maximal cross-sectional dimen-sions of approximately 155 µm by 120 µm in the transverseplane. In contrast to dzD neurons, the labeled somata in Mwere smaller, more ovoid in shape, and predominantlybipolar.

In one intracellular fill of a dzD crest cell, we were ableto trace a labeled process from this neuron to a smaller,more ovoid, bipolar soma, located adjacent to the dzD crestcell layer and within the medial tip of the medial octavalnucleus (Fig. 3B, inset and overlay). This dye-couplingbetween neurons suggests that there are lateral connec-

Fig. 2. A: Photomicrograph of the sacculus after injection ofneurobiotin into the dorsomedial zone of the descending octavalnucleus (dzD; shown in D). In this and each similar figure that follows,an icon of a horizontal brain section is provided to show the grosslocation of the injection site (caudal brain shown in black, medullaryinjection; rostral brain shown in black, midbrain injection), as well asthe approximate plane of sectioning (black/white transition). Rostral,dorsal, ventral, medial, and lateral axes (R, D, V, M, and L) are shownin the lower left corner of this and each following figure. Note saccularnerve root with labeled fibers branching as they enter the distalsensory epithelium (unlabeled arrow). B: Camera lucida drawing of asingle nerve fiber filled with neurobiotin after physiological recording(C). B corresponds to a similar section shown for another fish in D and

shows the course of the single filled afferent through the nerve intofirst-order nuclei. Note extensive branching and ramification withinizD and dzD. Fiber diameter decreases from 7 µm at entry to 2–4 µmwithin izD to 1–2 µm within dzD. Vertical dashed line marks midlinein this and all other figures. C: Physiological responses of single nervefiber (B) to tone bursts (30 msec rise/fall, 120 dB re 1 µPa) presented atbest frequency and 10 dB above threshold. Note sharp onset andsustained activity in raster diagram (panel 1), and precise synchroni-zation demonstrated by interspike interval (arrow indicates period oftone stimulus) and period histograms (panels 2, 3). D: Photomicro-graph of primary auditory inputs from nVIII to dzD, and izD afterinjection of neurobiotin into dzD. For abbreviations, see list.


Fig. 3. A: Photomicrograph of midline nucleus dzD, and the morelateral medial octaval nucleus (M). 1: Midline dzD is comprised ofmultipolar, polygonal cells with major and minor dimensions of 14µm 6 3.6 µm SD and 9 µm 6 2 µm SD. 2: The medial tip of M is labeledcontralateral to the midbrain injection, and is comprised of smallercell bodies (12 µm 6 1.9 µm SD by 8 µm 6 1.3 µm SD) which are moreovoid in shape. Photomicrographs in A–1 and A–2 were made at ahigher magnification and at a different focal plane from that of themain panel. D, dorsal; M, medial. B: Photomicrograph of a single dzDcrest cell filled intracellularly with neurobiotin after physiologicalrecording. The cell body is pyramidal with smooth apical dendritesprojecting into the crista cerebellaris. In addition, a second dye-coupled cell body (inset upper left, same scale) was labeled in the left

nucleus M (area 2 outlined in A). Note superimposed camera lucidadrawing, showing additional basal dendrites and both labeled somata(small V-shaped arrows). C: Physiological responses of dye-filledneuron shown in B to tones (606 Hz, 106 dB). Note gradual onset andoffset with sustained peristimulus activity in raster (panel 1) andprecise temporal synchronization demonstrated by interspike interval(ISI) histograms (panel 2, arrow indicates period of tone stimulus) andperiod histogram (panel 3). Each inset drawing marks the positions oflabeled terminals or cell bodies in the corresponding photomicro-graphs with a star; rough sketches of labeled fibers are also includedfor orientation purposes. For abbreviations, see list.


tions between the auditory crest cells of the dzD and M,perhaps mediated by gap junctions.

Additional primary octaval populations. In addi-tion to a significant portion of dzD, smaller areas of twoadditional cell groups were labeled after injections intoMD. The first of these two, the intermediate zone of thedescending octaval nucleus (izD), constitutes a seconddivision of the descending octaval nucleus based on obser-vations in thin sections (Fig. 1B,C). Cells in the izD sentterminating processes into dzD and received input fromdzD by means of a distinct tract (Fig. 4A,B). Cells in izDlabeled after injections into MD extended approximately450-µm rostrally from the point at which the saccularnerve entered the brain and formed a tight, sphericalcluster, approximately 150-µm in diameter, located imme-diately lateral to the decussating electrosensory lemniscal

fiber tract (Fig. 4D). The second cluster of labeled cellswere located approximately 500-µm anterior to the izDcluster (Fig. 6A-1) and represent the medial portion of theanterior octaval nucleus (Ant). Cells in Ant were seen intransverse sections at the same level as the dorsal second-ary octaval population (dSO; Fig. 6A-2). Many of thesomata in Ant were slightly larger than in izD, and nodirect connection between Ant and the nearby dzD popula-tion was visible. Neurons in Ant sent projection fibers intothe dll; these fibers branched within the subventricularregion of the dll and sent axon collaterals to each of theintermediate secondary octaval populations (iSO; Fig. 6A).

Secondary octaval nuclei. In addition to the cellgroups that have been identified as primary targets ofauditory input from the sacculus, injections into both themidbrain and medulla revealed extensive dendritic fields

Fig. 4. A: Photomicrograph of midline nuclei dzD and izD afterinjection of neurobiotin into dzD. Unlabeled arrow indicates a densebilateral tract connecting dzD and izD. B: The dzD-izD tract shown athigher magnification, including labeled terminals (arrows) in bothnuclei. C: Multi-unit activity recorded from within dzD during presen-tation of 20 tone bursts at 105 dB re 1 µPa, varying in frequency from105 Hz to 2.2 kHz. For each tone burst frequency, a responsenormalized to the maximum was plotted. The baseline response(spontaneous activity) is plotted as a solid horizontal line at 22%maximum. Dotted lines show the position of peaks in the spectrum of

natural Pollimyrus communication signals (Crawford et al., 1997a).The peaks in multi-unit activity fit closely to these frequencies,indicating increased overall firing activity at these frequencies, in-creased synchronization of multi-unit activity, or both. D: Photomicro-graph of izD contralateral to an injection of biocytin into midbrainnucleus MD. Note that the tract connecting izD to dzD is not labeledafter midbrain injections as in A and B. Neurons in izD average 10.3µm 6 2.2 µm SD by 6.8 µm 6 1.4 µm SD along their major and minoraxes. For abbreviations, see list.


and somata that we have interpreted as second-orderprocessing areas within the medulla. These areas receivedinput from primary auditory centers in the medulla as wellas descending input from the midbrain.

The secondary octaval populations in mormyrids andother teleosts include several cytoarchitecturally distinctdivisions. In goldfish (Carassius) and catfish (Ictalurus),three divisions are recognized: a dorsal Purkinje-like cellregion (dSO), an intermediate spherical cell region (iSO),and a ventral fusiform cell region (vSO; McCormick andHernandez, 1996). We labeled all three of these areas inPollimyrus after injections into the midbrain auditorynucleus MD. The dorsal and intermediate SO divisionswere strongly labeled after midbrain injections (Figs. 5,6A,A-2,B), whereas only a few fusiform cells in vSO werelabeled ipsilateral to injections into MD along the perim-eter of the lateral lemniscus (Fig. 6A).

Injections into the midbrain yielded extensive bilaterallabeling of the iSO beginning within the same transversesections as dzD, but approximately 320-µm ventral to thecenter of dzD, and approximately 175-µm lateral to mid-

line (Fig. 5A). Extensive terminal fields in iSO were visibleboth ipsilateral and contralateral to the midbrain injec-tion, and originated from labeled dzD, dSO, and Antpopulations and possibly from descending MD projections.The projection from dzD into iSO in Pollimyrus wasstriking in that a large number of highly varicose fiberscoursed directly from the ventrolateral boundaries of dzDinto both ipsilateral and contralateral iSO, both afterinjections into MD (Fig. 5A–D) and into dzD (Fig. 5E). Thelabeled fibers showed distinct outswellings along theirentire length and formed a transverse sheet connectingventrolateral dzD neurons to the contralateral iSO. Thusprojections from dzD to iSO are extensive, bilateral, andform a lattice of fibers that spans the midline and is visiblein the transverse plane.

Injections into MD labeled iSO somata more stronglyipsilaterally than contralaterally both in terms of numberof cells labeled and the density of label within individualneurons (Fig. 5C,D). The iSO somata were spheroid andmonopolar in transverse sections, and no differences in cellmorphology were observed between the two sides. Aside

Fig. 5. A: Photomicrograph of midline nucleus dzD labeled after aninjection into the right midbrain nucleus MD. Projections into iSO aredense, and marked by unlabeled arrows. Terminals in ipsilateral vSOare also visible. B: Photomicrograph of projections 60 µm anteriorfrom A reveal projections into iSO as well as dense terminal fieldswithin an additional dorsal portion of the secondary octaval area(dSO). Decussating fibers are present at this level, forming diagonal

links from dzD to contralateral iso (iSOc) and ipsilateral iso (iSOi).Contralateral to the injection site (C) terminals and faintly stainedsomata are visible in iSO, whereas many densely stained somata andterminals are usually seen in the ipsilateral iSO (D). E: Afterinjections into dzD, decussating fibers appear with en passant outswell-ings also visible (arrows). For abbreviations, see list.


from differences in labeling, the overall number of labeledprofiles appeared to be approximately fourfold greater onthe ipsilateral side, based on iSO cell profile ratios.

The dSO was also labeled by injections into MD. It waslocated rostral to the most strongly labeled part of iSO(Fig. 5A) and within the same rostrocaudal column as dzD(Fig. 1B–G). The dSO thus appeared to replace dzDrostrally. The morphology and pattern of inputs associatedwith cells in dSO was clearly different from those in dzD.The dSO somata were elongate, or fusiform, with largeapical dendrites extending dorsally (Fig. 6A,A-2,B) intothe overlying crista cerebellaris. This anatomical organiza-tion of the rostral medulla appears strikingly similar tothat observed in goldfish and other teleosts (McCormick,1998).

Terminals were seen distributed among the fusiformsomata of the dSO after MD injections (Figs. 5B, 6B).These terminals originated from forward projecting pro-cesses of dzD neurons visible in more caudal sections.Injections into dzD revealed terminal fields in dSO, but nolabeled somata (Fig. 6C). Labeled fibers originating in dSOalso decussated and contributed more caudally to thedense iSO terminal field (Fig. 5B). Thus, these resultsindicate that the dSO forms part of a second, indirectpathway between dzD and iSO in the medulla, and be-tween dzD and MD in the midbrain.

Lateral lemniscus and perilemniscal nucleus. Theascending lemniscal pathway carries sensory informationfrom the auditory medulla to the midbrain in Pollimyrus.In mormyrids, the lateral lemniscus contains fibers project-

Fig. 6. A: Photomicrograph of the anterior octaval nucleus (Ant),and dSO labeled after an MD injection, 120 µm anterior to 5B. At thislevel, dSO neurons are completely labeled, revealing elongated, fusi-form cell bodies with apical dendrites projecting into the cristacerebellaris (panel 2). Ant is comprised of spherical cell bodies(panel 1), with average major and minor dimensions of 12.5 µm 6 2.6

µm SD and 8.4 µm 6 2.0 µm SD. B: Photomicrograph taken at a levelbetween that of 5B and 6A, revealing fusiform dSO neurons togetherwith terminals originating in dzD. C: After an injection into dzD,terminals alone are visible, indicating separate projections from dSOinto MD and from dzD into dSO. For abbreviations, see list.


ing from the inner ear, lateral line, and electrosensorysystems. Projecting fibers of each modality form separatelemniscal sheets, with ascending auditory axons confinedto the medial perimeter of the lateral lemniscus (Bell,1981b; Haugede-Carre, 1983). In transverse sections, theaxons labeled by injections into MD formed a darklystained crescent within the ipsilateral lemniscus (Fig. 7A,arrows). At the level of the auditory midbrain, axonsascending from the medulla through the lateral lemniscusentered MD at its dorsomedial edge, to terminate amongcells in the dorsomedial portion of this nucleus (ie. nucleuscentralis; Fig. 8D). After injections into dzD, terminals inMD appeared globular, with a single labeled fiber appear-ing to give rise to a series of large boutons (Fig. 8D, arrow).

A small cluster of spherical somata was also labeled nearthe lateral lemniscus contralateral to midbrain injections(Fig. 7B). This nucleus, referred to as the IRN in previousstudies (Bell, 1981b; Haugede-Carre, 1983) was approxi-mately 120 µm in its rostrocaudal extent, 130-µm across,and was located approximately 1-mm rostral to the centerof medullary nucleus dzD (Fig. 1H). The ipsilateral IRNhad no labeled somata after MD injections, althoughterminals in this nucleus were labeled (Fig. 7A). Many ofthese terminals appeared to originate from descendingMD projections that traversed the toro-preeminential tractin distinct fascicles (Fig. 9A, lower arrows). Axons from thecontralateral IRN were seen decussating approximately

300-µm anterior to IRN (Fig. 7C); some joined the ascend-ing lateral lemniscus and others projected caudally intothe ipsilateral IRN terminal field. The circuits connectingMD and IRN, thus, form a complex bilateral loop ofascending and feedback projections.

Midbrain

Mediodorsal toral nucleus. The primary target ofauditory inputs in the mormyrid torus semicircularis isthe mediodorsal toral nucleus (MD; Fig. 8A). MD has beenthe subject of several anatomical (Haugede-Carre, 1983;Bell, 1981b) and neurophysiological studies and is knownto contain neurons with both simple and complex receptivefield properties (Crawford, 1993, 1997a). MD is made up ofdorsomedial and ventrolateral subdivisions that are clearlydifferent in cresyl violet stained thin sections. The dorsome-dial and ventrolateral cytoarchitectural divisions becomeclear at a point approximately one third of the way alongthe caudal to rostral extent of MD (Fig. 1H). McCormick(1998) has suggested that these two divisions of MDcorrespond to the auditory nucleus centralis (dorsomedialMD) and the mechanosensory nucleus ventrolateralis (ven-trolateral MD) in other teleosts. We observed in thinsections that Nissl bodies in the dorsomedial and caudaldivision of MD were larger than those in the ventrolateraland rostral division. At lower magnification, the density of

Fig. 7. A: Photomicrograph of lateral lemniscus ipsilateral to theinjection site in midbrain nucleus MD. Labeled fibers are concentratedin the medial portion of the tract (unlabeled arrows). A terminal fieldin IRN originating in MD, contralateral IRN, and medullary regions isalso indicated. B: 60-µm anterior to A, contralateral to the injectionsite, the cell bodies in the IRN appear along the medial edge of ll.C: 360-µm anterior to B, decussating projection fibers from IRNappear in the ventral periventricular region (arrows). These fibers jointhe lateral lemniscus and project to MD or terminate in the contralat-eral IRN. For abbreviations, see list.


Nissl substance appeared more diffuse in the dorsomedialregion of MD than in the densely packed ventrolateraldivision (Fig. 1H–J; cf. Haugede-Carre, 1983; Crawford,1993). Focal injections were made into the dorsomedialand caudal division of MD, nucleus centralis (Fig. 8A).

Injections were made after recording auditory responsesfrom one or more single neurons at the injection site. Thephysiology and anatomical positions of recorded neuronsin this study corresponded well to results from previouscharacterizations of MD in Pollimyrus (Fig. 8B; Crawford,

Fig. 8. A: Auditory midbrain after injection of neurobiotin into thedorsomedial division of MD, nucleus centralis. Injection site (IS) wascentered in the dorsomedial region of caudal MD, within a transverseplane located approximately one third of the distance along the caudalto rostral extent of MD. Tracer labeled a dense plexus of fibers andsomata within this dorsomedial part of MD. A transition was visiblefrom dense to weaker staining, corresponding to the cytoarchitecturalborder separating the two regions of MD visible at this level in normalhistology (dashed line and unlabeled arrow; see Fig. 1H–J).B: Physiology of a single unit at the injection site. The response to

tones (97 dB re 1 µPa) is sustained (raster plot, panel 1), with the weakphase-locking (ISI and period histograms, panels 2 and 3) that ischaracteristic of many MD neurons. Raster plot (panel 4) shows weaksynchrony to clicks during presentations of 400-msec click trains (15msec ICI, onset at 50 ms). This neuron had a simple temporalreceptive field (non-selective; but see Crawford, 1997a). C: Terminalsare visible in contralateral MD (MDc, arrows) which originate from theinjections site in ipsilateral MD. D: Terminals which show a differentmorphology are also visible in dorsomedial MD (arrow) after aseparate injection into dzD. For abbreviations, see list.


1993, 1997a). Most MD neurons showed significantlyweaker temporal synchronization to tones (Fig. 8B, panels1–3) than is typical of neurons recorded in the medulla(Fig. 3C). Furthermore, click train stimuli (Fig. 8B, panel 4)also gave rise to poorly synchronized responses. Biocytinlabeled a dense plexus of fine processes, and afferentsentering dorsomedially from the lateral lemniscus (Fig. 8A).Labeled somata were small and primarily spherical, withprofiles ranging from 5–6 µm in diameter (Fig. 8A). Largermultipolar profiles were less common and ranged in diam-eter from 8 to 15 µm. MD efferents were observed exitingits ventral border. There were a few fibers and somatalabeled in the ventrolateral portion of MD, and the borderindicated by the density of stain (Fig. 8A) correspondedclosely with the border that is visible at this level innormal Nissl-stained material. Labeling in the ventrolat-eral zone probably resulted from connections between thetwo parts of MD or from fibers of passage.

Collateral projections to contralateral MD. Injec-tions into MD not only labeled medullary and lemniscal

sites, but also reveled a terminal field in the contralateralMD. These terminals (Fig. 8C) were finer and morepunctate than those filled after injections into dzD (Fig. 8D),suggesting that their role in auditory processing may befunctionally distinct from ascending medullary inputsfrom dzD. We observed a few labeled axons decussatingdorsal to the ventricle in the dorsal mesencephalic commis-sure, and these could form an inter-MD, homotopic connec-tion (cf. Bell, 1981b). However, because MD injections onlylabeled terminals in the contralateral MD, but never cellbodies, it seems probable that the neurons seen terminat-ing in the contralateral MD have their somata in anotherlocation, perhaps in the medulla. This pattern of labelingcould be explained if medullary or perilemniscal neuronshad branched projections, with terminal fields in bothMDs. We have observed branched axons within the dll, andMcCormick and Hernandez (1996) have suggested thatindividual medullary neurons similarly send bilateralprojections into midbrain auditory areas of the goldfish.

Fig. 9. A: Darkfield photomicrograph of a 15-µm section processedwith Bodian fiber staining, and counterstained with cresyl violet.Large bundles of MD output fibers are visible as they traverse MV(upper arrow), and the toro-preeminential tract in distinct fascicles(lower arrows). B: Terminals (arrows) originating from some of thesefascicles appear 480 µm caudal to the previous plane of sectioning (A)in the IG after injections of biocytin into MD. C: Smaller, and morenumerous synapses appear 240 µm anterior to A in the main mesence-phalic granular mass. D: Individual end bulb synapses in IG fromanother MD injection shown at higher magnification. For abbrevia-tions, see list.


Recipients of MD output

Isthmic granular nucleus. Auditory neurons withinthe midbrain nucleus MD sent axons ventrally whichtraversed the underlying medioventral toral nucleus (MV)and ventral posterior toral nucleus (VP) as sheets of fibers;these fibers then entered the medial toro-preeminentialtract (tp) and clustered into distinct fascicles (Fig. 9A,lower arrows). In some transverse sections these fasciclesappeared as a regularly spaced series of parallel verticalbands, extending from near the lateral lemniscus, laterallytoward the tectum. The tp has also been called the tectumMarklager (ml) in previous studies (Stendell, 1914; Bell,1981b). Approximately 300-µm ventral to MD, many ofthese output fibers formed rather extraordinary termina-tions within the IG (Fig. 9B–D).

The IG is a division of the mesencephalic granular cellmass (MGM) just ventral to the tp and extends from thecaudolateral edge of the optic tectum (OT) rostromediallyto the lateral lemniscus (Fig. 1H). Fibers coursing ven-trally from MD appeared to give rise to a sheet of boutonsin IG, each of which enveloped a single, unlabeled, spheri-cal cell body (cf. Bell, 1981b). Terminals were larger andmore sparse in lateral portions of IG (Fig. 9B,D), andbecame more dense and smaller as the nucleus extendedmedially into the main MGM throughout more rostraltransverse sections (Fig. 9C). Each labeled terminal ap-peared to originate from a single MD efferent fiber orefferent collateral because no branching was observed inany fibers leading to these terminals. Terminals wereapproximately 30- to 60-µm across in the caudolateral IG,and 10–40 µm in rostromedial IG. The largest of theseterminals (Fig. 9B, D) resembled the end bulbs of Held,described in birds (Carr and Boudreau, 1991) and in theanteroventral cochlear nuclear of mammals (e.g., Ryugoand Fekete, 1982).

Thalamic projections. Four distinct terminations offibers projecting from MD into the thalamus were identi-fied. At the level of the lateral toral nucleus (L, electrosen-sory), a projection originating in the dorsomedial region ofrostral MD gave rise to a terminal field in the periventricu-lar region of the thalamus (Fig. 10A). We have termed thisregion the central posterior nucleus (CP). The CP terminalfield extended 50 µm laterally from the perimeter of theventricle and 400 µm along the dorsoventral axis. Approxi-mately 100-µm rostral to CP, a separate set of MD fibersprojected ventrally to form a small terminal field approxi-mately 50 µm in diameter within the posterioventralthalamic nucleus (pvTH; Bell, 1981b; Bell and Szabo,1986). Terminals were finer and more punctate in pvTHthan in CP. A third terminal field appeared, as a densecrescent-shaped band, 100-µm rostral to pvTH along thedorsolateral edge of the large anterior thalamic nucleus ofBell (1981b), also known as the preglomerular complex(PG; Bell and Szabo, 1986; Fig. 10B). Finally in somepreparations, we also observed a group of fibers thatbypassed the ipsilateral PG, coursed 240-µm rostrally anddecussated (Fig. 10C) to give rise to a projection near thecontralateral PG.

Optic tectum. MD projections into the OT in Polli-myrus were observed primarily at the inner lamina of themost rostral extent of OT and consisted of coiled, parallel-coursing fibers (Fig. 11A). These fibers were regularlyspaced and appeared within a single deep layer of thetectum, called the stratum album centrale by Echteler

(1984) in his studies of the carp (Cyprinus). These tectalfibers continued dorsomedially within the rostral OT andexited at its dorsal edge. The rostral OT here refers to thatportion of OT that appears as a closed circular structure intransverse sections (Fig. 1L). At this level, the parallel-coursing fibers decussated within an intertectal commis-sure and the torus longitudinalis (TL; Fig. 11B). Granulecell bodies in TL were also labeled ipsilateral to MDinjections. Decussating tectal fibers reorganized to form anarray of parallel-coursing fibers within the horizontalplane of TL; each fiber within this array crossed themidline perpendicular to the rostrocaudal axis. The hori-zontal sheet of decussating fibers was approximately 400µm in rostrocaudal extent. Axons showed varicositiesalong their entire length, possibly constituting en passantsynapses within TL. Upon crossing the midline, the fibersreoriented to course caudally and enter the contralateralOT. The pattern of projection created within the contralat-eral OT was identical to the parallel-coursing fibers ob-served in ipsilateral OT (Fig. 11C). Thus, labeled fibers ineach OT presumably interdigitated with fibers originatingin the contralateral MD.

Anatomical models. The auditory pathway from theear to the mesencephalon that we have described (Fig. 12)includes compact sets of cell groups that represent a totalvolume which is less than 1% of total brain volume (brainvolume, approximately 70 µl). To develop a better macro-scopic picture of the auditory structures in Pollimyrus, wehave used the experimental material from midbrain injec-tions presented here to make a three-dimensional model ofthe auditory brainstem (Fig. 13) and to estimate relativebrain volumes occupied by each nucleus as discussedabove for dzD. Because only cell bodies labeled after MDinjections are rendered in this model, projections from MDinto the thalamus, IG, and OT are not represented.

DISCUSSION

Some of the most fundamental behaviors mediated byaudition depend on the time structure of natural acousticsignals, and the representation of this structure by thetiming of action potentials in the auditory nervous system.Temporal cues are critical for the interpretation of commu-nication sounds in many species, including humans (Ger-hardt, 1978; Myrberg et al., 1978; Thorson et al., 1982; Hoyet al., 1982; Suga, 1988; Cazals and Palis, 1991; Shannonet al., 1995), and for localizing sound sources (e.g., Yost,1974; Moiseff, 1989; Blauert, 1996). In mammals, theneural analysis of temporal information is superimposedon an exquisite spectral analysis delivered to the brain bylabeled lines originating from the organ of Corti (vonBekesy, 1947; Liberman, 1982). However, among verte-brates, the fishes are distinguished by the absence of suchan organ, or any other elongated auditory epithelium thatcould perform high-resolution mechanical frequency analy-sis (Popper and Fay, 1984; Rogers et al., 1988).

Despite the relative simplicity of the Pollimyrus ear, theanatomical studies presented here have revealed remark-ably complex circuitry in the auditory brainstem of thisanimal (Fig. 12), circuitry that appears capable of signifi-cant computation even at the level of the medulla. Both thestructure of the ear and our neurophysiological recordingsfrom primary afferents and first-order medullary neuronsindicate that this peripheral auditory system providesprecise information about temporal features of acoustic


signals by the timing of synchronized action potentials(Figs. 2C, 3C). Although spectral analyses have not beenruled out (see Lu and Fay, 1993), it seems very likely thatthese auditory circuits rely on temporal coding for behav-iorally significant computations such as recognition ofsounds made by potential mates and determining thelocations of sound sources. Understanding these computa-tions in fish may shed light on similar processes in othervertebrates in which temporal analyses are carried out inparallel with spectral analyses.

There are four distinct nuclear divisions in the medullathat receive auditory input from the ear (i.e., first order:dzD, izD, Ant, M; Bell, 1981a), and three second-ordernuclear divisions (iSO, dSO, vSO). These auditory regionsare extensively interconnected. The organization of theauditory medulla in this fish is similar to that of othervertebrates, including mammals.Auditory afferents branchupon entering the brain providing primary input to dis-tinct first-order nuclei. First-order nuclei communicatewith second-order nuclei, and auditory information isrelayed bilaterally to the midbrain (in mammals, theinferior colliculus).

The primary midbrain target of medullary projections isnucleus centralis (MD), accessed by means of the largebundle of auditory axons in the lateral lemniscus project-ing into the torus semicircularis. The torus semicircularisis apparently homologous to the mammalian inferior

colliculus (Sarnat and Netsky, 1981). Auditory regions inthe torus correspond to the dorsomedial part of the medio-dorsal toral nucleus (MD) described previously by Bell(1981b) and Haugede-Carre (1983). MD is now recognizedas consisting of two nuclei, the dorsomedial auditorynucleus centralis, and the ventrolateral mechanosensorynucleus ventrolateralis (McCormick, 1998).

We found no evidence for first- or second-order saccularinput to the lemniscal nucleus IRN in this study. McCor-mick (1998) has concluded that the IRN may not be part ofthe auditory pathways in teleosts, but instead appears insome studies due to labeling of fibers of passage, orinadvertent labeling of tectal or midbrain mechanosensorylateral line neurons. Although these are possible explana-tions for our results, we labeled IRN consistently even insome of our most focal injections into dorsomedial MD. Weconclude that, although IRN may not receive direct saccu-lar nerve or first-order octavolateral input, this regionforms an important link between auditory regions, espe-cially between the dorsomedial divisions of MD.

The circuits formed by projections from the medulla todorsomedial MD, and between the IRN and the midbrain,suggest important bilateral processing in this species(Fig. 12). Outputs of the midbrain include extensive arraysof parallel-coursing fibers within the toral output pathway(tp; Fig. 9A) and within the optic tecta (Fig. 11). Thesearrays provide additional neuroanatomical substrates for

Fig. 10. Projections from midbrain nucleus MD into the thalamus.A: Thalamic projection to the periventricular CP from mediodorsalMD. Projection fibers enter dorsolaterally from MD (upper arrow) tocreate an elongated terminal field bordering the ventricle. Some fiberscontinued on to more ventral thalamic nuclei (lower arrow). B: MDprojection into the ventral thalamic PG; note crescent-shaped termi-nal field (arrows). C: In this same preparation, 240-µm anterior to C,fibers are clearly visible originating near the ipsilateral PG anddecussating within the toro-diencephalic commissure where theyproject to the region of contralateral PG. For abbreviations, see list.


bilateral processing, and perhaps spatial hearing. Addi-tional outputs into three auditory thalamic nuclei mayrepresent a separate pathway for acoustic feature analysisof communication sounds in this species. In the followingsections, we discuss our anatomical findings in terms ofpotential function.

Temporal feature extraction and the roleof medullary processing

Previous research has identified interval selective neu-rons in the auditory midbrain of Pollimyrus (nucleuscentralis, within MD), which extract temporal featuresfrom complex naturalistic sounds (Crawford, 1997a). Eachof these interval-selective neurons responds with its maxi-mal spike rate (spikes/second) to its own best temporalperiodicity, or temporal feature. Sounds with differenttemporal periodicities will activate different neurons withinthe subpopulation of interval selective midbrain neurons,creating a rate-place code for temporal features. In compari-son to auditory neurons in the medulla, the interval-selective neurons have low spike rates and weak synchro-nization of spikes to the temporal structure of the stimulus.Thus, it is not synchronization, but the relative firing rateof these selective midbrain neurons that represents tempo-ral features of auditory stimuli. Because these physiologi-cal properties of midbrain selective neurons are not ob-

served in medullary neurons, they must be derived, orcomputed, from medullary input to the midbrain.

We have proposed a simple model that may describe theessence of the neural computations that yield temporalselectivity in the midbrain (Crawford, 1997a). The modelrequires as input a faithful cycle by cycle representation ofthe acoustic stimulus in the form of synchronized actionpotentials. We have shown that medullary neurons in dzDprovide precisely this kind of input to circuitry in themidbrain (Fig. 3C). The model processes its synchronizedinput with an array of temporal filters. Each filter is builtfrom parallel excitatory inputs to each of two neurons. Oneof these neurons, an inhibitory interneuron, causes inhibi-tion and then rebound excitation in the second postsynap-tic output neuron. The temporal dynamics of inhibitionand postinhibitory rebound together with excitation deter-mines the timing of inputs to the circuit that will give riseto a selective pattern of output (additional details inCrawford, 1997a). We propose that these interval-selectivecomputations are performed by neurons in the midbrain,with possible contributions from the IRN.

One of the functions of the medullary circuitry presentedin this paper may also be to enhance the temporal represen-tation of sound provided by the primary afferents before itis delivered to the midbrain by dzD output neurons. Theproposed midbrain temporal filtering mechanism works

Fig. 11. A: Photomicrograph at the inner face of the anterior extentof the ipsilateral optic tectum (OTi) after biocytin injection into MD.Fibers course dorsally in an ordered array of parallel axons (arrows).These fibers originate in MD and leave the tectum at its dorsalboundary. B: Additional fibers cross the midline at a more rostral level(1420 µm) in an intertectal commissure (arrows) within the toruslongitudinalis. C: Contralateral to the MD injection site, similarparallel-coursing fibers (arrows) are found coursing into the anteriortectum (OTc), presumably originating in the intertectal commissure.For abbreviations, see list.


optimally if every cycle of the stimulus is represented byan action potential. Preliminary results using tones asstimuli (in progress) suggest that when representation isdefined as precise phase-locking (high coefficient of synchro-nization) with each stimulus cycle eliciting a single spike(low coefficient of variation in the interspike intervals),dzD neurons show better temporal representation thanprimary afferents. Some of the primary afferents skipstimulus cycles or may generate bursts of spikes for somecycles, whereas dzD neurons show extremely precise cycleby cycle representation over a wide range of stimulusperiodicities. This enhancement of temporal representa-tion may result from convergence of multiple primary

afferents on each dzD neuron, but the specific mechanismsare not yet known.

Medullary substrates for spatial hearing

Many terrestrial vertebrates use interaural time differ-ences and interaural intensity differences to localize sounds(e.g., Kuwada and Yin, 1983; Takahashi et al., 1984;Irvine, 1986; Konishi et al., 1988; Carr and Konishi, 1990).Mormyrid fishes are unique in that they have separate,paired ear bladders, although there are other groups offishes in which paired extensions of a single swim bladdermake direct contacts with portions of the inner ear(reviewed by Popper and Coombs, 1982; Schellart andPopper, 1992). The bilaterally paired ear bladders inmormyrids lead one to consider possible binaural analyseslike those used by terrestrial vertebrates. However, inter-aural differences in arrival times and ongoing transientdisparities cannot exceed 1 µsec in Pollimyrus due to theanimal’s small size. It would be impressive if these ani-mals were able to exploit sub-microsecond disparities fordirectional hearing. Significant interaural intensity differ-ences are also unlikely, because even at the highestfrequencies used in communication, the dimensions of theentire fish represent only a minute fraction of a wave-length, and the acoustic impedance of most of the animal’stissue is close to that of the water. Thus, the intensity ofsound incident at the two ear bubbles should be essentiallyequivalent regardless of source direction. Clearly, thereare severe physical constraints on binaural processing ofpressure signals in these animals even though they havepaired pressure receivers.

The bilateral projection pattern of saccular nerve fibersinto dzD (Fig. 2B) in Pollimyrus suggests that auditoryinputs from each ear may be combined early in auditoryprocessing at this major first-order nucleus. This projec-tion pattern into dzD could be functionally organized in atleast three different ways: (1) to form the basis of animmediate binaural comparison by individual dzD neu-rons, (2) to create separate but anatomically overlappingpopulations of monaural dzD neurons, or (3) to integrateinformation from the two ears so as to achieve a singlerepresentation of acoustic pressure. We suspect that thebilateral projection pattern of primary afferents into dzDmay have evolved to integrate information from the twoears and to create a fused representation of the pressurecomponent of the sound field. Any small differences be-tween the output of the two pressure receiving systems,thus, would be eliminated within the dzD population ofbinaural neurons. The highly interconnected organizationof dzD (Fig. 3A) and extensive local projections of indi-vidual dzD neurons (Fig. 3B overlay) also could contributeto the generation of a uniform representation of pressurewithin the population of dzD neurons. This single represen-tation of the pressure component of the sound field in turnmay be advantageous for computing sound source direc-tion underwater.

It has long been recognized that the pattern of activationof inner ear accelerometers (i.e., otolithic organs) in fishescould be used to compute the axis along which a soundsource lies (Fay, 1984; Lu et al., 1996; Wubbels andSchellart, 1997). However, additional information is re-quired to resolve the 180° axial ambiguity in determiningthe true source direction. This ambiguity can be elimi-nated with the addition of information about sound pres-sure (Buwalda, 1981; Schuijf, 1981; van den Berg and

Fig. 12. Schematic diagram of auditory pathways from medulla todiencephalon. For abbreviations, see list.


Schuijf, 1983; Rogers et al., 1988). In this computation,source direction can be found by calculating the relativephase between pressure and particle motion. In mor-myrids, this would mean comparing the output of acceler-ometers such as the utriculi with the output of a pressuremeasuring system such as the saccular system discussedabove. A uniform midline representation of pressure withindzD might be optimal for this computation, because itwould provide phase comparator circuits with a single,reliable representation of this parameter of the sound.

In ostariophysines, which possess a single midline pres-sure receiver (i.e., the swim bladder) coupled to bothsacculi by means of the Weberian ossicles, saccular inputsto all first-order medullary nuclei display a unilateralprojection pattern (McCormick and Braford, 1994). Such apattern may be preserved in these fish because a fusedrepresentation of pressure is already provided by thesingle midline pressure transducing organ, and thus thephase of the pressure signal is more or less constantthroughout the medulla. In both the clupeiformes (McCor-mick, 1997) and mormyriformes (Bell, 1981a), however,bilateral projections to pressure coding regions of themedulla have been demonstrated. Interestingly, both ofthese groups of fishes show bilateral pressure transducers,although in the clupeids, these transducers are continuouswith and thus joined by means of the main swim bladder.It would be interesting to look for bilateral projectionpatterns or fused midline organization within pressurecoding medullary nuclei in other fishes such as the squir-relfish (Holocentridae) or tarpon (Elopidae), which alsoshow bilateral forked projections of the swim bladder thatmay also serve a pressure-transducing mechanism (Schel-lart and Popper, 1992).

According to Bell (1981a), the region of M labeled in thisstudy after midbrain injections receives primary afferentinput from both the lagena and utriculus. The neurons inM were labeled both by intracellular filling of crest cells indzD (Fig. 3B), and by injections into the auditory midbrain(Fig. 3A-2). Similar labeling patterns have been reportedin the mormyrid Gnathonemus after injections into themidbrain (Bell, 1981b). Extracellular injections into MDcould have labeled M neurons either by direct retrogradetransport from dorsomedial auditory regions of MD, bymeans of transynaptic spread from labeled dzD crest cells,or possibly by inadvertent labeling of M due to tracerspread to the mechanosensory part of MD (nucleus ventro-lateralis). This last route of labeling seems unlikely giventhe focal nature of injections and the apparent limitedspread of dye within the dorsomedial and caudal regions ofMD (Fig. 8A).

The finding that some of the crest cells in dzD aredirectly coupled to a small cluster of medial octaval (M)neurons just lateral to the midline suggests a possiblesubstrate for the integration of acoustic pressure informa-tion from the saccule with particle motion informationfrom the lagena and utriculus. The only direct input to thecrest cells in dzD appears to be acoustic pressure inputfrom the sacculus, whereas projections from both thelagena and utricle overlap in M (Bell, 1981a). Crest cells indzD produce spikes that are entrained precisely to acousticstimuli (Fig. 3C). The anatomy indicates that these cellsmay also receive particle acceleration information fromthe lagena or utriculus, but this input is indirect andmediated by gap junctions with the nearby neurons in M(Fig. 3B). The timing of spike generation by some of the

crest cells in dzD may be modulated by the gap junctioninput from M, and therefore may be sensitive to the phaserelationship between acoustic pressure and particle accel-eration. The circuitry formed by M and dzD neurons, thus,could result in a temporal code for spatial hearing thatmight be further analyzed in the midbrain and tectum.

Descending modulation of auditory centersin the medulla

The local circuitry formed by crest cells, deeper cells inthe dzD, and the overlying crista cerebellaris in the octavalpathways of fishes has led several authors to comparecrest cells of fishes with cerebellar Purkinje cells of mam-mals and to pyramidal cells in the electrosensory lateralline lobe of weakly electric fish (e.g., New et al., 1996; Belland Szabo, 1986; Bastian, 1986; McCormick, 1998). Thecrista cerebellaris is composed of parallel fibers originat-ing in the more rostral eminentia granularis of the cerebel-lum (Maler, 1974). In mormyrids, this granule cell massreceives primary afferent input from most octavolateralendorgans with the marked exception of the sacculus(Bell, 1981a). The parallel fibers of the crista cerebellariscontact crest cell apical dendrites, suggesting that primaryauditory processing in the crest cell layer may be modu-lated by these other octavolateral systems, and perhaps byother forms of descending input. In all other systemsfollowing this general pattern of organization, the apicaldendrites of pyramidal cells are spiny (Montgomery et al.,1995), but we have not observed spines in the apicaldendrites of dzD neurons of Pollimyrus (Fig. 3B). Golgistudies of these neurons will be required to confirm thisapparent difference.

The dSO of Pollimyrus is also organized much like dzDcrest cell layer at more caudal levels. At this level (Fig.1F,G), a close association exists between the dorsal den-drites of the dSO neurons and the parallel fibers of thecrista cerebellaris (Fig. 6A-2). Neurons in dSO are secondorder and form an important alternate link between dzDand both iSO and MD; thus, descending neuromodulationof these neurons may serve a different function from thatof dzD crest cells. In electrosensory systems, descendinginput to the apical dendrites of pyramidal cells is known toplay a role in gain control (Bastian, 1986, 1995) and in thecancellation of self-generated reafference (Montgomery etal., 1995; Duman and Bodznick, 1996; VonderEmde andBell, 1996; Bell et al., 1997). Similar circuitry may beinvolved in auditory processing in the medulla of mam-mals (reviewed in Warr, 1992; Montgomery et al., 1995).The crista cerebellaris, thus, could have profound influ-ences on auditory processing in both the crest cells of dzDand in second-order dSO neurons.

Male Pollimyrus actively swim during sound produc-tion. Females also swim while searching for males andduring the sonic phase of courtship. Self-generated motioncould possibly stimulate saccular afferents at low frequen-cies (Fay, 1984; Fay et al., 1994; Edds-Walton and Fay,1995; Lu et al., 1996), potentially interfering with auditorysignal analysis. The cerebellar-like organization of theauditory pathway described here could play an importantrole in the cancellation of any noise generated in theauditory system from swimming motion. Additionally, asfemales explore the territories of available males (Craw-ford, 1997b; Crawford et al., 1997b), they will experience awide range of sound pressures as their distance fromvarious males changes; during the sonic phase of court-


ship, females may approach to within 5 cm of a soundproducing male. The cerebellar-like organization of thissystem may also serve to control of gain in the ascendingauditory system under these variable stimulus conditions(see Piddington, 1971b).

Secondary octaval nuclei of fish and themammalian superior olive

Secondary octaval cell groups like those described forPollimyrus have been observed previously in several otherteleost fishes. These areas have been termed the superiorolive (Bell, 1981b; Echteler, 1984; McCormick, 1992; McCor-mick and Braford, 1993; Bass et al., 1994), or moreconservatively the secondary octaval population (McCor-mick and Hernandez, 1996). One of the first studies tosuggest a ‘‘superior olive’’ in a teleost species was carriedout in carp (Echteler, 1984). Echteler proposed that thecase for a strong analogy between this auditory region infish and similar structures in the mammalian brainstemwould hinge on whether the SO of fish received bilateral(binaural) inputs from first-order auditory nuclei, as doesthe superior olivary complex in mammals. We have ob-served bilateral inputs into the iSO in Pollimyrus fromeach ventrolateral margin of dzD (Fig. 5) and from eachizD, both after injections into MD and into dzD. Asmentioned above, dzD does not appear to anatomicallysegregate its binaural inputs; izD, however, does appear toreceive some inputs from the ipsilateral sacculus only(Fig. 2B), and this finding cannot be ruled out for theventrolateral margin of dzD as well. Thus, althoughEchteler’s requirement seems fulfilled based on both ipsi-lateral and contralateral origins of SO inputs, it may bethat some convergence of binaural signals occurs in dzD,before reaching the SO in Pollimyrus. The role of SO inbinaural processing has yet to be determined.

Preliminary analysis of physiological recordings madeduring these studies in the auditory medulla indicatedthat neurons in iSO produce temporal response patternssimilar to the choppers of the mammalian superior olive(Kozloski and Crawford, 1998). These neurons producedsustained responses with characteristic interspike inter-vals that do not correspond faithfully to the stimulusperiod and which range from 3 to 12 ms. Thus, bothanatomical connectivity and auditory physiology of the SOin Pollimyrus appear similar in some respects to thesuperior olivary complex of other vertebrates. It is possiblethat the SO of teleost fishes may be functionally analogousto components of the mammalian superior olivary com-plex.

Reciprocal connections and feedbackbetween midbrain and medulla

Descending feedback by centrifugal pathways in thevertebrate auditory system is well known (e.g., Zhang etal., 1997) and has been implicated in the modification ofauditory responses in some physiological studies of fishes(Piddington, 1971a). Pollimyrus has feedback circuits fromthe midbrain nucleus MD to the perilemniscal nucleusIRN (Fig. 12) and possibly to the second-order medullarynucleus iSO. Similar feedback circuitry from MD to SO hasbeen shown in other mormyrids (Bell, 1981b) and inseveral otophysan fishes (Echteler, 1984; McCormick andBraford, 1993; McCormick and Hernandez, 1996). Thefunction of these circuits in fishes is not yet clear and needsto be examined further in mormyrids and in other fishes

with specializations of the auditory periphery (McCormickand Hernandez, 1996).

The indirect connections between the two MDs, and thereciprocal connections between MD and the contralateralIRN, form a complex local circuit (Fig. 12). Each IRNreceives no input from first-order auditory centers in themedulla, projects to the contralateral MD and IRN, andreceives descending input from the ipsilateral MD (Fig. 12).The circuitry connecting IRN and MD is essentially identi-cal to that connecting the dorsal nucleus of the laterallemniscus and the IC in mammals (reviewed in Schwartz,1992). Many of the lateral lemniscal nuclei projections andhomotopic projections into the mammalian IC are knownto be inhibitory (Gonzalez-Hernandez et al., 1996), andimmunohistochemical (Mugnaini and Maler, 1987) andneurophysiological studies (Crawford, 1993, 1997a) bothpoint to important inhibitory inputs into MD in mor-myrids. If projections onto MD neurons from the contralat-eral IRN or MD in Pollimyrus are found to be inhibitory,these projections may represent important components ofthe inhibitory gating model previously proposed to explainthe complex tuning (interval selectivity) in MD neurons(discussed above and in Crawford, 1997a).

Midbrain output to IG and thalamus

The IG was the most immediate recipient of MD efferentoutput. The enormous boutons surrounding each of theround IG somata (Fig. 9B–D) suggest that this pathway isinvolved in temporal processing. End bulbs of this kind arecharacteristic of time coding pathways in the auditorysystems of birds (Carr and Boudreau, 1991; Koppl, 1994),mammals (Rouiller et al., 1986), and within the electrosen-sory pathways of electric fish (Bell and Szabo, 1986;Friedman and Hopkins, 1998). It is likely that neurons inIG of Pollimyrus represent the temporal firing patternsprovided by MD output neurons precisely. It is possiblethat the subpopulation of MD neurons that showed sus-tained and highly phase-locked responses (Crawford, 1993)communicate by means of these large club endings withthe IG neurons to form an anatomically segregated path-way for higher level temporal analyses. Projection targetsfrom IG include the valvula (Bell and Russel, 1978; Fingeret al., 1981) as well as the OT (Wullimann and Northcutt,1990). By comparison with other systems, we might expectthe involvement of the OT projection pathway in spatialhearing.

Output from MD in thalamic areas may be important inthe temporal analysis of communication sounds. Becausethere has not yet been any physiological study of theauditory thalamus in mormyrids, little can be said regard-ing the three diencephalic projections we observed fromMD. Recent studies have explored responses to tones inthe goldfish CP and suggest that neurons in this thalamicregion, or the axons projecting from the midbrain into CP,have broader tuning to simple acoustic stimuli than lower-order neurons in the pathway (Lu and Fay, 1995).

Midbrain output to the optic tectum

Perhaps the most interesting anatomical feature ob-served among MD projections in Pollimyrus was theextraordinary pattern of parallel-coursing fibers formedwithin the optic tecta. These arrays of parallel-coursingfibers appeared to give rise to convergent input from thebilateral MD nuclei by means of axons as long as 3 mm.These axon lengths could translate into quite long delays


between the appearance of an action potential in ipsilat-eral OT and its appearance at the ventral border ofcontralateral OT within a single axon (i.e., approximately15 msec, assuming conduction velocity of 5 m/sec). Theselong axons could potentially function within the tecta asdelay lines, generating tuning in OT neurons to temporaldisparities between activity in the right and left MD.Given that many responses recorded in MD were phasicand marked the onset of tones with great precision, thiscircuit, when coupled to other mechanism, could serve as acomparator between phasic responses in each MD. Thephysiology of OT neurons remains to be studied.

Additional comparisons of midbrain outputin Pollimyrus and in other species

In several instances we have noted differences betweenthe anatomy of Pollimyrus and that of other mormyrids.Previous work on mormyrids has shown strong projectionsfrom MD into the valvula (Bell, 1981b; Haugede-Carre,1983), and into midbrain nucleus preeminentialis ventra-lis (Bell, 1981b). Our results indicate that if these projec-tions exist in Pollimyrus, they are quite weak. Only in afew preparations did we observe one or two labeled fibersfrom MD penetrating the valvula, and these may haveoriginated from fibers of passage in the valvular peduncle.We observed no terminals in preeminentialis ventralis. Avery small and weakly labeled terminal field was observedat the ventral border of nucleus preeminentialis but not inpreeminentialis ventralis, and only a few labeled fiberswere observed traversing preeminentialis ventralis. Itseems most likely that the projections into the valvula andnucleus preeminentialis from MD in other species oc-curred because both nucleus centralis (auditory) andnucleus ventrolateralis (mechanosensory lateral line) werelabeled in these earlier studies during MD injections.

We also observed contralateral labeling of OT in Polli-myrus by means of an intertectal commissure. In previousstudies of the Mormyrid mesencephalon, projections fromMD into the optic tectum were described, but only ipsilat-eral to the injection site (Bell, 1981b; Haugede-Carre,1983). In our own unpublished observations of anothermormyrid species, Gnathonemus petersii, the projectionsinto OT were also ipsilateral only. These projections werebranched and seemed to traverse laterally the same re-gions occupied by dorsoventrally oriented parallel-cours-ing fibers in Pollimyrus. In carp, a decussating OT projec-tion pattern similar to that seen in Pollimyrus was alsoobserved; parallel-coursing fibers projected not only to theipsilateral OT, but also to the contralateral OT by means ofan intertectal commissure (Echteler, 1984). BecauseEchteler used the same tracer (horseradish peroxidase) asBell, and because we obtained the same result as Bell didin our own biocytin studies of Gnathonemus, it seemsreasonable to accept these differences in tectal projectionpatterns as true species differences within the mormyrids.The intertectal commissural projection in Pollimyrus andcarp (Echteler, 1984) were both marked by periodic swell-ings, which Echteler termed en passant synapses.

The CP terminal field in the thalamus of Pollimyrus hasnot been described previously in mormyrids. The positionof this periventricular projection is similar to that of CP inother fish (e.g., Striedter, 1991), although the terminalfield within this nucleus in Pollimyrus appears far morerestricted and limited to the periventricular region. Thecrescent shaped projection which we observed in the

preglomerular complex of the thalamus corresponds to theanterior thalamic projection of Bell (1981b), and wassubsequently termed the preglomerular complex, as inother teleost species (Bell and Szabo, 1986). This regionhas also been shown to receive extensive inputs frommidbrain auditory nuclei in the catfish (Striedter, 1991).

Pathways and local circuitry

In studies such as this one, conclusions focus on connec-tions between major nuclei involved in sensory processing.However, it is clear that important local circuits are alsopresent within the structures described here, and there-fore that not all auditory neurons in this system can belabeled after injections into MD or dzD. These localconnections and neuronal populations cannot be includedin our circuit diagram (Fig. 12) and space filling model(Fig. 13). Nevertheless, we expect that these local connec-tions play a crucial role in auditory processing. It is ourhope that with further behavioral, neurophysiological, andneuroanatomical studies of this system, we will begin tounderstand better the computations performed within andbetween the structures depicted here and by the entirePollimyrus auditory system.

Fig. 13. A: Rostrolateral view of a three dimensional space fillingmodel of medullary cell profiles labeled after a midbrain injection intoMD (see Materials and Methods section). Orange rod indicates therostrocaudal axis. Rendering made from the perspective of the MDinjection site; thus, only contralateral MD is visible (white), asreconstructed from camera lucida outlines of this nucleus. B: Dorsalview of the same medullary space filling model. MD from thisperspective is very large and far dorsal and rostral to the medullarynuclei, and thus is not rendered within this field of view. Three-dimensional white scale bars originate at the most dorsal extent of themodel, M (orange), and extend 500-µm rostral, caudal, and ventralfrom the origin.


ACKNOWLEDGMENTS

Some of the histological protocols described here werebased on methods developed by generous colleagues:Curtis Bell, Masashi Kawasaki, G. Kennedy, and WalterHeiligenberg. We are indebted to Curtis Bell for lendingslides of his original horseradish peroxidase studies ofmormyrids. David Sparks and James Saunders kindlyloaned equipment, and Peter Sterling aided with prelimi-nary interpretation of anatomical results. Xiaofeng Huangassisted with the histology, and Camille A.N. Henry helpedwith the preparation of the manuscript. Matthew Fried-man and Catherine McCormick provided valuable com-ments on a draft of the manuscript. We are particularlyindebted to Catherine McCormick for her critical input onthe research presented here, for lending histological mate-rial, and for helping us to place our results in an appropri-ate comparative context. James Kozloski received a predoc-toral NRSA from the NIMH.

LITERATURE CITED

Bass, A.H., M.A. Marchaterre, and R. Baker (1994) Vocal-acoustic path-ways in a teleost fish. J. Neurosci. 14:4025–4039.

Bass, A.H., D.A. Bodnar, and J.R. McKibben (1997) From neurons tobehavior: Vocal-acoustic communication in teleost fish. Biol. Bull.192:158–160.

Bastian, J. (1986) Gain control in the electrosensory system: A role for thedescending projections to the electrosensory lateral line lobe. J. Comp.Physiol. A 158:505–515.

Bastian, J. (1995) Pyramidal-cell plasticity in weakly electric fish: Amechanism for attenuating responses to reafferent electrosensoryinputs. J. Comp. Physiol. A 176:63–78.

Bell, C.C. (1981a) Central distribution of octavolateral afferents andefferents in a teleost (mormyridae). J. Comp. Neurol. 195:391–414.

Bell, C.C. (1981b) Some central connections of medullary octavolateralcenters in a mormyrid fish. In W.N. Tavolga, A.N. Popper, and R.R. Fay(eds): Hearing and Sound Communication in Fishes. New York: Springer-Verlag, pp. 383–392.

Bell, C.C. and C.J. Russel (1978) Termination of electroreceptor andmechanical lateral line afferents in the mormyrid acousticolateral area.J. Comp. Neurol. 82:367–382.

Bell, C.C. and T. Szabo (1986) Electroreception in mormyrid fish: Centralanatomy. In T.H. Bullock and W. Heiligenberg (eds): Electroreception.New York: John Wiley & Sons, pp. 375–421.

Bell, C.C., D. Bodznick, J.C. Montgomery, and J. Bastian (1997) Thegeneration and subtraction of sensory expectations within cerebellum-like structures. Brain Behav. Evol. 50:17–31.

Blauert, J. (1996) Spatial Hearing: The Psychophysics of Human SoundLocalization. Cambridge, MA: MIT Press.

Braford, M.R., Jr., E. Prince, and C.A. McCormick (1993) A presumedacoustic pathway from the ear to the telencephalon in an osteoglosso-morph. Soc. Neurosci. Abstr. 19:160.

Buwalda, R.J.A. (1981) Segregation of directional and nondirectionalacoustic information in the cod. In W.N. Tavolga, A.N. Popper, and R.R.Fay (eds): Hearing and Sound Communication in Fishes. New York:Springer-Verlag, pp. 139–186.

Capranica, R.R. (1965) The Evoked Vocal Response of the Bullfrog - A Studyof Communication by Sound. Cambridge, MA: MIT Press.

Carr, C.E. and R.E. Boudreau (1991) Central projections of auditory nervefibers in the barn owl. J. Comp. Neurol. 314:306–318.

Carr, C.E. and M. Konishi (1990) A circuit for detection of interaural timedifference in the brainstem of the owl. J. Neurosci. 10:3227–3246.

Cazals, Y. and L. Palis (1991) Effect of silence duration in intervocalic velarplosive on voicing perception for normal and hearing-impaired subjects.J. Acoust. Soc. Am. 89:2916–2921.

Coombs, S. (1994) Nearfield detection of dipole sources by the goldfish(Carassius auratus) and the mottled sculpin (Cottus-bairdi). J. Exp.Biol. 190:109–129.

Coombs, S. and R.R. Fay (1989) The temporal evolution of masking andfrequency selectivity in the goldfish (Carassius auratus). J. Acoust. Soc.Am. 86:925–933.

Crawford, J.D. (1993) Central auditory neurophysiology of a sound-producing mormyrid fish: The mesencephalon of Pollimyrus isidori. J.Comp. Physiol. 172:1–14.

Crawford, J.D. (1997a) Feature-detecting auditory neurons in the brain of asound-producing fish. J. Comp. Physiol. A 180:439–450.

Crawford, J.D. (1997b) Hearing and acoustic communication in the mor-myrid electric fishes. Mar. Freshwater Behav. Physiol. 29:1–21 .

Crawford, J.D., A.P. Cook, and A.S. Herberlein (1997a) Bioacoustic behaviorof African fishes (Mormyridae): Potential cues for species and individualrecognition in Pollimyrus. J. Acoust. Soc. Am. 102:1200–1212.

Crawford, J.D., P. Jacobe, and V. Benech (1997b) Field studies of a stronglyacoustic fish in West Africa: Reproductive ecology and acoustic behaviorof Pollimyrus isidori, Mormyridae. Behaviour 134:677–725.

Doupe, A.J. and M. Konishi (1991) Song-selective auditory circuits in thevocal control-system of the zebra-finch. Proc Natl Acad Sci USA88:11339–11343.

Dowben, R.M. and J.E. Rose (1953) A metal-filled electrode. Science 118:22.Duman, C.H. and D. Bodznick (1996) A role for GABAergic inhibition in

electrosensory processing and common mode rejection in the dorsalnucleus of the little skate, Raja erinacea. J. Comp. Physiol. A 179:797–807.

Echteler, S.M. (1984) Connections of the auditory midbrain in a teleost fish,Cyprinus carpio. J. Comp. Neurol. 230:536–551.

Echteler, S.M. (1985a) Organization of central auditory pathways in ateleost fish, Cyprinus carpio. J. Comp. Physiol. A 338:387–391.

Echteler, S.M. (1985b) Tonotopic organization in the midbrain of a teleostfish. Brain Res. 338:387–391.

Edds-Walton, P.L. and R.R. Fay (1995) Regional differences in directionalresponse properties of afferents along the saccule of the toadfish,Opsanus tau. Biol. Bull. 189:211–212.

Fay, R.R. (1984) The goldfish ear codes the axis of acoustic particle motionin three dimensions. Science 225:951–954.

Fay, R.R. (1995) Perception of spectrally and temporally complex sounds bythe goldfish (Carassius auratus). Hearing Res. 89:146–154.

Fay, R.R. and T.J. Ream (1986) Acoustic response and tuning in saccularnerve fibers of the Goldfish (carassius auratus). J. Acoust. Soc. Am.79:1883–1895.

Fay, R.R., P.L. Edds-Walton, and S.M. Highstein (1994) Directional sensitiv-ity of saccular afferents of the toadfish to linear acceleration at audiofrequencies. Biol. Bull. 187:258–259.

Feng, A.S., J.C. Hall, and D.M. Gooler (1990) Neural basis of sound patternrecognition in anurans. Prog. Neurobiol. 34:313–329.

Fine, M.L. (1981) Mismatch between sound production and hearing inoyster toadfish. In W.N. Tavolga, A.N. Popper, and R.R. Fay (eds):Hearing and Sound Communication in Fishes. New York: SpringerVerlag, pp. 257–263.

Finger, T.E., C.C. Bell, and C.J. Russel (1981) Electrosensory pathways tothe valvula cerebelli in mormyrid fish. Exp. Brain Res. 42:23–33.

Friedman, M.A. and C.D. Hopkins (1998) Neural substrates for speciesrecognition in the time-coding electrosensory pathway of mormyridelectric fish. J. Neurosci. 18:1171–1185.

Furukawa, T. and Y. Ishii (1967) Neurophysiological studies on hearing ingoldfish. J. Neurophysiol. 30:1377–1403.

Gerhardt, H.C. (1978) Discrimination of intermediate sounds in a syntheticcall continuum by female green tree frogs. Science 199:1089–1091.

Gonzalez-Hernandez, T., B. Mantolan-Sarmiento, B. Gonzalez-Gonzalez,and H. Perez-Gonzalez (1996) Sources of GABAergic input to theinferior colliculus of the rat. J. Comp. Neurol. 372:309–326.

Haugede-Carre, F. (1983) The mormyrid mesencephalon. II. The medio-dorsal nucleus of the torus semicircularis: Afferent and efferent connec-tions studied with the HRP method. Brain Res. 268:1–14.

Heiligenberg, W. (1991) The neural basis of behavior: A neuroethologicalview. Annu. Rev. Neurosci. 14:247–267.

Horikawa, K. and W.E. Armstrong (1988) A versatile means of intracellularlabelling: Injection of biocytin and its detection with avidin conjugates.J. Neurosci. Methods 25:1–12.

Hoy, R.R., G.S. Pollack, and A. Moiseff (1982) Species recognition in the fieldcricket, Teleogryllus oceanicus: Behavioral and neural mechanisms.Am. Zool. 22:597–607.

Irvine, D.R.F. (1986) The auditory brainstem: A review of the structure andfunction of auditory brainstem processing mechanisms. In D. Ottoson(ed): Progress in Sensory Physiology. Berlin: Springer-Verlag, pp.1–279.

Kawasaki, M. (1997) Sensory hyperacuity in the jamming avoidanceresponse of weakly electric fish. Curr. Opin. Neurobiol. 7:473–479.


Kawasaki, M. and Y.X. Guo (1996) Neuronal circuitry for comparison oftiming in the electrosensory lateral line lobe of the African wave-typeelectric fish Gymnarchus niloticus. J. Neurosci. 16:380–391.

Konishi, M. (1993) Neuroethology of sound localization in the owl. J. Comp.Physiol. A 173:3–7.

Konishi, M., T.T. Takahashi, H. Wagner, W.E. Sullivan, and C.E. Carr(1988) Neurophysiological and anatomical substrates of sound localiza-tion in the owl. In G.M. Edelman, W.E. Gall, and W.M. Cowan (eds):Auditory Function. Neurobiological Bases of Hearing. New York: JohnWiley & Sons, pp. 721–745.

Koppl, C. (1994) Auditory nerve terminals in the cochlear nucleus magnocel-lularis: Differences between low and high frequencies. J. Comp. Neurol.339:438–446.

Kozloski, J. and J.D. Crawford (1995) Auditory pathways of a sonic fish(Pollimyrus). Soc. Neurosci. Abstr. 21:361.7.

Kozloski, J. and J.D. Crawford (1996) Time coding in the auditory medullaof a sonic fish. Soc. Neurosci. Abstr. 22:178.3.

Kozloski, J. and J.D. Crawford (1997) Physiology of primary afferents andfirst order auditory neurons in the medulla of a sonic fish: Pollimyrusadspersus. Assoc. Res. Otolaryngol. Abstr. 20:566.

Kozloski, J. and J.D. Crawford (1998) Chopper responses in the auditorymedulla of a sonic fish. Assoc. Res. Otolaryngol. Abstr. 21:403.

Kuwada, S. and T.C.T. Yin (1983) Binaural interaction in low-frequencyneurons in the inferior colliculus of the cat. I. Effects of long interauraldelays, intensity, and repetition rate on interaural delay function. J.Neurophysiol. 50:981–999.

Liberman, M.C. (1982) The cochlear frequency map for the cat: Labelingauditory-nerve fibers of known characteristic frequency. J. Acoust. Soc.Am. 72:1441–1449.

Lu, Z. and R.R. Fay (1993) Acoustic response properties of single units inthe torus semicircularis of the goldfish, Carassius auratus. J. Comp.Physiol. A 173:33–48.

Lu, Z. and R.R. Fay (1995) Acoustic response properties of single neurons inthe central posterior nucleus of the thalamus of the goldfish, Carassiusauratus. J. Comp. Physiol. A 176:747–760.

Lu, Z., A.N. Popper, and R.R. Fay (1996) Behavioral detection of acousticparticle motion by a teleost fish (Astronotus ocellatus): Sensitivity anddirectionality. J. Comp. Physiol. A 179:227–233.

Lugli, M., G. Pavan, and P. Torricelli (1996) The importance of breedingvocalizations for mate attraction in a freshwater goby with a compositesound repertoire. Ethology, Ecology, & Evolution 8:343–351.

Maler, L. (1974) The acousticolateral area of bony fishes and its cerebellarrelations. Brain Behav. Evol. 10:130–145.

Maler, L., H.J. Karten, and M.V.L. Bennet (1973a) The central connectionsof the anterior lateral line nerve of Gnathonemus petersii. J. Comp.Neurol. 151:67–84.

Maler, L., H.J. Karten, and M.V.L. Bennet (1973b) The central connectionsof the posterior lateral line nerve of Gnathonemus petersii. J. Comp.Neurol. 151:57–66.

McCormick, C.A. (1982) The organization of the octavolateralis area inactinopterygian fishes: A new interpretation. J. Morphol. 171:159–181.

McCormick, C.A. (1992) Evolution of central auditory pathways in anamni-otes. In D.B. Webster, A.N. Popper, and R.R. Fay (eds): The Evolution-ary Biology of Hearing. New York: Springer-Verlag, pp. 323–350.

McCormick, C.A. (1997) Organization and connections of octaval andlateral line centers in the medulla of a clupeid, Dorosoma cepediamum.Hearing Res. 110:39–60.

McCormick, C.A. (1998) Anatomy of the central auditory pathways of fishand amphibians. In R.R. Fay and A.N. Popper (eds): Fish and Amphib-ians. New York: Springer-Verlag.

McCormick, C.A. and M.R. Braford, Jr. (1993) The primary octaval nucleiand inner ear afferent projections in the otophysan Ictalurus punctatus.Brain Behav. Evol. 42:48–68.

McCormick, C.A. and M.R. Braford, Jr. (1994) Organization of inner earendorgan projections in the goldfish, Carassius auratus. Brain Behav.Evol. 43:189–205.

McCormick, C.A. and D.V. Hernandez (1996) Connections of octaval andlateral line nuclei of the medulla in the goldfish, including the cytoarchi-tecture of the secondary octaval population in goldfish and catfish.Brain Behav. Evol. 47:113–137.

Moiseff, A. (1989) Bi-coordinate sound localization by the barn owl. J.Comp. Physiol. 164:637–644.

Montgomery, J.C., S. Coombs, R.A. Conley, and D. Bodznick (1995) Hind-brain sensory processing in lateral line electrosensory, and auditorysystems: A comparative overview of anatomical and functional similari-ties. Auditory Neuroscience 1:207–231.

Mugnaini, E. and L. Maler (1987) Cytology and immunocytochemistry ofthe nucleus exterolateralis anterior of the mormyrid brain: Possible roleof GABAergic synapses in temporal analysis. Anat. Embryol. 176:313–336.

Myrberg, A.A., Jr (1997) Underwater sound: its relevance to behavioralfunctions among fishes and marine animals. Mar. Freshwater Behav.Physiol. 29:3–21.

Myrberg, A.A., Jr, E. Spanier, and S.J. Ha (1978) Temporal patterning inacoustical communication. In E.S. Reese and F.J. Lighter (eds): Con-trasts in Behavior. New York: John Wiley & Sons, pp. 136–179.

Narins, P.M. (1995) Frog communication. Sci. Am. 273:78–83.New, J.G., S. Coombs, C.A. McCormick, and P.E. Oshel (1996) Cytoarchitec-

ture of the medial octavolateralis nucleus in the goldfish Carassiusauratus. J. Comp. Neurol. 366:534–546.

Piddington, R.W. (1971a) Central control of auditory input in the goldfish. I.Effect of shocks to the midbrain. J. Exp. Biol. 55:569–584.

Piddington, R.W. (1971b) Central control of auditory input in the goldfish.II. Evidence of action in the free-swimming animal. J. Exp. Biol.55:585–610.

Popper, A.N. and S. Coombs (1982) The morphology and evolution of the earin actinopterygian fishes. Am. Zool. 22:311–328.

Popper, A.N. and R.R. Fay (1984) Sound detection and processing by teleostfish: A selective review. In L. Bolis, R.D. Keynes, and S.H.P. Mandrell(eds): Comparative Physiology of Sensory Systems. Cambridge: Cam-bridge University Press, pp. 67–101.

Rogers, P.H., A.N. Popper, M.C. Hastings, and W.M. Saidel (1988) Process-ing of acoustic signals in the auditory system of bony fish. J. Acoust. Soc.Am. 83:338–349.

Rouiller, E.M., R. Cronin-Schreiber, D.M. Fekete, and D.K. Ryugo (1986)The central projections of intracellularly labeled auditory nerve fibersin cats: An analysis of terminal morphology. J. Comp. Neurol. 249:261–278.

Ryugo, D.K. and D.M. Fekete (1982) Morphology of primary axosomaticendings in the anteroventral cochlear nucleus of the cat: A study of theendbulbs of Held. J. Comp. Neurol. 210:239–257.

Sarnat, H.B. and M.G. Netsky (1981) Evolution of the Nervous System.New York: Oxford University Press.

Schellart, N.A.M. and A.N. Popper (1992) Functional aspects of theevolution of the auditory system of actinopterygian fish. In D. Webster,R.R. Fay, and A.N. Popper (eds): The Evolutionary Biology of Hearing.New York: Springer-Verlag, pp. 295–322.

Schuijf, A. (1981) Models of acoustic localization. In W.N. Tavolga, A.N.Popper, and R.R. Fay (eds): Hearing and Sound Communication inFishes. New York: Springer-Verlag, pp. 267–230.

Schwartz, I.R. (1992) The superior olivary complex and lateral lemniscalnuclei. In D.B. Webster, A.N. Popper, and R.R. Fay (eds): The Mamma-lian Auditory Pathway: Neuroanatomy. New York: Springer-Verlag, pp.117–167.

Shannon, R.V., F.G. Zeng, V. Kamath, J. Wygonski, and M. Ekelid (1995)Speech recognition with primarily temporal cues. Science 270:303–304.

Simmons, A.M., Y. Shen, and M.I. Sanderson (1996) Neural and computa-tional basis for periodicity extraction in frog peripheral auditorysystem. Auditory Neurosicence 2:109–133.

Stendell, W. (1914) Die Faseranatomie des Mormyridengehirns. Abh.Senckenb. Naturforsch. Ges. 36:3–40.

Striedter, G.F. (1991) Auditory, electrosensory, and mechanosensory lateralline pathways through the forebrain in channel catfishes. J. Comp.Neurol. 312:311–331.

Suga, N. (1988) Auditory neuroethology and speech processing: Complexsound processing by combination-sensitive neurons. In G.M. Edelman,W.E. Gall, and W.M. Cowan (eds): Functions of the Auditory System.New York: John Wiley & Sons, pp. 679–720.

Suga, N. (1990) Biosonar and neural computation in bats. Sci. Am.262:60–68.

Szabo, T. and S. Libouban (1979) On the course and origin of cranial nervesin the teleost fish Gnathonemus determined by ortho- and retrogradehorseradish peroxidase axonal transport. Neurosci. Lett. 11:265–270.

Takahashi, T., A. Moiseff, and M. Konishi (1984) Time and intensity cuesare processed independently in the auditory system of the owl. J.Neurosci. 4:1781–1786.

Thorson, J., T. Weber, and F. Huber (1982) Auditory behavior of the cricket.II. Simplicity of calling-song recognition in Gryllus, and anomalousphonotaxis at abnormal carrier frequencies. J. Comp. Physiol. 146:361–378.


van den Berg, A.V. and A. Schuijf (1983) Discrimination of sounds based onthe phase difference between particle motion and acoustic pressure inthe shark Chiloscyllium griseum. Proc. R. Soc. Lond. B Biol. Sci.218:127–134.

Vaney, D.I. (1993) The coupling patterns of axon bearing horizontal cells inthe mammalian retina. Proc. R. Soc. Lond. B Biol. Sci. 252:93–101.

von Bekesy, G. (1947) The variations of phase along the basilar membranewith sinusoidal vibrations. J. Acoust. Soc. Am. 19:452–460.

VonderEmde, G. and C.C. Bell (1996) Nucleus preeminentialis of mormyridfish, a center for recurrent electrosensory feedback. I. Electrosensoryand corollary discharge responses. J. Neurophysiol. 76:1581–1596.

Warr, W.B. (1992) Efferent pathways. In D.B. Webster, A.N. Popper, andR.R. Fay (eds): Springer Handbook of Auditory Research, Vol. 1: TheMammalian Auditory Pathway: Neuroanatomy. New York: Springer-Verlag.

Wong, C.J.H. (1997) Afferent and efferent connections of the diencephalic

prepacemaker nucleus in the weakly electric fish, Eigenmannia vire-scens: Interactions between the electromotor system and the neuroendo-crine axis. J. Comp. Neurol. 383:18–41.

Wubbels, R.J. and N.A.M. Schellart (1997) Neuronal encoding of sounddirection in the auditory midbrain of the rainbow trout. J. Neuro-physiol. 77:3060–3074.

Wullimann, M.F. and R.G. Northcutt (1990) Visual and electrosensorycircuits of the diencephalon in mormyrids: An evolutionary perspective.J. Comp. Neurol. 297:537–552.

Yan, H.Y. and A.N. Popper (1993) Acoustic intensity discrimination by thecichlid fish Astonotus ocellatus (Cuvier). J. Comp. Neurol. 173:347–351.

Yost, W.A. (1974) Discriminations of interaural phase differences. J. Acoust.Soc. Am. 55:1299–1303.

Zhang, Y., N. Suga, and J. Yan (1997) Corticofugal modulation of frequencyprocessing in bat auditory system. Nature 387:900–903.


functional neuroanatomy of auditory pathways in the sound-producing fishpollimyrus

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