Anatomy and Physiology of Principal Cells of the Medial Nucleus of the Trapezoid Body (MNTB) of the Cat

Abstract

Anatomy and Physiology of Principal Cells of the Medial Nucleus of the Trapezoid Body (MNTB) of the Cat

Philip H. Smith¹, Philip X. Joris²^,³, and Tom C. T. Yin²

¹ Department of Anatomy and ² Department of Neurophysiology, University of Wisconsin Medical School, Madison, Wisconsin 53706; and ³ Division of Neurophysiology, Medical School, KULeuven, B3000 Leuven, Belgium

The Journal of Neurophysiology Vol. 79 No. 6 June 1998, pp. 3127-3142.

ABSTRACT

Smith, Philip H., Philip X. Joris, and Tom C. T. Yin. Anatomy and physiology of principal cells of the medial nucleusof the trapezoid body (MNTB) of the cat. J. Neurophysiol. 79:3127-3142, 1998. We have recorded from principal cells of themedial nucleus of the trapezoid body (MNTB) in the cat's superiorolivary complex using either glass micropipettes filled with Neurobiotinor horseradish peroxidase for intracellular recording and subsequentlabeling or extracellular metal microelectrodes relying on prepotentialsand electrode location. Labeled principal cells had cell bodiesthat usually gave rise to one or two primary dendrites, whichbranched profusely in the vicinity of the cell. At the electronmicroscopic (EM) level, there was a dense synaptic terminal distributionon the cell body and proximal dendrites. Up to half the measuredcell surface could be covered with excitatory terminals, whereasinhibitory terminals consistently covered about one-fifth. Thedistal dendrites were very sparsely innervated. The thick myelinatedaxon originated from the cell body and innervated nuclei exclusivelyin the ipsilateral auditory brain stem. These include the lateralsuperior olive (LSO), ventral nucleus of the lateral lemniscus,medial superior olive, dorsomedial and ventromedial periolivarynuclei, and the MNTB itself. At the EM level the myelinated collateralsgave rise to terminals that contained nonround vesicles and, inthe LSO, were seen terminating on cell bodies and primary dendrites.Responses of MNTB cells were similar to their primary excitatoryinput, the globular bushy cell (GBC), in a number of ways. Thespontaneous spike rate of MNTB cells with low characteristic frequencies(CFs) was low, whereas it tended to be higher for higher CF units.In response to short tones, a low frequency MNTB cell showed enhancedphase-locking abilities, relative to auditory nerve fibers. Forcells with CFs >1 kHz, the short tone response often resembledthe primary-like with notch response seen in many globular bushycells, with a well-timed onset component. Exceptions to and variationsof this standard response were also noted. When compared withGBCs with comparable CFs, the latency of the MNTB cell responsewas delayed slightly, as would be expected given the synapse interposedbetween the two cell types. Our data thus confirm that, in thecat, the MNTB receives and converts synaptic inputs from globularbushy cells into a reasonably accurate reproduction of the bushycell spike response. This MNTB cell output then becomes an importantinhibitory input to a number of ipsilateral auditory brain stemnuclei.

Introduction

Principal cells of the medial nucleus of the trapezoid body (MNTB) play a pivotal role in the processing of binaural informationby brain stem auditory circuitry. As a result of their glycinergicnature (Bledsoe et al. 1990; Sanes et al. 1987; Wenthold et al.1987; Zarbin et al. 1981), these cells convert an excitatory signal,received from globular bushy cells (GBCs) in the contralateralcochlear nucleus, to an inhibitory signal. The inhibition is projectedchiefly to the ipsilateral lateral superior olive (LSO), whichalso receives excitatory input from spherical bushy cells (SBCs)of the ipsilateral cochlear nucleus. Many LSO cells, then, areexcited by stimulation of the ipsilateral ear and inhibited bystimulation of the contralateral ear. As a consequence, they becomethe initial point at which sensitivity to interaural level disparitiesis processed.

The excitatory GBC input to the MNTB is in the form of large somatic terminals known as the calyces of Held (Banks and Smith1992; Friauf and Ostwald 1988; Glendenning et al. 1985; Held 1893;Lenn and Reese 1966; Morest 1968; Smith et al. 1991; Spirou etal. 1990; Tolbert et al. 1982; Warr 1972). The size and somaticlocation of the calyx of Held recently has made it the focus ofa considerable amount of interest in the cellular/biophysicalneuroscience community where, for the first time in the CNS, wholecell patch-clamp recordings have been made from a presynapticterminal, sometimes while simultaneously recording from the postsynapticcell as well (Borst and Sakmann 1996; Borst et al. 1995; Forsythe1994; Takahashi et al. 1996). The calyceal recordings showed thepresence of specialized potassium and calcium conductances thatwould be appropriate for a synapse that has to accurately processhigh rates of spike activity (Borst et al. 1995; Forsythe 1994)but also indicated the existence of presynaptic metabotropic glutamatereceptors that may act to modify the output of this terminal (Takahashiet al. 1996). Both in vitro sharp and patch electrode recordingsfrom rodent MNTB cells (Banks and Smith 1992; Banks et al. 1993;Borst et al. 1995; Brew and Forsythe 1995; Forsythe and Barnes-Davis1993a,b; Wu and Kelly 1991) indicates that the mature calycealinput acts primarily on non-N-methyl-D-aspartate glutamatergicreceptors to generate a large, fast suprathreshold synaptic response.The rapid repolarization of the synaptic response, allowing thesecells to follow their inputs at high rates, and the tendency ofthese cells to fire once to sustained depolarizing current are,in part, due to a dendrotoxin sensitive, low-threshold potassiumconductance. A similar conductance has been reported for the bushycells that provide the calyceal input to MNTB (Manis and Marx1991; Oertel 1983; Wu and Oertel 1984). A second fast, high-thresholdpotassium conductance also is present in MNTB cells serving torapidly repolarize the action potential (Brew and Forsythe 1995).Thus MNTB is part of an afferent chain with morphological andphysiological specializations for temporally precise transmissionof signals.

As described above, a rather extensive investigation of both the anatomic and physiological features of the calyceal inputand the MNTB cell has been made in brain stem slices. However,several major gaps remain in our knowledge of the function ofthese cells in vivo. First, the response features of MNTB principalcells to simple auditory stimuli have not been unequivocally established.The only published study of recordings from positively identified $---$ andsubsequently labeled $---$ principal cells comes from the rat MNTB (Sommeret al. 1993). In this paper, the authors recorded intraaxonallyfrom 11 MNTB principal cells and successfully injected them withhorseradish peroxidase (HRP), labeling cell body and dendritictree as well as much of the axonal field. However, the brief recordingtimes allowed only a very limited measurement of the responsesof these cells to auditory stimuli: the characteristic frequency(CF, the frequency at which the threshold intensity was the lowest)of only six cells was determined, and only 10 stimulus trialswere used to generate short tone responses to the CF (STCF responses),making it impossible to determine their physiological responsetype, i.e., primarylike (PL; a response resembling auditory nervefibers), primarylike-with-notch (PL_N: a response resembling theglobular bushy cell) or phase-locked (cells of low CF with spikesoccurring at a particular phase of a low-frequency stimulus cycle).The remaining in vivo studies of MNTB have used extracellularmetal electrodes (Guinan and Li 1990; Guinan et al. 1972a,b; Liand Guinan 1971; Tsuchitani 1994, 1997) and indirect evidence,the presence of a large prepotential, to identify the recordedcells. With metal electrode recordings, many cells in the vicinityof the MNTB exhibit spikes with complex waveforms, so-called prepotentialcells. Based on similar recordings from the cochlear nucleus thatwere proposed to arise from the large auditory nerve endbulb ofHeld terminal/bushy cell complex (Pfeiffer 1966), Guinan and Li(1990) postulated that these waveforms arose from the calyx/MNTBprincipal cell complex and consisted of a prepotential in thepresynaptic calyx followed by a postsynaptic spike. Responsesto short tones from cells displaying such spike waveform wereeither primarylike, primarylike-with-notch or phase-locked. SubsequentlyTsuchitani, (1994, 1997) categorized cells in the vicinity ofMNTB as being MNTB principal cells based on the presence of aprepotential and/or a PL or PL_N short tone response. Given thelarge number of fibers in the trapezoid body coursing directlythrough the MNTB with PL and PL_N responses (Smith et al. 1991,1993b), we believe the presence of a prepotential is essentialfor positive identification of extracellular recordings from MNTBcells. Furthermore, while it is generally thought that MNTB cellsare monaurally driven by the contralateral ear, there are no documentedinteraural level difference functions in the literature for MNTBcells documenting this characteristic.

A second gap exists regarding the surprising observation in several rodents, bats, and in cat (Adams and Mugnaini 1990; Banksand Smith 1992; Kuwabura and Zook 1992; Kuwabura et al. 1991;Smith 1995; Sommers et al. 1993) that MNTB cells project to themedial superior olive (MSO), a nucleus containing cells that arethought to be comparing the time of arrival of the excitatoryinputs from the two ears (Yin et al. 1997). All but one of thesestudies were in vitro so the auditory response features (CF, STCFresponse, spontaneous rate) of these MSO-projecting MNTB cellscould not be assessed. In the one in vivo study (Sommers et al.1993), no auditory responses were recorded from the one labeledMNTB cell that projected to MSO. The excitatory input to the MSOcomes bilaterally from the spherical bushy cells of the anteroventralcochlear nucleus (AVCN), cells that exhibit enhanced synchronizationas compared with their auditory nerve input to low CF tones (Joriset al. 1994a). It is essential to know the character of the inhibitoryinput to these cells from the MNTB if we are to understand theprocessing of interaural time disparities in the MSO and interaurallevel differences in the LSO.

A third gap in our knowledge of the characteristics of these cells is in the anatomy of their synaptic input and output. Forthe input, early electron microscopic studies (Jean-Baptiste andMorest 1975; Lenn and Reese 1966; Morest 1968, 1973) describedthe calyceal ending and mentioned that there were other "noncalycealterminals" on the cell body, but no measures were made of theextent of these terminals, and, because of the unlabeled natureof the cells, a description of the synaptic input to only themost proximal dendrites could be given. For the MNTB output, severallight microscopic studies have described the projection patternof MNTB axons in various species (Banks and Smith 1992; Kuwaburaand Zook 1991, 1992; Schofield and Cant 1992; Sommer et al. 1992),but no electron microscopy was done to confirm or describe theterminal morphology. Cant (1984) reported that in the LSO (a majorrecipient of MNTB axon collaterals), almost three-fourths of thesurface of the LSO principal cell body and proximal dendritesare covered with synaptic terminals that are almost exclusivelythose containing small vesicles many of which are flattened orcylindrical. She proposed that these terminals arose from MNTBaxons but admitted that it remained to be demonstrated experimentally(Cant 1984). Thus the ultrastructural identification of the axonterminals of identified MNTB principal cells has never been unequivocallymade.

Our goal in these in vivo experiments was to answer the questions described above by characterizing the basic response featuresof cat MNTB cells, with glass electrodes, and to subsequentlylabel the cell with either HRP or neurobiotin. Labeled cells couldbe studied at the light and electron microscopic level to examinethe morphology of the axonal and dendritic tree as well as thedistribution of synaptic inputs and features of the output terminals.

Methods

Many of the methods used for these experiments have been described in detail elsewhere (Smith and Rhode 1985; Smith et al.1991, 1993b) and are summarized here. Animals were maintainedin an American Association for Accreditation of Laboratory AnimalCare (AAALAC)-approved animal care facility, and all methods havebeen approved by the University of Wisconsin Institutional AnimalCare And Use committee.

Surgical procedure

Young adult cats were anesthetized and maintained in an areflexive state with pentobarbital sodium (35 mg/kg). Pinnae wereremoved, and the external auditory meati cut transversely forinsertion of metal ear pieces through which acoustic stimuli,calibrated from 60 Hz to 40 kHz, were delivered. The MNTB wassurgically approached from the ventral surface of the brain stem.A small hole was drilled in the basioccipital bone just lateralto the pyramidal tract, and a slit was made in the dura. The smallrootlets of cranial nerve VI served as an external landmark forthe more deeply located MNTB.

Acoustic stimuli and data collection

Calibrated acoustic stimuli generated by a computer-controlled digital stimulus system (Rhode 1976) were delivered from Telex140 earphones or Radioshack supertweeters. Spike-triggered pulseswere sent to a unit event timer and stored for on-line and subsequentanalysis. As the electrode was advanced toward the MNTB, a searchstimulus of short tone bursts with variable frequency was presentedto both ears until an axon or a cell body was encountered.

Glass electrodes $---$ recording and injection

Intracellular glass electrodes were filled either with a buffered (pH 7.6), filtered 5% HRP (Sigma) solution in 0.5 M KClor a 2% Neurobiotin (Vector Labs) solution in 0.5 M KCl. The electrodewas lowered over the hole drilled in the skull and advanced in1-µm steps. Extracellular and intracellular signals were monitoredusing standard techniques for DC monitoring, amplification, filtering,and display. Recordings from MNTB cells using the glass microelectrodescould only be verified by subsequent location and identificationof the labeled cells. However, some features of axonal responseswere used to distinguish presumed MNTB axonal responses from thoseof other axons: 1) because the primary excitatory input to a MNTBcell arises from a globular bushy axon from the contralateralcochlear nucleus, only units driven from the contralateral earwere considered. 2) Because of the interposing synapse betweenglobular bushy axon and MNTB cell, first spike latencies takenfrom peristimulus time histograms (PSTHs) to STCFs should be slightlylonger for a MNTB cell axon than for a bushy cell axon with thesame characteristic frequency.

While tones at the unit's CF were presented and the response monitored, entry into the axon, as signaled by a DC shift offrom $-$ 30 to $-$ 60 mV, was accomplished with 100-ms current pulses.After physiological characterization of the cell (see further)HRP or neurobiotin was injected for 2-10 min using 100 ms, 1-5nA current pulses while CF tones were presented continuously.Current injection was terminated if the response during pausesin the injection changed, which would indicate that the electrodehad slipped into a different cell. After data collection, theelectrode was withdrawn, the DC shift noted and a 10-mV calibrationpulse recorded on the intracellular channel of the tape monitor.

Metal electrodes $---$ recording

Commercially available tungsten metal electrodes (Microprobe, 10-20 µM exposed tips, 5 M $Omega$ impedances) were used in separateextracellular experiments. Electrode placement, method of advance,stimulus presentation, and unit response characterization wereas described above for glass electrodes. Metal electrode recordingsfrom MNTB cells were distinguished by their prepotentials (Guinanand Li 1990; Guinan et al. 1972a,b; Li and Guinan 1971) and bysubsequent histological location of the recording site from lesionsmade at several points in each penetration (except for 4 cellsin 1 animal where histology was not available).

Tissue processing

For metal electrode experiments, we perfused the animal with formalin shortly after a lethal dose of pentobarbital sodiumwas administered after the last penetration. For the HRP/Neurobiotinexperiments, the animal was maintained in an areflexive statefor 24-36 h after the last penetration using a drip solution ofsodium pentobarbital (3% in 5% dextrose) while heart rate, respiration,and withdrawal reflexes were checked every 20 min by one of theexperimenters. Wound areas were swabbed at 2-h intervals withlidocaine. After a lethal dose of sodium pentobarbital, the catthen was perfused transcardially with saline followed by two concentrationsof phosphate-buffered, calcium-containing glutaraldehyde:paraformaldehydefixative (0.01:0.01 and 0.02:0.01), and the brain stored eitherin sucrose buffer (for frozen sectioning) or 1:1 glutaraldehyde:paraformaldehydefixative (for vibratome sectioning).

For the HRP injections, the following steps were taken. Coronal or horizontal sections were cut at 60- or 70-µm thicknesswith a vibratome or after freezing and then were reacted usingthe 3,3'-diaminobenzidine (DAB)-nickel/cobalt intensificationmethod (Adams 1981). For light microscopy, sections were mountedon glass slides, counterstained with cresyl violet, and coverslipped.For electron microscopy, vibratomed, HRP-reacted sections werefixed in 2% osmium tetroxide, dehydrated, and flat-embedded inplastic resin. After a camera lucida drawing of the injected axonwas made, thin sections were taken, counterstained with uranylacetate and lead citrate, and observed with a JOEL 100CX electronmicroscope.

For the Neurobiotin injections, the following steps were taken. Vibratomed 70-µm section were cut into 0.1 M phosphate buffer,washed, incubated in 0.5% H₂0₂ in phosphate buffer, then reactedovernight in ABC reagent (Vector ABC kit, in 0.1 M phosphate buffer,pH 7.4 containing 0.3% TritonX, 2% bovine serum albumin, 2 dropsof A and 2 drops of B reagent of ABC kit/10 ml). The next daythe sections were rinsed in 0.1 M phosphate buffer and reactedusing the Adams (1981) DAB-nickel/cobalt intensification methodthen prepared for light and/or electron microscopy as describedabove.

Data analysis

PHYSIOLOGY. When a cell or axon was isolated, stimuli were presented to each ear separately to determine which ear(s) drove the unit.For all cells, we determined the characteristic frequency (CF,the frequency at which the threshold intensity was the lowest),spontaneous discharge rate, and Q₁₀ (CF/bandwidth at 10 dB abovethreshold) using an automated threshold tuning curve program.We also measured the STCF at 10- to 20-dB intervals from nearthreshold to 50-70 dB above threshold using 100 or 200 repetitionsof 25-ms tones presented every 100 ms with a 3.9-ms rise/falltime. Amplitude-modulated and click stimuli also were used butthe data will not be presented here.

From the STCF response, we derived the mean and standard deviation of first spike latency (see Smith et al. 1991), mean coefficientof variation over the interval 12-18 ms (Young et al. 1988), andthe sustained discharge rate and synchronization coefficient (Goldbergand Brown 1969) during the last 15 ms of the tone burst. To correctfor spontaneous activity in the computation of the mean and standarddeviation of the first spike latency, we eliminated all spikesin the time window from 0 to 2.4 ms after stimulus onset and alltrials in which a spike occurred in the last half of this window.

ANATOMY. Labeled axons were drawn in the coronal or horizontal plane using a camera lucida with ×63 oil objective. Measurements ofthe features of terminals on MNTB principal cell bodies and dendriteswere quantified using a Summagraphics bit pad Two graphics tabletinterfaced to the same microVax computer used to generate auditorystimuli.

Results

General

The recording configurations used to collect the MNTB cell data described in this paper as well as to collect data from theglobular bushy cells, which have been reported previously (Joriset al. 1994a; Smith et al. 1991) and which are used here for comparativepurposes, are illustrated in Fig. 1, top.

Using metal electrodes, we have recorded from 37 cells that displayed complex waveforms (Guinan et al. 1972a) and that weresubsequently localized to the MNTB by electrode-generated lesionsat the recording site. Typical complex waveforms recorded by extracellularmetal electrodes in the MNTB are shown in Fig. 1. They usuallyconsisted of a negative-going prepotential, that varied in amplitudefrom unit to unit, followed ~0.5 ms later by a larger bipolarspike, which was used to trigger the unit event timer. Althoughsound-driven spikes always were preceded by a prepotential, spontaneouslyoccurring spikes (those not generated by our sound system) unaccompaniedby a prepotential were occasionally seen (n = 3 cells; see Fig.1, bottom 2 panels).

When recording extracellularly with metal electrodes in the vicinity of the MNTB, we used the presence of the prepotentialas an indicator that we were recording from a MNTB principal cell.Invariably, "prepotential" cells were driven monaurally from thecontralateral ear and did not respond to stimulation of the ipsilateralear. In all cases, this was confirmed by subjective comparisonof the responses (over the audio monitor) to monaural stimulationof the left and right ears as well as binaural stimulation, at60-70 dB SPL to each ear. In a small number of cases, we verifiedthe lack of binaural input by stimulating the contralateral earwith CF tones at a constant level while varying the ipsilateralSPL over 30-80 dB (Fig. 2). In Fig. 2, the results from four MNTBcells are plotted as interaural level difference (ILD) functions,where ILD is defined as the contralateral minus ipsilateral SPL.For comparison the ILD functions of nine binaural IE cells (cellsthat were inhibited by contralateral stimulation and excited byipsilateral stimulation) in the neighboring LSO are shown. Thisfigure clearly illustrates that the response of the MNTB cellis independent of ipsilateral SPL.

With glass electrodes, we have recorded from and injected eight MNTB principal cells. On the basis of features typically usedto identify an electrode penetration site (flocculent extracellularreaction product and/or axonal swelling at the point of electrodeentry) and the monopolar shape of the action potential, recordingsfrom injected units have thus far been judged as intra-axonal.Extracellular spikes displaying prepotentials were never seenusing glass microelectrodes. Axonal penetration, while short tonesat CF were presented, was signaled by a DC shift of 30-60 mV.Four of the injected cells were well labeled with the dendritictree and the axonal projection field readily distinguishable.The other four were lightly labeled. Although the cell body shapeand location as well as primary dendritic tree and primary axoncollaterals of these cells were distinguishable, finer detailsof these could not be examined.

Anatomy

CELL BODIES. Previous extracellular studies of the cat superior olivary complex (Guinan et al. 1972b) indicated that the MNTB is tonotopicallyorganized, with low CF cells placed laterally and higher frequenciessituated progressively more medial with isofrequency strips angleddorsomedially to ventrolaterally. We found the absolute locationof our labeled MNTB cell bodies within the boundaries of the nucleushard to quantify. The MNTB boundaries are often difficult to distinguishespecially when sections are embedded in plastic but, in general,our data would seem to concur with this map. One labeled cellwith a very high CF was situated very medially, embedded in VIthnerve axon bundles. A single, labeled low best-frequency cellwas located considerably laterally and our other labeled cellswith intermediate CFs were located between these two extremes.

DENDRITIC TREE. Figure 3 illustrates the cell bodies and dendritic trees of our four well-labeled MNTB cells. Typically, the dendritic treearose from one or two large main dendrites that branched ratherprofusely within a single compact area much like the dendritictrees of globular bushy cells in the cochlear nucleus, which providethe main excitatory input to these cells. In one instance, however,(Fig. 3, soma at middle left), a single MNTB cell body gave riseto two main dendrites, one with the compact morphology and theother branching occasionally over a fairly extensive area. Thiscell also displayed the highly unusual feature of two main axonsarising from the cell body.

TERMINAL DISTRIBUTION. At the electron microscopic (EM) level globular bushy cell calyceal terminals form the major input onto the MNTB principalcell body. Figure 4 illustrates one such calyx that has been labeledby intraaxonal injection of the globular bushy cell axon givingrise to the calyx, synapsing on an unlabeled MNTB principal cell(Fig. 4, A and B) and another unlabeled calyx synapsing on a MNTBcell labeled by intraaxonal injection of its axon (Fig. 4C). Inselected EM sections, we measured the fraction of cell body anddendritic tree surface that was covered by synaptic profiles forthree of the well-labeled, plastic-embedded cells (top 3 cellsin Fig. 3). For the three cell bodies, 60.9, 66.6, and 68.4% ofthe total surface was covered with synaptic terminal profiles;39.6, 43.8, and 48% of the surfaces were covered with terminalscontaining round vesicles, whereas 21.3, 22.8, and 20.4% of thesurface were covered with terminals containing nonround vesicles.The remaining 39.1, 33.4, and 31.6% were terminal-free zones.The total amount of the surface of the proximal dendritic trees,within 100 µm of the cell body were also equally innervated with65.9% of the total surface covered and 35.5% of the surface coveredwith terminals containing round vesicles. Synaptic coverage droppedprecipitously on the distal dendritic tree (>100 µ from the cellbody) where only 15% of the surface was terminal-covered and only2% covered with round-vesicle containing profiles.

AXON. Our well-injected axons indicate that the projection of an individual MNTB cell axon can be quite extensive innervating anumber of auditory nuclei in the ipsilateral brain stem only.Figures 5-8 illustrate examples of projection patterns for labeledaxons. The primary sites of innervation of our MNTB cell axonswere the ipsilateral LSO and ventral nucleus of the lateral lemniscus(VNLL). In addition, collaterals often were seen with terminalswellings in periolivary regions, medial to the dorsal and ventralaspect of the MSO, which have been designated dorsomedial andventromedial periolivary regions (DMPO and VMPO) (Spangler etal. 1985, 1987). In some cases, these axons also had collateralsto MSO and the MNTB itself.

Cells whose axons were labeled sufficiently so that they could be followed as far lateral as the LSO (6/8) appeared to innervatethis structure. In these cases, the axon usually entered the LSOthrough either the dorsal or ventral hilus and branched withina fairly confined mediolateral space but rather extensively rostrocaudally(Figs. 5 and 8). In our one example of an injected low-frequencyMNTB cell, the axon coursed ventral and lateral to the LSO inthe fiber bundle surrounding the LSO until it reached the laterallow-frequency limb where it branched (Fig. 6). In another case,it was unclear whether the axon actually innervated the body ofthe LSO. This cell had a CF of 27,000 Hz and was cut in the horizontalplane (Fig. 7, left cell). It had a collateral branch that ranventral to the LSO and then headed dorsally immediately beneaththe LSO to branch just ventral to the area where the first signsof the ventral aspect of the LSO would appear in the next-mostdorsal section.

Of the cells with axons labeled darkly enough to reliably follow through the body of the MSO (6/8) three showed collateralswith terminals in the MSO (Figs. 6-8). Although our population issmall, this innervation appeared to be frequency dependent inthat the three cells with collaterals to MSO had CFs of 635, 6,300,and 13,000 Hz, whereas those without obvious collaterals had CFsof 13,500, 17,680, and 27,000 Hz. Innervation of MSO by MNTB collateralswas much less elaborate than that seen in the LSO with fewer branchesinnervating a smaller region of the nucleus. The cells that innervatedMSO did so in a tonotopic fashion with the low-frequency MNTBcell sending collaterals to the dorsal most aspect of the MSO,the intermediate CF MNTB cell innervating the proposed midfrequencyregion of the MSO, and the highest CF unit innervating the MSOvery ventrally. It is impossible to know, however, whether theCF of the MSO region innervated exactly matched the CF of theMNTB cell innervation.

Cells with well-labeled axons that could be followed for a considerable distance rostrally (5/8) all sent a collateral tothe VNLL (Figs. 6-8). This collateral would typically head rostrally,under the VNLL, and then head dorsally, directly beneath or rostralto the VNLL in the lateral lemniscus, sending multiple branchesinto the body of the VNLL.

We made measurements of the axon along its path at the light microscopic level. As described in a previous publication (Smithet al. 1993b), these represent measurements of the axoplasm anddo not include the myelin sheath and may be complicated by a numberof factors. In the vicinity of the cell body, the axon diameterappeared to be quite large. Before beginning to give off its majorcollaterals, the axon diameter was usually between 4 and 6 µm.After beginning to branch, the major collaterals that, for example,headed laterally to LSO or rostrally toward VNLL were typically3-4.5 µm and maintained this diameter until approaching near tothe nucleus. Subsequent secondary branching of one of these maincollaterals, for example when the major collateral projectingto the LSO began giving off major branches soon after enteringthe hilus, were usually 1.5-3 µm.

At the electron microscopic level, the main MNTB axon typically myelinated 30-40 µm from the cell body. The swellings alongand at the ends of myelinated collateral branches were terminalscontaining nonround synaptic vesicles. Figure 9 illustrates twosuch labeled terminals on the primary dendrite and cell body ofa principal cell in the LSO and represents the first direct evidenceof the terminal configuration of the MNTB principal cell axon.

The primary excitatory inputs to MNTB cells are the large somatic calyceal terminals that arise from GBC axons. Thus it wouldseem appropriate to make comparisons of MNTB cell responses withthose of GBCs that we have previously reported (Smith et al. 1991).

The CFs of our MNTB cell population ranged from 300 Hz to 36 kHz and thresholds at CF from 6 to 63 dB SPL (mean = 21 dB SPL)This compares with 300 Hz to 31 kHz and $-$ 1 to 53 dB for GBCs.Short tone responses were, in general, similar to GBC responseswith some noticeable variations. Figure 10 shows PSTHs from representativeMNTB cells over the entire frequency range and may be comparedwith the globular bushy cell response (Fig. 1 of Smith et al.1991). More than 1 kHz the onset component of the MNTB responseoften would be well timed at higher stimulus intensity levelsgiving the response a PL_N appearance. In some instances, however,the onset portion did not become well timed at any level (Fig.10, bottom left and 4th trace in right column). We also occasionallynoted what appeared to be a slight "sag" in the sustained portionof the short tone response (Fig. 10, bottom left and middle tracein right column).

Figure 11 shows the spontaneous spike rates (top) as well as the latency (middle), and standard deviation (bottom) of the shorttone-induced first spike of the extracellularly ( $open circle$ ) and intraaxonally( $square$ ) recorded MNTB cell population compared with the same datafrom our labeled globular bushy cell population. A number of comparisonsmay be made between populations. First, only a few of the MNTBcells had CFs <3 kHz (2/45) while this CF was represented muchmore extensively in our GBC population [~4% for MNTB cells vs.12/35 (34%) for GBCs]. Those two MNTB cells with CFs <3 kHz hadspontaneous rates of 0 spikes/s as did most of the GBCs populationwith such CFs (8/12 with 0 spikes/s and 11/12 <5 spikes/s). Morethan 3 kHz, the spontaneous rates tended to be higher for bothMNTB cells and GBC populations (mean = 27 spikes/s vs. 12.4 spikes/s,respectively). If the boundaries chosen in the auditory nerveby Liberman (1978) are used for distinguishing high, medium, andlow spontaneous rates (SRs) (18 and 1 spikes/s, respectively),the percentages of high, medium, and low SRs in MNTB cells were55, 27.5, and 17.5%, respectively, compared with 23, 46, and 31%for GBCs and 65, 20, and 15% for auditory nerve (AN).

As with GBCs, the latencies of MNTB cells decreased with increasing CF and, as might be expected given a requisite synapticdelay between GBC axons and MNTB cell axons, the MNTB cell latenciestended to be longer than GBC latencies for cells with similarCFs (Fig. 11B). Finally, as expected from the sharp onset responseof many MNTB cells, the timing of the first spike is quite precise(mean first spike standard deviation = 0.76 ms) although not asprecise as the GBC input (0.34 ms, Fig. 11C).

Discussion

Summary

We labeled eight MNTB cells intraaxonally after obtaining their physiological response properties. In agreement with previousqualitative anatomic descriptions (Jean-Baptiste and Morest 1975;Lenn and Reese 1966; Morest 1968, 1973), our measurements showthat calyceal coverage can take up one-fourth to one-half of thetotal surface of the cell body and primary dendrites. In addition,we have shown that nonexcitatory inputs also concentrate on thecell body and consistently cover around one-fifth of the surface,but the source of this input or means of activation is unknown.Our data also show that inputs with both excitatory and inhibitoryfeatures are rare on the distal dendritic tree. We also have shownfor the first time at the ultrastructural level that the terminalsof these MNTB axons contain nonround vesicles and, when observedin the LSO, terminate on the somata and primary dendrites of LSOprincipal cells.

As has been previously described in cat and other species (Banks and Smith 1992; Kuwabura and Zook 1991, 1992; Schofield andCant 1992; Sommer et al. 1992; Spangler et al. 1985), MNTB cellssend their axonal projections only to areas of the ipsilateralbrain stem, namely (in cat) the ventral nucleus of the laterallemniscus, the lateral and medial superior olives, the dorsomedialand ventromedial periolivary nuclei, and the MNTB. The only previousin vivo intracellular study (Sommer et al. 1992) indicated thatthe rat LSO receives MNTB inputs with best frequencies from 47kHz down to $>=$ 14 kHz. Our labeled cells show that, in the cat,this frequency range extends down to $>=$ 635 Hz. In addition, despitean admittedly small sample, our labeled cell data provides evidencethat the MNTB projection to the MSO may be frequency specific-cellswith CFs below <13 kHz innervate the MSO, cells with CFs >13 kHzdo not.

Physiologically our short tone data from cells, positively identified by intracellular staining, show that MNTB cell responsesare like those of globular bushy cells with similar best frequencies.We also have included physiological data from cells in the MNTBrecorded extracellularly that displayed prepotentials. Prior tothis, the only evidence that the complex spikes seen in the MNTBarose from the MNTB principal cell and its calyx was providedby Guinan and Li (1990). By stimulating either LSO or DMPO whilerecording a complex spike unit in the MNTB, they could antidromicallyactivate a spike, designated the "A" spike. This A spike resembledthe portion of the complex waveform, designated the "C2" component,that was believed to be the postsynaptic MNTB cell spike. Whenthe complex spike was activated by stimulation of the trapezoidbody at the midline, thus driving the globular bushy axon/calycealsynapse and presumably activating the MNTB principal cells, theantidromic A spike was refractory and could not be driven for~1 ms, implying that the A spike and C2 component were both generatedby the MNTB cell. We felt that this evidence, together with ourpositive identification of MNTB principal cells with physiologycomparable to the complex waveform units, was sufficient to permitour inclusion of the extracellular data.

Globular bushy cell input $---$ a comparison of responses

The data available from our MNTB cell sample show the results of what is apparently a good transmission of temporal informationacross the synapse. Globular bushy cells carry a variety of accuratelytimed information about low- as well as high-frequency auditorystimuli. Our previous reports (Joris et al. 1994a; Smith et al.1991) have shown that low-frequency globular bushy cells can phaselock to the stimulus waveform of a short tone at their CF withremarkable precision (as measured by the synchronization coefficient)and often will entrain (generate a spike for every cycle of thestimulus waveform). Because our sample of MNTB cells with CFs<1 kHz is so small, it is difficult to compare GBC and MNTB cellpopulations in terms of phase locking capabilities. Neverthelessthe positively identified MNTB cell with a CF <1 kHz showed "enhanced"synchrony, i.e., its maximum synchrony (0.89) was high relativeto that of most auditory nerve fibers at that frequency. Also,maximum synchronization for the one MNTB cell in our sample witha CF between 1 and 3 kHz was at the lower edge of the range observedin the auditory nerve. These observations are consistent withsynchronization in GBCs, which also show enhanced synchronizationat CFs <1 kHz and poor synchronization at intermediate CFs (1kHz < CF < 3 kHz). GBCs with CF >3 kHz show enhanced synchronizationto tones in the low-frequency tail of the tuning curve (Joriset al. 1994b), but we did not test this in MNTB cells. As seenin Fig. 10, the onset component of the MNTB cell responses waswell timed, although as a population not as well timed as globularbushy cells (Fig. 11C). Finally, GBC and MNTB cells show a slightimprovement in envelope phase-locking compared with the auditorynerve, and overall the two populations are indistinguishable intheir envelope phase-locking properties (Joris and Yin, unpublisheddata).

As described in RESULTS, the percentage of MNTB cells we recorded from, with CFs <3 kHz, was significantly lower (4%) thanthe percentage of globular bushy cells (34%) we previously recordedfrom with similar CFs. We feel that, even though the frequencymap of Guinan et al. (1972b) indicates that low frequencies areless well represented than higher frequencies in MNTB, 4% is probablynot a true reflection of the actual number of MNTB cells withCFs <3 kHz but is probably biased by our surgical approach andelectrode placement. In these experiments, the electrode was insertedinto the brain stem just lateral to the pyramidal tract. In someinstances, the electrode entered the brain near the rootlets ofthe abducens nerve perpendicular to the surface of the brain.Using this approach the more laterally situated, lower frequencyregions of the MNTB are not traversed. Even when we angled theelectrode so that it diverged from the midline with increasingdepth the lateral aspect of the MNTB was often still not reliablysampled. This would probably also explain the disparity in meanspontaneous rates between our GBC (12.4 spikes/s) and MNTB cell(27 spikes/s) populations. Many of the low CF globular bushy cellswe labeled with CFs <3 kHz had spontaneous rates of 0 spikes/s.Our two examples of MNTB cells with CFs <3 kHz also had spontaneousrates of 0 spikes/s, and it is likely that, had we sampled a higherpercentage of low CF MNTB cells, the means would be closer.

Globular bushy cell calyceal and other inputs

In our previous report (Smith et al. 1991), we noted that the labeled calyx of a globular bushy cell axon covered ~24% ofits postsynaptic MNTB cell body surface, while the total (labeledand unlabeled) round vesicle coverage of the cell body was 26.4%.In this case, the great majority of the excitatory input was thelabeled calyx, providing evidence that this cell received onlyone calyx. In the present study, the total round vesicle terminalcoverage for three of our labeled MNTB cell bodies was 39.6, 43.8,and 48%. What does this say about the excitatory synaptic inputto the MNTB cell body? 1) In the four cases, we have examinedin detail the extent of somatic coverage by excitatory terminalshas varied considerably, ranging from slightly more than one-fourthto slightly less than one-half. Thus if each MNTB cell is receivingonly one calyx, then there may be considerable variation in amountof surface of MNTB cells covered by one globular bushy calycealterminal. 2) Alternatively, some MNTB cells receive two calycesor 3) some MNTB cells receive one calyx and a lot of other noncalycealround vesicle inputs. These could potentially arise from the small"precalycean" terminals of other globular bushy cell axons thatmake smaller terminal endings in the MNTB (Morest 1968; Smithet al. 1991). The axons of other projection neurons of the cochlearnucleus, with round vesicle containing terminals, do not ventureinto the MNTB so they are unlikely sources for additional excitatoryinputs. These include the spherical bushy cells (Smith et al.1993b), the stellate cells the axons of which exit the cochlearnucleus via the trapezoid body and that respond in a chopper fashionto short tones (Smith et al. 1993a), the octopus cells of theposteroventral cochlear nucleus (PVCN) and the giant and fusiformcells of the dorsal cochlear nucleus (DCN) (Joris et al. 1992).Little information is available on the other potential sourcesof excitatory inputs to MNTB in the brain stem. Labeled MSO principalcells in the guinea pig (Smith 1995) never gave off collateralsto MNTB, and there is only one report of an occasional projectionto MNTB from labeled rat LSO principal cell the axon of whichcrossed the midline (Banks and Smith 1992).

We also measured the nonround vesicle coverage of the same four cell bodies. The nonround vesicle coverage of the cell receivingthe labeled calyx reported previously (Smith et al. 1991) was21.6%, and 21.3, 22.8, and 20.4% for the three cells in the presentstudy. Thus it would appear that the presumably inhibitory "nonroundvesicle" input to the soma is quite heavy and, at least in termsof surface coverage, is constant from cell to cell. Anatomically,it has been shown in cat and guinea pig that glycine immunoreactiveterminals are rather sparse in the MNTB (Adams and Magnaini 1990;Helfert et al. 1989; Wenthold et al. 1987) as is glycine receptorlabeling (Zarbin et al. 1981) while $gamma$ -aminobutyric acid (GABA)immunoreactive terminals are quite numerous (Adams and Magnaini1990). This agrees with our observations here and similar observationsin the rat (Banks and Smith 1992) that the MNTB cells, which arethe major source of glycinergic innervation to the auditory brainstem, only give off occasional sparsely branching collateralswithin the home nucleus. Little evidence is available as to thesource of any of the GABAergic terminals nor is there evidenceas to whether they are activated by auditory stimuli. Kuwaburaet al. (1991) described axon collaterals of cells in the rodentventral nucleus of the trapezoid body that could branch in MNTB,but it is not known what transmitter these cells manufacture.In vitro physiology in the mouse has shown that bath applicationof either GABA or glycine decreases the input resistance in onlyabout half of the mouse MNTB cells tested (Wu and Kelly 1995).Further in vitro data indicated that shock stimulation of thetrapezoid body at the midline or ipsilateral to the MNTB in therat (Banks and Smith 1992) but not the mouse (Wu and Kelly 1991)would occasionally evoke inhibitory postsynaptic potentials (IPSPs)that could be blocked by the glycine antagonist, strychnine. Ourin vivo data on the response of MNTB cells to binaural stimulationas the sound to the contralateral ear was held constant whilethe sound to the ipsilateral ear was increased (Fig. 2) showedno inhibitory affects on the contralaterally driven output. Inother in vivo experiments, Guinan and Li (1990) also could notdemonstrate any sort of inhibitory affect on the calyceal drivenMNTB cell output either by auditory stimulation to either earor by shock stimulation of the trapezoid body. Some of our MNTBshort tone responses show an apparent sag in the sustained response(Fig. 10), but it would be premature to propose an inhibitory inputas the cause. Thus inhibitory inputs on MNTB cells appear to bepresent and functional but there is no evidence of activationby simple auditory stimuli.

Calyceal and postsynaptic physiology

In addition to the anatomic specializations, some physiological specializations also apparently have developed both pre- andpostsynaptically to aid in the secure and rapid transfer of accuratelytimed information. On the basis of whole cell patch recordingsfrom calyceal terminals, the calyx possesses a large, high-voltage-activatedcalcium conductance with fast activation and deactivation kinetics(Borst et al. 1995) that would be required for rapid synaptictransmission. In addition, preliminary reports (Forsythe 1994)showed that, in current clamp, the presynaptic terminal can spikeat high rates in response to depolarizing current pulses becauseof a fast delayed rectifier potassium current that allows rapidspike repolarization. Such a rapid response capability also mightbe requisite for rapid synaptic transmission. The synaptic currentgenerated postsynaptically by the calyx is fast and very large,averaging ~5 nA (Borst and Sakmann 1996), but this current amplitudediminished significantly during repetitive stimulation such thatthe calyceal synapse could not make the MNTB cell fire reliablyto a 100-Hz shock stimulation of the bushy cell input. It wouldappear, however, that such poor postsynaptic following is probablya function of the immature preparation used (8- to 10-day-oldrats) because shock rates of the bushy cell input generating MNTBcell spikes were reliably followed to well over 500 Hz in a maturemouse slice preparation (Wu and Kelly 1993). Postsynapticallythe MNTB cell has specialized potassium conductances (Banks andSmith 1992). One has been designated I_TEA, which might allow forrapid spike repolarization, the other I_DTX, which might permitonly a single spike to occur in response to the huge calycealinput (Brew and Forsythe 1995).

MNTB cell output

Our anatomic data on the projections of MNTB principal cells correspond well with Spangler et al. (1985). With one exception,all axons that could be followed to LSO innervated that structurein a fashion that was consistent with the proposed frequency mapof the LSO (Guinan et al. 1972b). Our one example of a low-frequencyMNTB cell sending a projection to the low frequency limb supportsthe contention (Finlayson and Caspary 1991; Joris and Yin 1995)that some low-frequency LSO cells are inhibited by contralateralstimulation.

The terminals we examined at the EM level had nonround vesicles and terminated on large dendritic profiles or the cell bodiesof principal cells. Cant (1984) reported that almost three-fourthsof the surface of the LSO principal cell body and proximal dendritesare covered with synaptic terminals that are almost exclusivelythose containing small vesicles many of which are flattened orcylindrical. Our labeled synaptic terminals from identified MNTBprincipal cells correspond to the description both in locationand vesicle content (Cant 1984) and provide the first direct evidencethat these terminals are derived from axons of MNTB cells. Inthe rat, Moore and Caspary (1983) showed that the contralaterallyevoked inhibition of ipsilaterally evoked excitation could beblocked by application of the glycine antagonist strychnine. Similarresults were obtained in the slice preparation, by Wu and Kelly(1991), with IPSPs evoked by electrical activation of MNTB. Thusour anatomic data, showing that the axon terminals of MNTB cellsto the LSO have nonround synaptic vesicles, provide an importantpiece of data linking the presumed inhibitory function of MNTBonto ipsilateral LSO to the anatomic data of Cant (1984) showingflattened vesicles on the soma and proximal dendrites of LSO cells.

For axons that could be followed through MSO the ones with the three lowest CFs sparsely innervated the nucleus in appropriatefrequency regions, whereas the three with the higher best frequenciesdid not. Clark (1969) and Schwartz (1980) have reported the presenceof terminals with flattened vesicles on principal cells in catMSO. The innervation of MSO by MNTB cells also has been notedanatomically in rat, bat, mouse, gerbil, and guinea pig (Banksand Smith 1992; Kuwabura and Zook 1991, 1992; Smith 1995), andIPSPs, evoked by MNTB shock, in MSO principal cells were blockedby strychnine (Grothe and Sanes 1993, 1994; Smith 1995), confirmingthe glycinergic nature of the input. The function of such an activelydriven inhibitory input to the MSO from MNTB remains speculative.Several possibilities include a MNTB input the CF of which isslightly off the CF of the innervated MSO cell could serve tosuppress coincident excitatory inputs at non-CF frequencies; mediationof precedence-like effects; and changing the MSO cells restingmembrane potential during contralateral ear stimulation.

All MNTB neurons with darkly labeled axons had a collateral that could be followed rostrally to VNLL. Unfortunately nothingis known about the distribution of synaptic terminals on cellsin the VNLL or their ultrastructure nor is there any informationon why VNLL cells might require a contralaterally driven inhibitoryinput when much of the excitatory input to this nucleus is alsofrom the contralateral side. Collaterals also were sent to thetwo nuclei medial to the MSO, the DMPO and VMPO. In the cat, GBCsoften send an excitatory collateral to synapse on cells in theDMPO (Smith et al. 1991), so, as with the VNLL, we again notea region receiving both a contralateral excitatory input fromcochlear nucleus and an inhibitory input from MNTB, a nucleusthat is driven by contralateral excitatory input. The rodent superiorparaolivary nucleus (SPN) is a large periolivary nucleus dorsomedialto the MSO that is considered the homolog of the cat DMPO. Itreceives input from both cochlear nuclei (Thompson and Thompson1991) as well as a strong topographic input from MNTB principalcells (Banks and Smith 1992), and its cells project primarilyeither to the inferior colliculus or cochlear nucleus (Schofield1991). Although some extracellular recordings were done in vivoon cells in the gerbil SPN and cat DMPO (Guinan et al. 1972a;Spitzer and Semple 1995), the physiology of a large populationof cells in this nucleus has never been explored in great detailand consequently the possible effects of the MNTB input, on auditoryresponse properties of cells here, are not known. We also occasionallynoted a collateral being given off within the MNTB and, as notedabove, inhibitory synaptic events can be elicited in rat MNTBcells by shocking their bushy cell input (Banks and Smith 1992),but it is not clear that these were due to collaterals of MNTBcells.

Miscellaneous

ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants DC-01999 and DC-00116 and National Science Foundation Grant BNS-8901993.

FOOTNOTES

Address for reprint requests: Philip Smith, Dept. of Anatomy, University of Wisconsin Medical School, Madison, WI 53706.

Received 3 November 1997; accepted in final form 20 February 1998.

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Figures

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Figure 1 Top: diagrammatic representation of the recording configurations from a medial nucleus of the trapezoid body (MNTB) principalcell or its globular bushy cell input. Globular bushy cell axonswere recorded extra or intra-axonally, on the same or oppositeside as the MNTB that they innervated, as previously described(Joris et al. 1994a; Smith et al. 1993b). MNTB cells were recordedfrom either intra-axonally with glass electrodes or extracellularly,with metal electrodes in the vicinity of the presynaptic calyx/postsynapticcell body complex. Bottom: extracellular metal electrode recordingsfrom the MNTB. Waveform showed a prepotential (P.P.) presumedto arise from the calyx, followed by a MNTB cell action potential.Lower 2 traces: sound-driven MNTB action potentials, precededby a prepotential, are superimposed on a trace, taken in the absenceof intentional sound stimuli, showing no prepotential (No P. P.).Scales at top of columns apply to all traces in that column.

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Figure 2 Normalized short tone interaural level difference (ILD) functions for 4 cells localized to the MNTB that displayed prepotentials(encompassed by shaded area below "MNTB") and 9 cells localizedto the lateral superior olive (LSO; encompassed by shaded areanext to "LSO"). Tones at CF were used for all cases shown. Forthe MNTB recordings, contralateral stimulus was held constant20-30 dB above threshold while the intensity of the ipsilateralstimulus was varied around 0 ILD. For the LSO cells, we held theipsilateral level constant at 20-30 dB above threshold and variedthe contralateral level.

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Figure 3 Camera lucida drawings of 4 labeled MNTB cells. Three cells on the left are drawn in the horizontal plane, whereas the cellon the right is drawn in the coronal plane. *, swelling wherethe electrode penetrated the axon and the axon distal to the recordingsite is much thinner than the axon between the injection siteand the cell body. Scale bar at top right applies to all cells.

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Figure 4 A: electron micrograph of an unlabeled MNTB principal cell receiving synaptic input from an intraaxonally labeled globularbushy cell axon. In thin section, the fingerlike tendrils of thecalyceal terminal are cut in cross-section (*). B: micrographof the same MNTB cell at a level where the labeled globular bushyaxon (curved dark arrows) loses its myelin sheath (curved openarrow) close to the origin of the calyx, then surrounds the cellin a "hand-gripping-a-ball"-like fashion, making multiple synapticcontacts (*). C: micrograph of MNTB cell labeled by intraaxonalinjection. A large unlabeled terminal (curved arrows) containinground vesicles is presumably part of the globular bushy calycealterminal. Scale bar in C = 5 µm and refers to all micrographs.

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Figure 5 Camera lucida recontruction of a high (17.7 kHz) characteristic frequency principal cell, in the right MNTB, sectioned inthe coronal plane (see also Fig. 3). Top left: higher power drawingof the cell body, dendritic tree and initial portion of the axon.*, penetration site of the electrode. Bottom left: low power reconstructionof the same cell and its axon collateral field. Location of someof the innervated nuclei appear out of place on this 2-dimensionaldrawing because of the large rostro-caudal distance traversedby the axon. Axon gave off numerous collaterals innervating anappropriate frequency region of the LSO [given the characteristicfrequencies (CF)], MNTB, dorsomedial and ventromedial periolivaryregions (DMPO and VMPO), and ventromedial region of the ventralnucleus of the lateral lemniscus (VNLL) before fading in the LL.Bottom right: short tone response of this cell at its CF (seealso Fig. 10).

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Figure 6 Right: camera lucida reconstruction of a low (635 Hz) characteristic frequency MNTB cell, located in the right MNTB, sectionedin the coronal plane. Cell body was lightly stained and only themain dendrite and 2nd-order branches could be seen so the dendritictree is not illustrated. Axon sent collaterals into the low-frequency,lateral limb of the LSO as well as into the dorsal, low-frequencyregion of the medial superior olive (MSO) at the 2 rostrocaudallocations illustrated. Branches were also seen in the VMPO. Anumber of collateral branches appeared to fade before termination,so this cell may have innervated other areas as well. Two profilesof the MSO represented are from 2 sections that contained collateralbranches in this nucleus at 2 rostrocaudal locations. LSO profilerepresents the outline of the LSO in the section that containedthe bulk of the collateral branches in the lateral limb of theLSO. Thus only these parts of the axon collateral branches areplaced accurately in the drawing relative to the illustrated locationsof the LSO and MSO. Scale bar = 500 µm. Inset, bottom left: shorttone response of this cell at its characteristic frequency (seealso Fig. 10).

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Figure 7 Camera lucida drawings of 2 MNTB cells in the horizontal plane illustrating the extent of the axon collateral fields. Cellbody of the cell on the left (CB) was located in the left MNTB.CF of the cell was 27 kHz. Dendritic tree of this cell has notbeen drawn in this figure, to eliminate confusion of dendriteswith axon collateral branches, but is illustrated in Fig. 3 (topleft cell). Destination of the main axon branches are indicated(to LSO, to VNLL). Other collaterals are in MNTB and VMPO. Cellbody of the cell on the right (CB) was located in the right MNTB.CF of the cell was 13 kHz. Destination of main collaterals areindicated (to LSO, to VNLL). Collateral branches between the cellbody and the to-LSO label are primarily in the MSO. Enlarged versionof the cell body and dendritic tree of this cell are illustratedin Fig. 3 (bottom left). Scale bar = 1 mm.

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Figure 8 Camera lucida of another MNTB cell in the horizontal plane. CF of the cell was 6,300 Hz. Left: drawing of cell body (CB) dendritictree and the axon collateral system. Cell body and dendritic treeof this cell also are illustrated in Fig. 3. Scale bar = 500 µm.Middle: low power reconstructions of 3 horizontal sections ofthe right superior olivary complex representing the dorsal, intermediate,and ventral aspects of the LSO (top to bottom). Asterisk and arrowsin top drawing indicate the dorsal course of the MNTB cell collateralas it enters the LSO. Middle and bottom: shaded portions of theLSO indicate the location of MNTB collateral branching and terminalswellings within the LSO. Scale bar = 2 mm. Right: higher powerreconstruction of the MNTB axon collateral and branches withinthe LSO. Scale bar = 100 µm.

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Figure 9 A: electron micrograph of an unlabeled cell in the LSO (lso cell) receiving a labeled MNTB synaptic terminal on the cell body(curved arrow) and a primary dendrite (d, curved arrow). B: micrographof the myelinated MNTB axon collateral (a) that gave rise to thelabeled terminal (*) on the LSO cell dendrite (d). C: labeledMNTB axon terminal (*) on the cell body of the LSO cell (cb).Scale bar in A = 5 µm. Scale bar in B = 1 µm and applies to Band C.

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Figure 10 Representative short tone responses from axons of positively identified MNTB cells (*) or cells showing prepotentials in extracellularrecordings localized to the MNTB, for a wide range of characteristicfrequencies. Responses are to 200 tones (25 ms each) at the characteristicfrequency of the unit, which is indicated at the top right ofeach panel. *, cell was labeled. Decibel value below the CF isthe level above threshold that the tones were presented. Bin width= 0.1 ms.

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Figure 11 Comparison of the spontaneous activity (top), 1st spike latency (middle), and 1st spike standard deviation (bottom) betweenlabeled globular bushy cells, that we have previously reported(Smith et al. 1991) and MNTB cells. Labeled MNTB cells (MNTB)were recorded from intraaxonally. Unlabeled MNTB cells recordingsusing metal electrodes (PP) were identified based on the presenceof a prepotential (see Fig. 1) and subsequent location of an electrodegenerated lesion site within the confines of the MNTB. Globularbushy cells axons were either recorded on the side ipsilateralor contralateral to their parent cell body.

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