such as "Introduction", "Conclusion"..etc
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.
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.
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.
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.
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 PLN 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 () and intraaxonally() 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).
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