Spectral Integration by Type II Interneurons in Dorsal Cochlear Nucleus

George A. Spirou,1 Kevin A. Davis,2 Israel Nelken,3 and Eric D. Young2

 1Department of Otolaryngology, West Virginia University School of Medicine, Morgantown, West Virginia 26506-9200;  2Department of Biomedical Engineering and Center for Hearing Sciences, Johns Hopkins University, Baltimore, Maryland 21205; and  3Department of Physiology, Hebrew University-Hadassah Medical School and the Interdisciplinary Center for Neural Computations, Hebrew University, Jerusalem, Israel


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Spirou, George A., Kevin A. Davis, Israel Nelken, and Eric D. Young. Spectral Integration by Type II Interneurons in Dorsal Cochlear Nucleus. J. Neurophysiol. 82: 648-663, 1999. The type II unit is a prominent inhibitory interneuron in the dorsal cochlear nucleus (DCN), most likely recorded from vertical cells. Type II units are characterized by low rates of spontaneous activity, weak responses to broadband noise, and vigorous, narrowly tuned responses to tones. The weak responses of type II units to broadband stimuli are unusual for neurons in the lower auditory system and suggest that these units receive strong inhibitory inputs, most likely from onset-C neurons of the ventral cochlear nucleus. The question of the definition of type II units is considered here; the characteristics listed in the preceding text define a homogeneous type II group, but the boundary between this group and other low spontaneous rate neurons in DCN (type I/III units) is not yet clear. Type II units in decerebrate cats were studied using a two-tone paradigm to map inhibitory responses to tones and using noisebands of varying width to study the inhibitory processes evoked by broadband stimuli. Iontophoresis of bicuculline and strychnine and comparisons of two-tone responses between type II units and auditory nerve fibers were used to differentiate inhibitory processes occurring near the cell from two-tone suppression in the cochlea. For type II units, a significant inhibitory region is always seen with two-tone stimuli; the bandwidth of this region corresponds roughly to the previously reported excitatory bandwidth of onset-C neurons. Bandwidth widening experiments with noisebands show a monotonic decline in response as the bandwidth increases; these data are interpreted as revealing strong inhibitory inputs with properties more like onset-C neurons than any other response type in the lower auditory system. Consistent with these properties, iontophoresis of inhibitory antagonists produces a large increase in discharge rate to broadband noise, making tone and noise responses nearly equal.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Neurons of the dorsal cochlear nucleus (DCN) are notable for the complexity of their response maps, especially their prominent inhibitory inputs (Evans and Nelson 1973; Greenwood and Maruyama 1965; Young and Brownell 1976). Many of the sound-evoked responses of the principal cells of the DCN, especially those classified physiologically as type IV units, are attributable to activity in a particular inhibitory interneuron called a type II unit (Voigt and Young 1980, 1990). The type II unit exhibits a unique set of physiological responses, principally defined by its near zero spontaneous activity and its robust response to tonal stimuli relative to broadband stimuli (Davis et al. 1996; Joris 1998; Young and Brownell 1976; Young and Voigt 1982).

The most likely anatomic correlate of type II units is the vertical, or tuberculoventral, cell in DCN. Vertical cells are located in the deep DCN, in a thick band beneath the pyramidal cell layer (Lorente de Nó 1981; Zhang and Oertel 1993b). They are characterized by small somata, vertically oriented dendrites, and immunocytochemical staining for the inhibitory neurotransmitter glycine (Kolston et al. 1992; Osen et al. 1990; Saint Marie et al. 1991). Vertical cell axons project within their isofrequency sheet in DCN and also send a collateral to the ventral cochlear nucleus (VCN) (Lorente de Nó 1981; Zhang and Oertel 1993b). The projections in DCN make inhibitory contacts on principal cells, as inferred from the patterns of inhibitory postsynaptic potentials (IPSPs) evoked by stimulation of the auditory nerve (Zhang and Oertel 1993a, 1994). In addition, both bushy and multipolar cells of the VCN have been shown to receive a glycinergic input from the region containing vertical cells in the DCN (Wickesberg and Oertel 1990), so that inhibitory effects from vertical cells are probably also important in VCN. Although DCN structure varies considerably across species, the vertical cell system seems to be a stable feature of its organization (e.g., cat, Osen et al. 1990; guinea pig, Kolston et al. 1992; Saint Marie et al. 1991, bat, Kemmer and Vater 1997; and baboon, Moore et al. 1996), suggesting that this inhibitory system is a fundamental part of signal processing in the mammalian cochlear nucleus.

The correspondence between type II units and vertical cells is based on three lines of evidence: the first is their common identity as inhibitory neurons (Davis and Voigt 1997; Voigt and Young 1980, 1990); the second is the fact that type II units do not project out of the nucleus through the dorsal acoustic stria (Joris 1998; Young 1980) but can be antidromically activated from the VCN (Young 1980); the third is the location of type II recording sites in the deep DCN in the general location of vertical cells (Ding and Voigt 1997; Voigt and Young 1990; Young and Voigt 1982; this paper). Attempts to identify type II units by dye filling have given mixed results (Ding et al. 1994; Joris and Smith 1995; Rhode 1999) but are generally consistent with the type II-vertical cell link.

The weak responses of type II units to broadband noise has led to the hypotheses that they are themselves the targets of substantial inhibitory input and that this input is more responsive to broadband than narrow band sounds (Spirou and Young 1991; Young and Voigt 1982). A similar inhibitory input is needed to account for certain properties of DCN principal cells (Nelken and Young 1994, 1997), and it is parsimonious to assume that the two inhibitory inputs come from the same neuron, the so-called wideband inhibitor. Neurons with properties appropriate to the wideband inhibitor, called onset-C units, have been recorded in the VCN (Joris 1998; Smith and Rhode 1989; Winter and Palmer 1995). The temporal properties of onset-C units have been shown to be appropriate for an inhibitory input to both type II and type IV neurons in DCN (Joris and Smith 1998). However, the spectral integration properties of type II units and onset-C units have not been critically compared as a test of the hypothesized inhibitory relationship.

In this paper, the spectral integration properties of type II units are described and compared with those of onset-C units, as reported by Palmer and colleagues (Jiang et al. 1996; Palmer et al. 1996). First, the definition of type II units is considered, in relation to a similar group of units, called type I/III. It is shown that a homogeneous type II group can be defined using conservative criteria, but that a definitive separation of the two unit types is not possible using the physiological measures that have been employed. Type II units that fit the conservative definition are shown to be more strongly inhibited by stimuli as the bandwidth is increased; this property parallels the increase in response observed in onset-C units as bandwidth increases. The broadband inhibition of type II units is shown to be blocked by either strychnine or bicuculline; with both antagonists, type II units give more nearly equal tone and noise responses. The results are generally consistent with an inhibitory input to type II neurons from onset-C neurons, or from essentially identical neurons.


    METHODS
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INTRODUCTION
METHODS
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Animal preparation

The data in this paper were collected along with other data that have been published previously (Davis and Young 1997; Nelken and Young 1994; Shofner and Young 1985; Spirou and Young 1991; Spirou et al. 1993; Sydorenko 1992; Voigt and Young 1990; Young et al. 1995); some data from unpublished experiments on iontophoresis and two-tone suppression in the auditory nerve also are included. The preparation and experimental methods were essentially identical across those studies. All protocols were approved by the Animal Care and Use Committee at the Johns Hopkins University.

Cats with clean external ears were decerebrated under ketamine anesthesia, usually after receiving xylazine as a tranquilizer. Atropine was given to control mucous secretion. No additional anesthetic was given after the decerebration. A tracheal cannula was inserted to maintain a clear airway, and the bulla was vented with a length of small-bore polyethylene tubing. The cats' temperatures were maintained between 36 and 38°C, and lactated Ringer solution was given regularly to preserve fluid volume. Some animals were respired artificially to maintain adequate respiration or with a pneumothorax to increase the stability of recording. These animals were paralyzed with gallamine triethiodide and maintained at 4% end-tidal CO2.

The DCN was exposed in one of two ways: a hole was drilled through the bone lateral to the foramen magnum (Young and Brownell 1976) or the cerebellum was aspirated and the DCN was approached from the contralateral side (Spirou et al. 1993). In the first approach, the electrode is placed directly on the surface and travels perpendicular to the DCN's layers but roughly parallel to an isofrequency lamina. In the second approach, the electrode travels parallel to the pyramidal cell layer and perpendicular to the DCN's isofrequency laminae.

Single neurons were recorded using platinum-iridium electrodes. In some experiments, a multibarrel glass electrode was piggybacked on the metal electrode to allow iontophoresis of glycine or GABAA antagonists in the vicinity of the recording site (similar to Havey and Caspary 1980). One barrel was filled with strychnine hydrochloride, a second was filled with bicuculline methiodide (each 10 mM, pH 3.5-4.0), and the third barrel was filled with pH-balanced buffer (potassium hydrogen phthalate, pH 4.0). A constant current generator passed -20 nA between each drug barrel and the buffer barrel as a retention current and +50 nA to eject the drug. In these experiments, control data were taken first; then the iontophoresis was turned on and test data were taken with the current on; then the ionophoresis was turned off and data were taken during recovery to control conditions. In all cases where units were held long enough (4/8), complete recovery from both strychnine and bicuculline was observed.

In one experiment, the auditory nerve was exposed by retraction of the cerebellum after the decerebration. Glass micropipettes were used to record from auditory nerve fibers.

Acoustic stimuli and experimental protocol

Sound was delivered using a closed system connected to the ipsilateral ear through an earbar. The system was calibrated in situ using a calibrated probe tube placed ~2 mm from the eardrum. Examples of the acoustic calibration have been shown previously (Rice et al. 1995; Spirou and Young 1991). The calibration is flat with fluctuations of <10 dB at frequencies <= 30-40 kHz.

When a unit was isolated, it was characterized based on its responses to tones at best frequency (BF) and to broadband noise. Rate versus level functions were constructed for BF tones and broadband noise by presenting 200-ms stimulus bursts once per second over an 80- to 100-dB range of levels. Sound level was changed in 1-dB steps and each level was presented once. These data were used to define the neuron's response type using the criteria described previously (Young 1984) and elaborated in this paper. Additional studies varied with the goals of the particular experiment. For many units, response maps were constructed by recording the responses to 200-ms tone bursts presented over a range of frequencies (generally 2-5 octaves interpolated logarithmically to 100 points) and a range of sound levels (generally 40-70 dB in 8- to 10-dB steps). Again, each frequency/sound-level combination was presented once. Response maps also were taken in the presence of a second tone, a fixed BF tone of 3-8 dB above threshold, which served to produce a steady level of background activity. This two-tone paradigm allowed inhibitory responses to be seen in the absence of spontaneous activity.

For some units, responses to noisebands of varying bandwidth were obtained. The noisebands were generated by multiplying a low-pass filtered noise (bandwidth fl) by a tone of frequency f0, which results in a noiseband of width 2fl centered at f0. To eliminate temporal fluctuations, two independent low-pass filtered noises were multiplied by quadrature tones (i.e., tones of the same frequency 90° out of phase) and added (Spirou and Young 1991). In later experiments, noisebands were generated digitally by inverse Fourier transforming the desired spectrum and playing the resulting signal through a 16-bit D/A converter (Nelken and Young 1997).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Properties of type II units

Data from a total of 201 units are included in this paper. Because type II units are difficult to isolate, recording times are generally not long and few units were studied with more than one or two different stimulus types. Most data are from a subset of 41 type II units that were studied with two or more stimulus paradigms.

Figure 1, A-C, shows examples of type II units. These units have no spontaneous activity, narrow V-shaped tuning, strong responses to tones with characteristically nonmonotonic rate behavior at BF, and weak or no response to noise. Figure 1A shows a typical response map for a type II unit. It consists of a V-shaped portion at low sound levels and a broad tail extending to low frequencies at high sound levels. The tuning of type II units is similar to that of auditory nerve fibers at 10 dB re threshold but is somewhat narrower, for units with BF >1 kHz, at 40 dB above threshold (Davis et al. 1996; Young and Voigt 1982); moreover, the low-frequency tail of type II units occurs at sound levels >60 dB re threshold, as opposed to 40-60 dB in auditory nerve fibers (Young and Voigt 1982).



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Fig. 1. Response characteristics of low spontaneous rate (<2.5/s) dorsal cochlear nucleus (DCN) units; most units of this type have 0 spontaneous rate. A: type II unit response map; the map shows plots of discharge rate versus frequency at 9 sound levels, determined from single presentations of 200-ms tone bursts. Sound level is given as dB attenuation at right; the sound level at 0-dB attenuation varies with frequency but is usually near 100 dB SPL. Horizontal lines in each plot are the spontaneous discharge rates (0 spikes/s in this case). Excitatory response regions are filled with black. B: rate vs. level curves for the unit in A. Rates are shown for 200-ms best frequency (BF) tone bursts and broadband noise bursts. Portion of the BF tone rate-level curve above the first inflection point was fit with a straight line by least squares. Slope of this line is a measure of nonmonotonicity. C-E: rate-level curves for 3 additional low spontaneous rate units. Unit in C is type II and the units in D and E are type I/III. Sound level axes are shown as dB attenuation; the noise spectrum level at 0 dB was ~40 dB re 20 µPa/Hz1/2.

Figure 1, B and C, shows plots of discharge rate versus sound level (rate-level curves) for two type II units (A and B are the same unit). These data are typical of type II units in that they show nonmonotonic responses to BF tones (---) and weak responses to broadband noise (- - -). Two measures of type II characteristics are shown. The degree of nonmonotonicity is characterized as the slope of a line fit (least squares) to the rate-level function at sound levels above the first inflection point in the curve. The fit was done only over the range of levels (>= 20 dB) showing a roughly linear behavior. The slopes of many type II units change at the highest levels (Fig. 1C), and these levels were not included in the fit. The second measure is the relative noise response, defined as the ratio of the maximum noise response (a in Fig. 1C) to the maximum tone response (b in Fig. 1C).

In addition to type IIs, the DCN contains other units without spontaneous activity; examples are shown in Fig. 1, D and E. These units have stronger responses to noise than type II units and show saturating (Fig. 1D) or monotonic (except for noise fluctuations; Fig. 1E) rate-level functions for BF tones. Units with low spontaneous rates and strong responses to noise have been classified as type I/III (Young 1984).

The examples of type II and I/III units in Fig. 1 are typical in that almost all low spontaneous rate DCN units respond vigorously to BF tones; the variability in their relative noise response comes mainly from differences in the strength of the noise response.

The definitions given in the preceding text do not provide quantitative criteria for distinguishing between type II and type I/III units. Figure 2A shows the distribution of low spontaneous rate DCN units along two dimensions, which often are used in the definition of type II units (Davis et al. 1996; Young and Voigt 1982). The ordinate shows the degree of nonmonotonicity, defined as the slope of lines like those shown in Fig. 1, normalized by the maximum discharge rate; the abscissa shows the relative noise response, defined as the ratio a/b in Fig. 1C. Type II units occupy the bottom left part of Fig. 2A, reflecting their weak noise responses and nonmonotonic BF tone rate-level functions (negative slopes). Type I/III units are in the top right part of this panel. There is a general correlation between weak noise responses and nonmonotonic BF rate-level curves (r = 0.42, P << 0.001). However, there is no evidence of a division of the low spontaneous rate units into two qualitatively different groups.



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Fig. 2. Distributions of type II and type I/III units in terms of the parameters defined in Fig. 1. A: scatter plot in a 2-dimensional space defined by relative noise response (a/b in Fig. 1C, abscissa) and nonmonotonicity (slope of the lines in Fig. 1, B-E). Slope is normalized by the peak rate to the tone (b) to remove effects on the slope due to differences in maximum discharge rate. Dashed lines outline a conservative definition of type II units, i.e., points with normalized slopes less than -0.0025 and relative noise responses <0.35. Square and star symbols show units having special properties, identified in the legend. These include units showing inhibitory cross-correlations (IT for inhibitory trough) with type IV units, units antidromically activated from the anteroventral cochlear nucleus (AVCN), and units excited by electrical stimulation of the somatosensory dorsal column or spinal trigeminal nuclei (MSN). Both BF and noise rate-level data exist for only 2 of the units antidromically activated from AVCN and these are shown in A; 2 others are known not to have responded to noise and are plotted in B only. One additional unit had a nonmonotonic BF rate level function, but the noise response is not known, and this unit is not shown here. B: stacked histogram of the distribution of relative noise response, showing units with special properties using the same symbols as in A. Note there is no overlap of the bars in this plot, bars for different unit groups are stacked on top of each other. C: histogram of the depth, relative to the center of the DCN pyramidal cell layer, of 49 type II unit recording sites (depth data from Spirou and Young 1991; Spirou et al. 1993; Voigt and Young 1980, 1990; Young 1980; Young and Voigt 1982).

Previously, type II units have been defined as having relative noise response <0.3-0.36 (Davis et al. 1996; Shofner and Young 1985; Young 1984), although this criterion was relaxed in one study (Sydorenko 1992). Figure 2B shows a histogram of relative noise response obtained from the population of Fig. 2A. These data do not form an obvious bimodal distribution, although there is a dip in the distribution at the 0.35-0.4 bin, which corresponds to the previously used criterion. The vertical dashed line in Fig. 2A is placed at a relative noise response of 0.35. Units to the left of this line all have nonmonotonic BF rate-level curves as well as weak noise responses. Units to the right of this line form a mixed population with both monotonic and nonmonotonic rate-level curves. Thus a conservative definition of type II units consists of units in the box in Fig. 2A, bottom left, as defined by the dashed lines (see also Davis et al. 1996).

Given the evidence reviewed in the INTRODUCTION that type II units are recorded from vertical cells, it is interesting to know to what extent this conservative definition captures units with the properties expected of vertical cells. The shaded boxes in Fig. 2, A and B, are units that were activated antidromically from the anteroventral cochlear nucleus (AVCN) (Young 1980). Because vertical cells are the only DCN neurons known to project an axon into the principal cell areas of the VCN, these neurons are by definition vertical cells. Data on these units are incomplete because they were characterized manually in some cases; however, in all cases where data were taken, the BF rate-level functions were nonmonotonic (3 cases) and the relative noise responses were <0.35 (4 cases). Thus units antidromically activated from the VCN all fit the conservative type II definition.

The filled black symbols in Fig. 2, A and B, show type II units that have inhibitory-type cross-correlations with type IV units (Sydorenko 1992; Voigt and Young 1980, 1990). Most of these (13/16) are within the conservative definition of type II, although there are three cases with relative noise responses outside the dashed-line box. The other properties of those three units were typical of type II units, low spontaneous rates, and nonmonotonic BF rate-level functions. Those three units show that the conservative definition excludes some units that share important properties with type II units and could be considered to be members of an extended type II family. However, because most units with inhibitory correlations fit within the conservative definition and because these units scatter throughout the boxed region, the conservative definition probably captures most type IIs and mostly type IIs.

A final property that may be useful in distinguishing type II units from other response types is the degree of activation by somatosensory inputs. Type II units within the dashed-line box of Fig. 2A are inhibited weakly by electrical stimulation of the somatosensory dorsal column or spinal trigeminal nuclei (Young et al. 1995). By contrast, units with characteristics like the examples in Fig. 1E are sometimes strongly excited by somatosensory stimulation. Units of this type are marked by stars and are located in Fig. 2A, top right. These units have strong noise responses and monotonic BF tone rate-level curves and are clearly different from type IIs.

Figure 2C shows a histogram of the depths of the recording sites of 49 type II units, with characteristics that meet the conservative definition. The recording depths were determined from histological reconstructions of recording tracks and are expressed as the distance, perpendicular to the free surface of the DCN, from the center of the pyramidal cell layer. Most type II recording sites are between 0 and 1 mm below the pyramidal cell layer, which is consistent with the location of the vertical cells in DCN (Lorente de Nó 1981; Saint Marie et al. 1991; Zhang and Oertel 1993b).

The evidence in Fig. 2 supports the idea of a type II unit class that has low spontaneous rate, strong but nonmonotonic responses to BF tones, and (usually) weak responses to noise; at least some of these units are recorded from DCN vertical cells and are inhibitory interneurons. At the same time, there are other units, called type I/III, that have different properties. Although it is not possible to draw a definitive boundary between the two types using the criteria considered, nonmonotonic units with relative noise responses less than ~0.35 appear to form a group with uniform type II properties. This group of units forms a proper or conservative type II group but may exclude the tail of the type II distribution. In the remainder of this paper, we describe mostly units with properties that place them within the conservative type II group; in some cases, results are shown also for units outside the conservative type II group to illustrate the similarities in their properties.

Two-tone response maps have inhibitory regions

The weak responses of type II units to noise and the nonmonotonicity of their BF rate-level curves suggest that these units receive substantial inhibitory input (Young and Voigt 1982). Figure 3 shows two-tone response maps of four type II units. These maps are in the same format as Fig. 1A, except that the horizontal lines are the rate produced by a fixed-level BF tone and the plots show the deviation from that rate produced by a variable tone. Excitatory areas are filled with black and inhibitory areas are shaded. Note that the term "inhibitory" is used here in a functional sense, meaning rate reduction. Inhibitory responses could be produced by actual inhibition in the DCN or by cochlear two-tone suppression (Sachs and Kiang 1968). Evidence regarding the relative amount of true inhibition versus cochlear suppression is offered in the next section.



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Fig. 3. Representative 2-tone response maps recorded from type II units. Fixed tone was at BF, 3-8 dB above threshold; fixed tone parameters are given in the caption above each map. Plots show rate vs. frequency for a 2nd tone, plotted relative to the rate produced by the fixed tone alone (horizontal lines). Excitatory regions (rate increases above fixed-tone rate) are filled with black; inhibitory regions are shaded. Sound level of the 2nd tone is given as dB attenuation.

The typical type II response map consists of a narrow excitatory region centered on BF and flanked by both upper and lower inhibitory sidebands (UISB, LISB). The ISBs often appear to reflect a single inhibitory region centered on BF with a bandwidth wider than that of the excitatory input (Fig. 3, C and D). In other cases, the BF of the inhibition appears to be centered away from the type II BF (Fig. 3, A and B). For most type II units, the UISB has a lower threshold and is more prominent than the LISB at low to medium levels. Generally at least one sideband has a threshold very similar to, or even lower than, the BF excitatory threshold.

The extent of the ISBs was revealed by measuring the bandwidths, in kilohertz, of both sidebands at approx 40 dB above BF threshold. Figure 4A shows the UISB and LISB bandwidths plotted versus BF along with the total bandwidth of the inhibitory region, assuming that it is a single V-shaped area. Bandwidths increase with BF, although the growth is less than a one-for-one increase; the slopes of regression lines to the three sets of data in the log-log plot in Fig. 4 are 0.69 (LISB), 0.29 (UISB), and 0.36 (total). Most of the total bandwidth, measured linearly in hertz, is in the UISB, as can be seen by the similarity of the total and UISB bandwidths in Fig. 4A. Figure 4B shows the UISB and LISB bandwidths in logarithmic terms, as the distance in octaves from BF to the lower side of the LISB or to the upper side of the UISB. These octave distances are smaller for higher BFs, which is the same effect as the slower-than-linear growth of bandwidth in Fig. 4A. The upper and lower sidebands are roughly the same width, in logarithmic terms. However the linear measure of width in Fig. 4A may be more relevant when considering responses to noise signals, in which there is a fixed power per linear bandwidth. It is evident from Fig. 4A that the total noise power in the UISB is significantly larger than the noise power in the LISB for a broadband noise with constant spectrum level.



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Fig. 4. Bandwidths of upper and lower inhibitory sidebands and the total inhibitory bandwidth are plotted vs. the BF for type II units; symbols are identified in the legend. A: bandwidths in linear terms, as kHz. ---, slope of 1 (note that the plot is log-log). Bandwidths were measured from 2-tone response maps at approx 40 dB relative to BF threshold. Edge of a band was taken as the point at which the rate crossed the background rate. Upper and lower inhibitory sideband (UISB and LISB) bandwidths were measured from the edge of the excitatory area to the edge of the inhibitory area. Total width is measured between the edges of the 2 inhibitory areas. Note that 2 cells had no lower sidebands in this range of sound intensities so neither lower side nor total widths are plotted for them. B: bandwidths measured logarithmically, as octaves from the lower edge (LISB) or the upper edge (UISB) to BF.

Two-tone suppression or neural inhibition?

Because the type II response maps were measured using a two-tone paradigm, the inhibitory responses in Fig. 3 could be produced either by cochlear two-tone suppression or direct inhibition in the cochlear nucleus. The relative contributions of these two sources were estimated in two ways: by determining the amount of suppression seen in auditory-nerve fibers using the same stimulus paradigm and by blocking local inhibition with iontophoretic application of inhibitory neurotransmitter antagonists.

The two-tone response map paradigm was presented to auditory-nerve fibers in one animal. Fifty-four fibers were isolated long enough to measure BF and spontaneous rate (SR); the proportions of low, medium, and high SR fibers was roughly the same as is usually found (Liberman 1978) (in our data: 8/54 low SR, 15.4%; 13/54 medium SR, 25%; 31/54 high SR, 59.6%). Twenty-one auditory-nerve fibers were held long enough to generate two-tone response maps.

Typical two-tone response maps for auditory nerve fibers are shown in Fig. 5; the shaded regions show two-tone suppression. In most cases (15/19 with two uncertain cases), below-BF suppression was not encountered in auditory-nerve fibers. Two-tone suppression below BF has been documented in auditory nerve fibers (Rhode and Greenberg 1994; Sachs and Kiang 1968; Schmiedt 1982), but it occurs at higher stimulus levels than were used routinely here. Suppression areas were seen in almost all fibers at frequencies above BF (20/21), as in these two examples.



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Fig. 5. Two-tone response maps for a high (A) and a medium (B) spontaneous rate auditory-nerve fiber. Stimulus paradigm and shading of the response maps are the same as in Fig. 3. Horizontal lines in the plots show the response rates to the fixed tone, the parameters of which are given in the legend above each plot. Note the absence of LISBs at these sound levels.

The thresholds for an inhibitory or suppressive response were compared between auditory nerve fibers and type II units. Figure 6 shows the distribution of these thresholds in auditory nerve fibers (A-D) and type II units (E and F). Over the range of BFs included in these data, the sideband thresholds vary systematically with BF (not shown). These BF trends were removed from the data by subtracting the BF trend line determined for the type II units' sidebands; as a result, the thresholds in Fig. 6 are shown relative to the average type II threshold at the same BF. Actual and extrapolated thresholds are shown ( and ), computed as described in the figure caption, as is the highest sound level presented in cases () where no suppression or inhibitory response was observed . Consistent with Fig. 5, there are few estimates of auditory-nerve suppression thresholds for frequencies below BF (Fig. 6, A and C). In contrast, all type II units had LISBs and the median type II threshold (Fig. 6E, down-arrow ) is below the lowest lower-bound values for the auditory nerve fibers.



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Fig. 6. Comparison of inhibitory and suppression thresholds between auditory-nerve fibers and type II units. Histograms show the distribution of the lowest sound level at which suppression or inhibitory responses were observed in two-tone response maps. Left: data from LISBs; right: data from UISBs. A and B: auditory-nerve high spontaneous rate (SR) fibers; C and D: auditory nerve low plus medium SR fibers; E and F: type II units. down-arrow , medians of the type II data. Shading indicates the nature of the threshold determination: , actual thresholds; , highest sound intensity presented to a cell with no suppression evident (i.e., a lower bound on the suppression threshold); and , thresholds that were estimated by extrapolation from inhibitory responses at >= 2 sound levels. For extrapolation, a plot of average rate near the peak inhibition versus level was constructed, fit with a straight line, and the threshold was taken as the point where the line intersected the background rate. In cases like Fig. 5B, where the threshold was not close to the lowest sound level presented, the extrapolation was judged unreliable and these cases are not included. Both UISB and LISB thresholds decreased with BF (slopes of -17.0 dB/log-unit for LISBs, P < 0.01, and -13.7 dB/log-unit for UISBs, P < 0.05, in type II units). These regression lines were subtracted from the data before making the histograms shown here, to eliminate the BF effect. BF range for auditory nerve fibers was 0.55-25.6 kHz ,and for type II units it was 0.42-20.2 kHz. In 4 auditory nerve fibers, it was not possible to measure either the LISB or the UISB threshold, so the populations of units in the 2 columns are slightly different. Note there is no overlap of the bars in this plot, bars for different threshold determination methods are stacked on top of each other.

Both auditory nerve fibers and type II units showed inhibitory responses for frequencies above BF (except for 1 auditory nerve fiber; Fig. 6, B, D, and F). Type II UISB thresholds (Fig. 6F) overlapped considerably with auditory-nerve suppression thresholds, especially for low and medium SR auditory-nerve fibers (Fig. 6D). In fact, the medians of the threshold distributions for low and medium SR auditory-nerve and type II units are not significantly different (P > 0.3, rank-sum test).

These threshold results suggest that at low to moderate sound levels, type II LISBs represent inhibitory processes in the DCN and cannot be explained by cochlear suppression. However, many UISBs could reflect two-tone suppression, especially of low and medium SR auditory-nerve fibers, if such fibers are the predominant source of the excitatory inputs to type II units.

Application of antagonists to inhibitory neurotransmitters while presenting the two-tone stimulus paradigm provides a direct test of inhibition. Figure 7 shows two-tone response maps for two type II units in control conditions (left) and during iontophoresis of either strychnine (Fig. 7B) or bicuculline (Fig. 7D). The control response map was repeated for comparison in the right column (- - - and · · ·). The inhibitory antagonists had the general effect of increasing the response to the low level BF tone, evident as an increase in the background rate shown by the horizontal lines. In fact, for all cases except the one shown in Fig. 7B (7/8 cases), there was an increase in rate to BF tones at all levels in the presence of strychnine or bicuculline. At all stimulus levels, except the highest level in Fig. 7D, the LISB was eliminated by drug application, suggesting that it results from neural inhibition. By contrast, the UISB was reduced in bandwidth but inhibitory responses to the second tone remained. The bandwidth changes in the UISB were small but reliable; the bandwidths were measured by determining the frequencies at which inhibition was halfway between the background rate and the minimum rate in the sideband. At 40 dB above threshold, the UISB bandwidth was reduced by an average of 28% (range 19-39%, n = 4) by strychnine and 40% (range 20-54%, n = 3) by bicuculline. This result suggests that the UISB consists of a mixture of inhibition and cochlear suppression.



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Fig. 7. Examples of 2-tone response maps before (left) and during (right) application of inhibitory neurotransmitter antagonists. For the unit in A and B, strychnine was iontophoresed in the vicinity of the recording electrode and for the unit in C and D, bicuculline was used. Control response map is repeated in the right column as the dotted and dashed lines. In the presence of the inhibitory antagonist, the background rate to the fixed tone increased (horizontal lines), most of the LISB was lost, and the UISB was weakened but remained present. For the unit in A and B, the fixed tone was slightly below BF (6.6 vs. 7.1 kHz).

The results shown in Fig. 7 are typical; essentially identical changes in two-tone response maps were obtained in four cases with strychnine and three cases with bicuculline. The effects of applying inhibitory neurotransmitter antagonists are thus consistent with the inferences drawn from the threshold data in Fig. 6.

Iontophoresis shows that inhibition by noise is strong

The weak responses of type II units to noise suggests that their inhibitory input is somehow stronger for noise than for tones. Pharmacological blockade of inhibitory neurotransmitters supports this hypothesis. Figure 8A shows rate-level curves for BF tones and noise before and during iontophoresis of strychnine or bicuculline. Control responses are shown by the light solid lines and responses during inhibitory blockade are shown by the dashed (bicuculline) and heavy solid (strychnine) lines. For both tones and noise there was a substantial increase in rate when either inhibitory antagonist was applied. However, the increase in noise response was much larger than the increase in tone response. During strychnine application, the saturation discharge rates were approximately the same for tones and noise even though the noise response was essentially zero in the absence of inhibitory blockade. For bicuculline, the effects were similar, although not as large. For the type I/III unit shown in Fig. 8B, application of strychnine also produced an increase in response, but in this case, the relative noise response was approximately the same (0.6) before and during strychnine application. The examples in Fig. 8, A and B, are typical in that inhibitory blockade did not produce spontaneous activity in any of the cases tested (6 tested with both antagonists, 2 others tested with 1 blocker only).



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Fig. 8. Effects of strychnine and bicuculline iontophoresis on rate-level curves. Legend in B identifies the conditions for each rate-level plot. A: type II unit studied before and during strychnine and bicuculline. Leftmost 3 rate-level curves: BF tones, rightmost 3: for noise. Note the control noise response is essentially 0. B: type I/III unit studied before and during strychnine. Spontaneous activity did not develop in either unit during iontophoresis. C: plot of relative noise response before (abscissa) versus during (ordinate) application of inhibitory antagonist. Antagonist is identified by the symbols, defined in the legend. Diagonal line shows equality of relative noise responses and the vertical dashed line separates type II from type I/III units at a relative noise response of 0.35.

Figure 8C shows a comparison of relative noise response before (abscissa) and during (ordinate) application of inhibitory antagonist for all the units tested. Type II units are the points to the left of the vertical dashed line, i.e., the units with relative noise response <0.35 before iontophoresis. In all cases, the relative noise response increased dramatically with inhibitory blockade; the effects were larger for strychnine than for bicuculline. Type I/III units are the points to the right of the dashed line. For two of these cases, there was no increase in relative noise response with strychnine, although there was an increase in discharge rate (Fig. 8B is an example); in the remaining cases, the relative noise response increased. Figure 8C shows that type II units, and to a lesser extent type I/III units, receive glycinergic and GABAergic inputs that are much more effective for broadband noise stimuli than for tones.

Noiseband widening suggests strong inhibitory inputs

The frequency extent of the inhibitory input to type II units can be examined using noisebands of varying bandwidth. Figure 9 shows examples of rate-level curves for BF tones, noisebands, and broadband noise for two type II units. The noisebands were centered arithmetically at BF, and the bandwidths are marked on the curves. Sound level is plotted on the abscissas as total stimulus power. As a result, the curves should shift to the right as the stimulus bandwidth exceeds the integrating bandwidth of the unit; inhibitory effects should show up as reductions in discharge rate. Generally the responses for the narrowest noisebands tested are similar to the BF tone responses at the same stimulus power. These two units are typical of most units in that the rate to the narrowest noiseband is somewhat less than the rate to the tone. For all units examined (15), there was a monotonic decline in the response rate as the bandwidth increased. This response profile is strongly suggestive of an inhibitory input that is recruited over a wide range of stimulus bandwidths.



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Fig. 9. Rate vs. level curves for BF tones, noisebands of various bandwidth, and broadband noise (BBN). Noise bandwidths are given next to the curves. Data are shown for 2 type II units. Notice the monotonic decline in response as the bandwidth of the stimulus increases. Sound level on the abscissa is total stimulus energy for the tones and noisebands. Broadband noise curves are aligned to have the same spectrum level as the widest noiseband used. Some of these data were shown previously in Fig. 14B of Nelken and Young (1994).

Figure 10A shows a population plot of the maximum discharge rate across sound level versus bandwidth for data like those in Fig. 9. Data are shown for 14 type II units, and 1 unit that had a noise response outside the conservative type II definition. Maximum rates to BF tones and broadband noise are shown at the left and right extremes of the abscissa. This plot shows that the steady decline in response rate with stimulus bandwidth illustrated in Fig. 9 occurs in the whole population. However, there is considerable variability across the population in the relative noise responsive and the effects of bandwidth. These effects are shown more clearly in the normalized plot of Fig. 10B, where maximum rate is plotted relative to BF tone rate and bandwidth is expressed relative to BF. The variability is only partly reduced; the relative bandwidth at which units' responses are reduced by 50%, for example, varies over an order of magnitude.



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Fig. 10. A: maximum rate across sound level plotted against bandwidth for 15 units, 14 of which meet the conservative type II definition. Data are shown for BF tones, noisebands, and broadband noise. Unit BFs range from 2.2 to 17.8 kHz. B: same data replotted in normalized form, as maximum rate relative to maximum BF tone rate vs. bandwidth relative to BF.

Figure 10B shows that noise affects type II units over a very wide bandwidth. In most units, there is a decline in rate between the widest noiseband used and broadband noise. For example, in seven units rate declined by an average of 44% (range 3-75%) between a noiseband approximately twice as wide as BF (range 1.8-2.3) and broadband noise. That is, the inhibitory inputs to these units integrate energy over a frequency range extending more than an octave above BF.

Figure 11 shows a comparison of the inhibitory effects of tones and noise. Data are shown from three type II units in which both a two-tone response map and noiseband responses were obtained. Response rate versus tone frequency is shown (solid lines) for the two-tone paradigm with the variable tone at approx 50 dB above threshold; the fixed tone was, as usual, near threshold and produced the rate shown by the horizontal line. Tone responses are excitatory when the rate is above the horizontal line. The dotted vertical lines show the limits of the excitatory region as inferred from these tone responses. The symbols and dashed lines show rates in response to noisebands of various bandwidths. Each point is plotted twice, once at the frequency of the lower edge and again at the frequency of the upper edge of the noiseband. The noiseband levels were chosen to keep the spectrum level constant as bandwidth changed at the level at which the narrowest noiseband had the same total power as a BF tone. Notice that as noise energy was added, the discharge rate decreased, indicating an inhibitory effect. The rate decrease began at the narrowest bandwidth tested, even though those bandwidths were well within the unit's excitatory response range as determined by tones. Thus energy at frequencies within the excitatory area causes a rate increase when presented as a tone, but a rate decrease when added to a noiseband. This result is consistent with the iontophoresis data in suggesting that the inhibitory inputs to type II units are more strongly activated by noise than by tones.



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Fig. 11. Comparison of spectral integration for tones versus noise for 3 type II units. Solid lines are 2-tone response maps at ~50 dB re threshold. Horizontal solid line is the rate to the fixed tone, and the vertical dotted lines show the limits of the excitatory area for tones. Dashed lines and symbols are rate in response to noisebands, plotted at the noiseband edges (open triangles, 1 for the lower and 1 for the upper bandedge). Filled triangles show the rate in response to broadband noise.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Type II and type I/III units

The type II response type first was defined on the basis of its low response to broadband noise (Young and Voigt 1982). Type IIs are the only units in the cochlear nucleus that have this property. However, a more important insight was the association of type II units with an inhibitory interneuron in DCN. The first evidence for this relationship was the finding of inhibitory-type cross-correlation between type II units and the response types associated with DCN principal cells (Voigt and Young 1980). There are several inhibitory interneurons in DCN (Osen et al. 1990), but the evidence reviewed in the Introduction from antidromic stimulation, from recording in the dorsal acoustic stria, from the locations of type II recording sites, and from intracellular dye filling points to the vertical cell as the source of type II recordings. Because response types are most useful when they can be related to functional and anatomic entities, we suggest that type II should be thought of as a response type given by vertical cells. That assumption will be made in the remainder of this discussion.

The data in Figs. 1 and 2 show that type II units can be defined with a reasonably homogeneous set of properties including low spontaneous activity, low relative noise response, vigorous tone responses with a characteristic nonmonotonicity at high sound levels, and narrowly tuned response maps. A conservative definition of type II can be given based on these properties, but some units that might be called type II lie outside the conservative boundaries where they are apparently intermingled with type I/III units. The problems of defining unit types on the basis of responses to sound have been discussed (Joris 1998), and those problems apply here. In particular, it is not possible to draw an absolute boundary between type II and type I/III units on the basis of the existing data because of overlap of the two groups in plots like Fig. 2A. Additional measures, such as width of dynamic range (Davis et al. 1996) and PST histogram shape (Shofner and Young 1985) were considered, but found not to be helpful. Despite the lack of a clear boundary between types II and I/III, it nevertheless seems clear that there is more than one neuron type involved, and it also seems safe to assume that the conservative definition in Fig. 2A defines a homogeneous, but not necessarily inclusive, set of type II neurons.

In intracellular recordings in gerbil (Ding and Voigt 1997), type II and type I/III units are generally similar, although they show some differences in action potential parameters and in the prevalence of hyperpolarizing responses to off-BF tones (type II units showed them, type I/III units usually did not). The latter finding is consistent with the inference from Fig. 2A that type II units show stronger effects of inhibition (i.e., weak noise responses and nonmonotonic tone responses) than do type I/III units. These results suggest that the main difference between the two unit types might be the amount and strength of inhibitory input.

The anatomic identity of type I/III units is a matter of question. Type I/III responses have been recorded from a few identified pyramidal cells (Ding and Voigt 1999), although these cells are much more likely to be type III or IV (Ding and Voigt 1999; Joris 1998; Rhode et al. 1983; Young 1980). One possibility, suggested by the general similarity of type I/III and type II responses and by the fact that they appear to form a continuum in plots like Fig. 2A, is that they are recorded from different subpopulations of vertical cells. Some heterogeneity exists within the vertical cell population; for example, vertical cells located more deeply in the DCN have greater tendency to form VCN projections and a minority of cells have axonal arborizations only in the deep DCN (Lorente de Nó 1981). Immunocytochemistry reveals variations of intensity of glycine immunolabeling among small cells in deep DCN and about one-third of the cells colocalize GABA immunoreactivity in cat (Osen et al. 1990). It may be that the position of a cell in deep DCN determines its synaptic influences and type I/III units are those receiving less broadband inhibitory input. The question of the identity of type I/III units can only be settled by further research.

It is important to recognize that type II units should be defined as having a conjunction of properties: low SR, weak response to noise, and vigorous, narrowly tuned, slightly nonmonotonic response to tones. Attempts to construct a tree diagram for classifying neurons in DCN (Joris 1998) inevitably lead to separate decisions on these classification variables. The result, as pointed out by Joris in his paper, is that obvious misclassifications occur. For example, the degree of nonmonotonicity and the spontaneous rate of type II units overlap with other response types. In classifying type II units, therefore, simultaneous consideration should be given to all aspects of the unit's response.

Sources of excitatory input to type II units

An interesting observation is that application of strychnine and bicuculline did not increase SR in type II units. One interpretation of this result is that the excitatory input to these units is from a source with little or no spontaneous activity. Vertical cells receive monosynaptic excitatory input from auditory nerve fibers (Oertel and Wu 1989; Zhang and Oertel 1993b). Taken together, these two results suggest that low SR auditory nerve fibers may form the major excitatory input to type II cells. If true, this hypothesis also could explain the finding that type II units have consistently higher thresholds than DCN principal cells (Young and Brownell 1976) because low SR auditory nerve fibers also have elevated thresholds relative to the lowest threshold auditory nerve fibers (Liberman 1978). However, patterns of auditory nerve innervation of the DCN are most consistent with high SR fiber innervation of vertical cell somata and low SR fiber innervation of dendrites (Liberman 1993). In that case, the low spontaneous rates and high sound thresholds of type II units might be caused by a high intrinsic electrical threshold (Hancock et al. 1997); this is consistent with the responses of vertical cells to intracellular current injection (Ding and Voigt 1997; Zhang and Oertel 1993b).

Vertical cells receive polysynaptic excitatory postsynaptic potentials (EPSPs) after stimulation of the auditory nerve root in slice preparations (Zhang and Oertel 1993b). EPSPs also are evoked from glutamate stimulation of a tonotopically appropriate region of the VCN. These polysynaptic inputs could be from T-stellate cells of the VCN, which make terminals with excitatory morphology (Smith and Rhode 1989) and project an axon collateral to the DCN (Oertel et al. 1990). The role of T-stellate excitatory terminals in DCN, relative to those of auditory nerve fibers and granule cells, remains to be defined, but it is possible that the excitatory areas of type II units are produced by convergent input from T-stellate and auditory nerve axons. T-stellate cells give chopper response patterns to sound (Smith and Rhode 1989), and these responses are similar to those of auditory nerve fibers with regard to many features, such as tuning and rate-level functions (Blackburn and Sachs 1989; Bourk 1976). However, chopper units also have significant inhibitory inputs (Blackburn and Sachs 1992; Rhode and Greenberg 1994), so the possibility exists that some of the inhibitory effects seen in type IIs are presynaptic.

Inhibitory inputs to type II units

The results reported in this paper clearly show that type II units receive inhibitory inputs. To characterize inhibition by tones, two-tone response maps were used. The apparent inhibition seen in these maps is generated partly by cochlear two-tone suppression. However, the comparisons of sideband thresholds (Fig. 6) and the effects of inhibitory neurotransmitter antagonists (Fig. 7) argue that the inhibitory responses seen below BF and part of the responses seen above BF are attributable to neural inhibition. Rhode and Greenberg (1994) came to the same conclusion based on comparison of a wider range of properties between auditory nerve and cochlear nucleus. In fact, when inhibitory antagonists were applied, the bandwidth of the inhibitory regions in two tone response maps became narrower, so that the inhibitory bandwidth measurements shown in Fig. 4 are accurate reflections of the bandwidth of the inhibitory input, with little or no contamination from cochlear suppression.

Caspary and colleagues (1994) have argued that inhibitory inputs to neurons in AVCN are centered on the excitatory tuning curve on the basis of results showing that GABA or glycine antagonists produce the largest increase in discharge rate for tones at or near BF. DCN type II units show the same behavior (results to be shown elsewhere); this effect is evident in Figs. 7 and 8 in the increase in background discharge rate (the horizontal line) produced by the fixed tone. Thus it seems reasonable to assume that the inhibitory inputs to DCN type II units form a single inhibitory area centered at or near BF.

For noise, the results are also clear. The increase in rate in response to broadband noise following bicuculline or strychnine application (Fig. 8) shows conclusively that the noise stimulus is evoking an inhibitory input. Moreover, the fact that the increase in rate for noise was much larger than for tones (as in Fig. 8, A and C) shows that the inhibitory source gives a stronger response to broadband noise than to tones. Further evidence for this point is the fact that narrow noisebands, the passbands of which are wholly contained within the excitatory tuning curve for tones, cause a reduction in rate relative to the responses to a BF tone and the narrowest noiseband (Fig. 11).

The possible contribution of cochlear suppression to reductions of rate with noiseband widening should be considered. Auditory nerve responses to bands of noise arithmetically centered on BF have been studied (Ruggero 1973; Shalk and Sachs 1980). Effects of suppression are seen, but they are weaker than those shown in Figs. 9-11 of this paper. For example, for narrow (200 Hz) noisebands, the rate in response to the noiseband is the same as the rate to an equal-energy tone, for all SR groups (Ruggero 1973; Shalk and Sachs 1980). By contrast, most (9/15) type II units gave lower rates to narrow noisebands than to tones (as in Fig. 9). For wider noisebands, there is a decline in the saturation rate of rate-level curves in low and medium SR fibers (Shalk and Sachs 1980) that is qualitatively similar to the decrease seen in Fig. 10. The effect is much smaller than that seen in the type II data; however; in the most extreme example shown by Shalk and Sachs, the saturation rate for a noiseband of bandwidth equal to BF was ~0.67 of the saturation rate for a tone. Type II units vary between 0.1 and 0.6 at the same relative bandwidth (Fig. 10B). Thus the inhibitory effects of noisebands are substantially stronger in type II units than auditory nerve fibers.

Is the inhibitory input from the wideband inhibitor?

There are several sources of inhibitory terminals within the cochlear nucleus (Kolston et al. 1992; Osen et al. 1990) as well as descending inhibitory inputs from more central auditory nuclei (Spangler and Warr 1991). The possibility that the inhibitory inputs to type II units involve descending inputs cannot be discounted; however, not enough is known about such inputs to evaluate their possible contributions, so they will not be considered further here. Of the inhibitory sources within the cochlear nucleus, three are known to project axons into the region of the DCN occupied by the vertical cells. Cartwheel cells of the superficial DCN immunostain for glycine and sometimes also for GABA (Gates et al. 1996; Osen 1990) and their axons extend down to the deep DCN (Berrebi and Mugnaini 1991). However, intracellular recordings from vertical cells do not show IPSPs with the particular pattern of spontaneous and burst firing characteristic of cartwheel cells (Golding and Oertel 1997), so cartwheel cell input can be ruled out.

Second, vertical cells themselves produce significant axonal terminals in the deep DCN (Zhang and Oertel 1993b). However, given the evidence of this paper that some inhibitory inputs to vertical cells must respond strongly to broadband noise, it seems clear that an inhibitory input other than vertical cells is present. In addition, no signs of inhibitory or any other interaction have been observed between pairs of type II units in cross-correlation studies (Voigt and Young 1980, 1990).

Third, D-stellate or radiate neurons of the VCN project axons to DCN that terminate in regions where vertical cell dendrites are located (Doucet and Ryugo 1997; Oertel et al. 1990). These neurons are glycine-immunoreactive (Doucet et al. 1999) and so could provide inhibitory inputs to vertical cells (Zhang and Oertel 1993b). These neurons have been shown to give the so-called onset-C response (Smith and Rhode 1989), a prominent feature of which is wideband facilitation, which causes them to respond more strongly as the bandwidth of the stimulus is increased (Jiang et al. 1996; Palmer et al. 1996; Winter and Palmer 1995). In particular, onset-C neurons respond more strongly to noise than to tones. On this basis, Winter and Palmer (1995) proposed that onset-C neurons form the inhibitory inputs to DCN type II units that produce weak type II noise responses in the presence of strong tone responses. Current models of the response properties of both type II and type IV units in DCN are based on inhibitory input of the kind provided by onset-C neurons, the so-called wideband inhibitor (Blum and Reed 1998; Davis et al. 1996; Hancock and Voigt 1999; Hancock et al. 1997; Nelken and Young 1994, 1997).

Figure 12 shows two comparisons of properties of type II units and onset-C units. The onset-C properties are taken from guinea pig data of Palmer and colleagues (Jiang et al. 1996; Palmer et al. 1996). In those papers, onset-C units were often not segregated from other onset unit types, when the properties of the different unit types were the same. Nevertheless, in the ensuing discussion we refer only to onset-C units because that subclass is the candidate inhibitory input to type II cells. Figure 12A shows the total bandwidth data from Fig. 4A repeated (+) along with the bandwidths of excitatory tuning curves for onset neurons (open circle ) (from Jiang et al. 1996). These data show that the two-tone inhibitory areas of type II units have roughly the same bandwidth as the excitatory tuning curves of onset (and onset-C) neurons. Thus if onset-C neurons are the source of the inhibitory inputs to type II units, little or no convergence across BF is necessary to produce the inhibitory regions of two-tone response maps. In fact, because onset-C neurons show wideband facilitation, the use of two tones in constructing type II response map should make the 40-dB bandwidths of presumed onset-C inputs wider than the single-tone bandwidths shown in Fig. 12A (Jiang et al. 1996).



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Fig. 12. Comparison of the properties of inhibition in type II units and excitation in onset-C neurons. A: bandwidth of the total inhibitory area of type II units (same data as the + in Fig. 4A) and the bandwidth of the excitatory tuning curves of onset neurons, both at approx 40 dB re threshold. Onset unit data were computed from the Q40 data in Fig. 5 of Jiang et al. (1996). B and C: plots of rate versus sound level for BF tones (open circle ) and for noisebands at fixed spectrum level (); in each noiseband curve, the overall level varies with the bandwidth of the noise. Bandwidth scale is shown (bottom left) for the -10 dB plot in C. Numbers next to each curve give the spectrum level in dB re 20 µPa/Hz1/2. Data are shown for 2 type II units. Unit in C is the same as in Fig. 9B. Unit in B is the same data as Fig. 13 of Nelken and Young (1994). For both units, the narrowest bandwidth used was 200 Hz, and the bandwidth was doubled for successive points. BFs of the units were 6.88 kHz (B) and 9.28 kHz (C). Insets: schematic versions of similar plots for an onset unit (similar to Fig. 14 of Palmer et al. 1996) and a nononset (other) VCN unit (similar to Fig. 13 of Palmer et al. 1996 or Fig. 1 of Greenwood and Goldberg 1970). In the insets, - - -, BF tone rate curves; ---, noiseband responses for fixed spectrum levels.

Figure 12, B and C, shows rate-level curves for BF tones (open circle ) and for noisebands (), plotted slightly differently than in Fig. 9. Each of the noiseband curves shows rate versus sound level for noisebands of fixed spectrum level; sound level is varied within a curve by varying bandwidth (see the bandwidth scale at the bottom of the figure). Figure 12C is exactly the same data as in Fig. 9B, with the points connected up a different way. Type II units exhibit a very strong inhibition in these plots, in the form of the near-monotonic decrease in discharge rate as bandwidth (and stimulus energy) increase. The only exceptions are for the narrowest bandwidths at low sound levels, where rate increases with bandwidth. These two examples are typical of all type II units studied.

Type II units and onset-C units behave very differently from other cochlear nucleus neurons (Greenwood and Goldberg 1970; Palmer et al. 1996) and auditory nerve fibers (Ruggero 1973; Shalk and Sachs 1980) in plots like Fig. 12, B and C. The insets between Fig. 12, A and B, show schematic plots typical of data from onset units and other VCN units. Other VCN neurons show an increase in rate for narrowbandwidths at all sound levels and a decrease in rate at wider bandwidths. The increase in rate at narrow bandwidths, which also is observed in some type II units, is interpreted as the summation of energy within the neuron's tuning curve; the decrease in rate at wider bandwidths is interpreted as an effect of suppression or inhibition. Onset neurons, by contrast, show only a monotonic increase in rate as bandwidth increases; the rate increase continues up to bandwidths at least equal to BF. Note that the rates in response to noisebands are higher than the rates in response to BF tones at the same sound level, as shown in the inset. The onset neuron behavior is, in fact, the exact inverse of the behavior shown by type II units in Fig. 12, B and C (except for the increase in rate at low levels, where the effect of summation of energy within the type II tuning curve apparently outweighs the inhibitory effect). Thus onset-C units have just the right behavior to explain the accumulation of inhibition with bandwidth in type II units, whereas other neuron types in the cochlear nucleus do not. Specifically, other neuron types show a decrease in rate at wider bandwidths, which is the opposite of the behavior required to account for the type II behavior.

The arguments above show that onset-C units have the properties needed to account for several aspects of inhibition in type II units. Thus if onset-C units are not the inhibitory input to type II units, then the actual inhibitor should have properties identical to the onset-C unit.

Synaptic organization: unresolved issues

Despite the coherent picture developed in the preceding text, there are some remaining uncertainties about type II synaptic organization. First, there are few inhibitory puncta on the somata of vertical cells, in comparison with DCN principal cells, (Osen 1990; Saint Marie et al. 1991) and little sign of disynaptic IPSPs in vertical cells after auditory-nerve stimulation in slice preparations (Zhang and Oertel 1993b). Thus current anatomic and in vitro studies do not provide a basis for the strong inhibition seen physiologically. Second, the radiate (D-stellate) neurons are glycinergic (Doucet et al. 1999), but the noise-driven inhibition of type II units is reduced significantly by bicuculline as well as by strychnine (Fig. 8). The sensitivity of DCN cells to GABA antagonists has been reported before (Caspary et al. 1987; Evans and Zhou 1993). It seems likely that there are GABAergic inhibitory inputs on type II units, in addition to the glycinergic inputs from radiate neurons. The source of these GABAergic inputs is not known, although there are many GABAergic cells in the cochlear nucleus (Kolston et al. 1992; Osen et al. 1990) as well as GABAergic projections from the superior olivary complex (Ostapoff et al. 1990, 1997). Third, the evidence from two-tone response maps, inhibitory blockade, and noiseband widening suggests that the inhibition in type II units is centered on or near BF; models of type II responses assume the same thing. However, it is not clear that D-stellate cells project in a tonotopic fashion; indeed, injections of tracer into circumscribed regions of the tonotopic map of DCN fill radiate cells across a wide range of the tonotopic map in VCN (Doucet and Ryugo 1997). Thus it is not clear how the apparent tonotopic projection of the wideband inhibitor would correspond to the apparently nontonotopic projection of radiate neurons. Because the details of the anatomic circuitry of the deep DCN are largely unknown, the relevance of these points cannot be properly evaluated at present. More information about the anatomic inputs to vertical cells, especially their dendritic inputs, is required.

Functional role of type II units

The role of type II units in spectral processing is that of a narrowband inhibitor. Responses of DCN principal cells are strongly inhibited by this narrowband source. As a result, DCN principal cells are inhibited by sharp spectral peaks close to their BF. These units also are inhibited by a wideband source, probably the onset-C neuron (Nelken and Young 1994), which produces the inhibition seen in principal cells in response to notches in the stimulus spectrum (Spirou and Young 1991; Young et al. 1992). Thus type II cells participate in the construction of the exquisite sensitivity of DCN principal cells to sharp spectral features, both peaks and notches.

Type II units also supply an inhibitory input to the VCN (Wickesberg and Oertel 1990), but the role of type II terminals in the VCN is less clear. Three different hypotheses have been raised. The first is that this projection modulates the response thresholds of VCN neurons (Paolini et al. 1998). This hypothesis is based on the finding that vertical cells provide a tonotopically organized inhibitory input to bushy and stellate neurons of the VCN (Wickesberg and Oertel 1990) and on the finding that injection of the GABA agonist muscimol into DCN produced a significant lowering of the average threshold of VCN units (Paolini et al. 1998). The advantage of having VCN thresholds elevated in this way by DCN activity is not clear; moreover, the properties of the threshold elevation do not correspond to those of type II units, which are not spontaneously active and have high-thresholds themselves. Thus the role of type II units in modulating VCN thresholds is not clear.

The second hypothesis is that the inhibitory projection from DCN to VCN produces monaural echo suppression (Wickesberg and Oertel 1990). This hypothesis is based on the finding that vertical cells supply a delayed, frequency specific input to the VCN. Two-click data (Wickesberg 1996) provide partial physiological support for this idea, but only 19% of VCN cells showed the expected increase in response to the second click after lidocaine injections in DCN. In general, the role of type II units in temporal processing is hard to interpret because their temporal properties are complex and nonlinear (Joris and Smith 1998).

The third hypothesis on the role of the type II projection to VCN is that it helps to reduce the effects of spectral notches caused by the acoustical properties of the pinnae (Rice et al. 1992) on the representation of complex sounds in the VCN (Nelken and Young 1996). Unlike DCN neurons, VCN cells are not inhibited by narrow spectral peaks or notches. Presumably the inhibition from type II units is too weak in VCN to produce the effects that are seen in DCN. Instead the tonotopic array of type II neurons weakly inhibits VCN principal cells except in the region of the notch itself. The result is an enhancement of the spectral information in the notch relative to its surround, essentially counteracting the effects of pinna acoustics in producing the spectral notch. In this way, information about the notch itself would be conveyed in the outputs of the DCN and information about the spectral shape of the stimulus would be conveyed by the outputs of the VCN.

Type II neurons may participate in all the functions proposed in the preceding text, but more information about their connections and responses to complex stimuli are required to fully evaluate these hypotheses. Nevertheless, the accumulated physiological and anatomic evidence indicate that type II neurons play an important role in establishing the response properties of a wide variety of cochlear nucleus neurons, and thus they play a crucial role in the transformations of the auditory representation that take place in the cochlear nucleus.


    ACKNOWLEDGMENTS

The technical assistance of C. Alesczczyk and P. Taylor is acknowledged. Helpful comments on the manuscript were received from B. May and R. Ramachandran.

This work was supported by Grants DC-00115 and DC-01387 from the National Institute on Deafness and Other Communication Disorders.


    FOOTNOTES

Address for reprint requests: G. A. Spirou, Dept. of Otolaryngology, P.O. Box 9200, Health Sciences Center, West Virginia University, School of Medicine, Morgantown, WV 26506-9200.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 26 February 1999; accepted in final form 30 April 1999.


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ABSTRACT
INTRODUCTION
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DISCUSSION
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society