]>HEARES3104S0378-5955(98)00140-310.1016/S0378-5955(98)00140-3Elsevier Science B.V.Fig. 1An example of click waveform recorded from animal's concha in a sound-attenuating chamber.Fig. 2The responses of a PL/Type I/III AVCN unit to clicks and click pairs. Upper right panel shows the PSTH of the response of this unit to tone bursts at characteristic frequency (CF). The coefficients of variation (CV) for this unit were 0.62 and 0.57 in the 2–14.9 and 20–39.9-ms time windows (called CV2–14.9 and CV20–39.9), respectively. Responses to single click and click pairs at interclick intervals (ICI) between 1 and 16 ms at 65 and 85 dB SPL are shown in the two left columns. Lower right panel shows the second click response as a function of ICI at 45, 65 and 85 dB SPL and represents the percentage of recovery of second-click responses.Fig. 3The responses of a Chop-S/Type III AVCN unit to clicks and click pairs. The format of this figure is the same as Fig. 2. CV2–14.9=0.1; CV20–39.9=0.14; Ronset/Rsust.=1.0.Fig. 4The responses of a Chop-S/Type III AVCN unit to clicks and click pairs. The format of this figure is the same as Fig. 2 except that click levels of 105 dB SPL are included. CV2–14.9=0.27; CV20–39.9=0.23; Ronset/Rsust.=1.7.Fig. 5The responses of an On-C/Type I/III AVCN unit to clicks and click pairs. The format of this figure is the same as Fig. 2. CV2–14.9=0.07; there were no spikes in the 20–39.9-ms window to calculate the CV and Ronset/Rsust..Fig. 6The responses of a Chop-T AVCN unit to clicks and click pairs. Upper right panel shows the PSTH of the response of this unit to tone bursts at CF. The format of this figure is the same as Fig. 2. CV2–14.9=0.34; CV20–39.9=0.61; Ronset/Rsust.=1.3. This unit was classified as Chop-T type based the CV-vs.-time pattern even though chopping was not visible in the PSTH.Fig. 7The responses of an On-L/Type I/III AVCN unit to clicks and click pairs. The format of this figure is the same as Fig. 2. CV2–14.9=0.14; there were insufficient spikes in the 20–39.9-ms window to calculate the CV; Ronset/Rsust.=4.5.Fig. 8The second-click recovery function for primary-like (left column), chopper (middle column) and onset (right column) AVCN units at the four stimulus levels (45, 65, 85 and 105 dB SPL). The thick line in each panel represents the mean second-click response recovery function. The number of units represented, N, is shown in each panel. Only those units with data available for three or more ICIs are included.Fig. 9Mean second-click response±standard error of means (S.E.M.) vs. ICI for the three main PSTH types (primary-like, chopper and onset; panels a–c) and the three AVCN EIA types (Type I, Type I/III and Type III; panels d–f) at four stimulus levels (45, 65, 85, and 105 dB SPL). Mean recovery function for Type I units at 45 and 105 dB SPL are not available due to the small sample size.Fig. 10Comparison of SR distribution of AN fiber and AVCN unit. The number of units represented, N, is shown in each panel. AN data are from Kim et al. (1991).Fig. 11Mean second-click recovery functions (±S.E.M.) for two populations of AVCN neurons at four stimulus levels. AVCN units are divided into a low-SR (SR<20 spikes/s) group and a high-SR (SR>20 spikes/s) group.Fig. 12Comparison of mean second-click recovery functions among low-SR and high-SR AN fibers and AVCN neurons at four stimulus levels (45, 65, 85 and 105 dB SPL). Data from AN fibers at 105 dB SPL are not available.Fig. 13Mean spike counts (a), absolute number of spikes (b) and mean response duration (c) in response to single clicks as a function of stimulus level among low- and high-SR AN fibers and AVCN units at four stimulus levels (45, 65, 85 and 105 dB SPL).Table 1Number of AVCN units in PSTH and EI-area classification schemesPSTH TypeEI-area typeII/IIIIIIII or IVUnclassifiedTotalPercentPL6918174134Chop02223395747On073141512Unusual0410387Total64245523121Percent53537419Responses of anteroventral cochlear nucleus neurons of the unanesthetized decerebrate cat to click pairs as simulated echoesKParhamH.-BZhaoYYeD.OKim*Division of Otolaryngology, Surgical Research Center, Department of Surgery, Neuroscience Program, The University of Connecticut Health Center, Farmington, CT 06030-1110, USA*Corresponding author. Tel.: +1 (860) 679-3690; Fax: +1 (860) 679-2451; E-mail: kim@neuron.uchc.eduAbstractTo elucidate the contribution of the anteroventral cochlear nucleus (AVCN) to `echo' processing, this study documents the responses of AVCN neurons to simulated echoes and compares them to those of auditory nerve (AN) fibers. Single unit discharges were recorded from 121 units in the AVCN of 21 unanesthetized decerebrate cats in response to click pairs with inter-click intervals ranging from 1 to 32 ms between 45 and 105 dB SPL re 20 μPa. Units were classified according to the post-stimulus time histogram (PSTH) and excitatory-inhibitory response area (EI-area) schemes. Based on their spontaneous rates (SR), units were subdivided into low- (<20 spikes/s) and high- (>20 spikes/s) SR groups. A majority of the units exhibited second-click responses whose recovery time courses were similar to those of AN fibers. These units included primary-like, chopper and onset units in the PSTH scheme and Types I, I/III and III units in the EI-area scheme. A minority of the units exhibited responses that were distinct from those of AN fibers, in that they had second-click response recovery times that were either markedly reduced or prolonged. This group of units included those with primary-like, chopper and onset PSTHs and Type I/III and III EI-areas. No significant difference was found in the second-click response among various PSTH or EI-area types. High-SR AVCN units exhibited a decrease in the second-click response with increasing level. In contrast, low-SR AVCN units showed little level-dependent change in the second-click responses. This SR-based difference was similar to that previously found among AN fibers. The present results suggest that, although a majority of AVCN units exhibit similar time courses of second-click response recovery to those of AN fibers, there do exist mechanisms in the cochlear nucleus that can substantially alter this representation. Furthermore, the difference between the second-click response recovery functions of low- and high-SR AVCN units and the consistency of this finding between AVCN and AN suggest that SR represents an important dimension for signal representation in the AVCN neurons.KeywordsAnteroventral cochlear nucleusEcho processingClick pairSpontaneous rate1IntroductionIn a reverberant environment, such as a lecture hall, an acoustic signal originating from one location is typically followed by its echoes that arrive at the ears from multiple directions. Nevertheless, a listener can correctly perceive a single auditory image and its correct direction. This phenomenon of the first wavefront taking precedence over later-arriving wavefronts has been referred to as the `law of the first wavefront' or `precedence effect' (Wallach et al., 1949; Haas, 1951; Von Bekesy, 1960; Blauert, 1997). To gain an understanding of the mechanisms involved in auditory processing of the echoes, it would be useful to study this phenomenon across multiple structures within the ascending auditory pathway.We recently reported on echo processing features of the auditory nerve (AN) fibers in the unanesthetized decerebrate cat (Parham et al., 1996). A main finding was that the recovery of the response to the trailing click of a click pair among AN fibers not only depended on stimulus parameters (i.e., interclick interval and click level) but also on fiber spontaneous rate (SR). High-SR fibers exhibited longer recovery times than their low-SR counterparts.The cochlear nucleus (CN) serves as the obligatory termination site of AN fibers. Anatomically, it can be subdivided into three main divisions: the anterior and posterior ventral cochlear nuclei (AVCN and PVCN, respectively) and the dorsal cochlear nucleus (DCN). Each subdivision is composed of populations of neurons that, by virtue of different combinations of intrinsic membrane properties and synaptic inputs, can express diverse responses to sound. As a result, the CN consists of an array of neurons whose responses fall on a continuum from a faithful preservation to a substantial transformation of the incoming afferent signals. These responses are conveyed to higher auditory centers along parallel pathways arising from the CN subdivisions.Based on their responses to sounds, CN neurons have been characterized according to two schemes: temporal discharge patterns reflected in the post-stimulus time histogram (PSTH) scheme (e.g., Pfeiffer, 1966) and the expressions of excitatory and inhibitory regions and responsiveness to broad-band noise reflected in the excitatory-inhibitory area (EI-area) scheme (e.g., Young, 1984). The response types within the two schemes appear to be distributed differentially within CN subdivisions and are correlated with different morphological classes of neurons in the CN. In the present context, the anatomical and physiological diversity of CN neurons gives rise to three important questions: (i) Is echo representation among CN neurons different from that among AN fibers? (ii) Do various physiological classes of CN neurons differ from one another with respect to echo representation? (iii) Are SR-based differences in echo processing of AN fibers also present in the CN?Recently, Wickesberg (1996)reported that ventral cochlear nucleus (VCN) units showed rapid recovery of the second-click response in the anesthetized chinchilla. Units characterized by primary-like and chopper PSTHs showed 50% recovery by 2 ms interclick interval and a nearly complete recovery by 4 ms. Wickesberg also reported some variability in the responses of VCN neurons, in that a subset exhibited little to no reduction or enhanced response to the second click.In this study we attempt to elucidate the contribution of AVCN to echo processing. We systematically investigated the behaviors of AVCN neurons regarding their response recovery functions under a click-pair stimulus paradigm in decerebrate unanesthetized cats. In this assessment of AVCN behavior in response to click pairs, neurons were characterized according to both PSTH and EI-area schemes and SR-based differences were investigated. Early results of this study were presented at a meeting of the Association for Research in Otolaryngology (Kim et al., 1992).2MethodsThe care and use of animals reported on in this study were approved by the University of Connecticut Health Center Animal Care Committee (protocol title: `Physiology of the Cochlear Nucleus'; protocol number: 91-014-97-2). The details of Section 2are described elsewhere (Parham et al., 1996). Briefly, we used unanesthetized decerebrate cats. We opened the left posterior fossa, aspirated a portion of the cerebellum, and exposed the CN. A micropipette was positioned over the AVCN which was visualized through an operating microscope. Single unit spike discharges were recorded using micropipettes filled with 3 M sodium chloride or potassium acetate that had resistances of 20–50 MΩ. In some penetrations the recording location was marked by iontophoretic injection of markers, 10% biotinylated dextran amine or 10% horseradish peroxidase in 0.5 M potassium acetate in Tris buffer. As the electrode was advanced, we applied a tonal search stimulus whose frequency was swept with a triangular modulating waveform over 0–50 kHz with a modulation repetition period of 2 or 4 s. The level of the swept tone was in the range of 40–60 dB SPL (re 20 μPa). For each AVCN unit encountered, we recorded the following: (i) spontaneous discharge rate based on a 10- or 20-s sample without acoustic stimulation; (ii) response area curve representing discharges induced by 200-ms tone bursts (two repetitions) at various frequencies spaced at 50 frequencies per decade on a logarithmic scale and at a constant dB SPL, typically 10–30 dB SPL; from the response area we estimated the characteristic frequency (CF) (the frequency to which a unit is most sensitive) of the unit; (iii) responses to 60-dB SPL CF tone bursts (50 ms duration, 500 ms repetition interval, 5 ms rise/fall time, 60 repetitions) from which a post-stimulus time histogram was constructed; (iv) responses to 200-ms noise bursts (three repetitions, 5 ms rise/fall; 1000 ms repetition interval) in 5-dB steps between 20–70 dB SPL (measured as rms level with A-weighting); and (v) responses to single rarefaction clicks and equilevel click pairs with inter-click intervals (ICI) ranging from 1 to 32 ms; repetition period of 500 ms, 40 repetitions, at 45, 65, 85 and 105 dB SPL peak. The rarefaction clicks were produced by applying 30-μs pulses to the earphone. The acoustic waveform of a single click recorded in the concha of the cat is shown in Fig. 1.Event times for spike discharges and stimulus markers were stored with 20 μs resolution in the data files such that subsequent `off-line' analyses would have access to full information about spike discharge and stimulus events. Single units were classified according to the PSTH and EI-area schemes. Discharges of all units were examined for regularity based on the behavior of the coefficent of variation (CV) of spike discharges as a function of time (e.g., Young et al., 1988; Parham and Kim, 1992). Chopper units were subdivided into three subtypes: Chop-S (CV<0.35); Chop-T (CV increased from <0.35 to >0.35 within the first 15 ms of the response) and Chop-O (CV>0.35). Based on PSTH of the responses to pure tone at CF, onset units were identified based on a quantitative criterion that the ratio of the discharge rate over the first 5 ms of the response (Ronset) to the last 40 ms of the response (Rsust.) be greater than 2.5.The procedure for analysis of single unit responses to click pairs is described in detail elsewhere (Parham et al., 1996). Briefly, we examined the response of each AVCN unit to single clicks and click pairs in the form of PSTHs with binwidths of 0.08 ms. For graphical illustrations binwidths of 0.2 or 0.4 ms were used. To quantify the results, we defined the time window that included all of the evoked spike discharges to the single click as W1. W1 was defined for each unit and at each click level because response latency and duration varied among units and as a function of stimulus level. W1 served as the reference time window for subsequent computations of the responses to a click pair. The time window of the response to the second click of a pair (W2) was defined as a window identical to W1 except for a delay by the amount of ICI. The second-click response (SCR) was expressed as percentage of the response to the first click. At short ICIs, the quantification of the SCR took into account the tail end of W1 which overlaps with W2 (for details see Parham et al., 1996).At each ICI the level dependent differences between SCRs of various unit types were statistically evaluated using analyses of variance (ANOVAs). Significant interactions were followed up by lower order ANOVAs and significant main effects were further examined with Tukey HSD tests of mean comparisons.3ResultsThis study is based on the responses of 121 AVCN units recorded in 21 cats. Table 1 shows the number of AVCN units subdivided according to the PSTH and EI-area schemes. In the PSTH scheme, the majority of the units (93%) exhibited either primary-like (PL, 34%), chopper (Chop, 47%) or onset (On, 12%) PSTHs. The remaining units (7%) had unusual or non-descript PSTH appearances. The PL group (N=41) included primary-like (98%) and primary-like-with-notch (2%) PSTH types. The Chop group (N=57) was further subdivided into sustained (Chop-S, 33%), transient (Chop-T, 53%) and other (Chop-O, 14%) chopper subtypes. The On group (N=15) consisted of units exhibiting onset without sustained discharges (On-I, 13%), onset chopping (On-C, 47%), onset with low level of sustained activity (On-L, 33%) and onset followed by inhibition of spontaneous activity (On-inh, 7%). In the EI-area scheme, the majority of the units (77%) were characterized as Type I (5%), I/III (35%) or III (37%) with a minority falling into other categories (Type II, 3%; Type IV, 1%). For the remaining 19% of the units, response to broadband noise was unavailable and these units were not classified in the EI-area scheme. Among the present PL units, 18 out of 41 units were EI-area Type III. This is a much greater tendency than a previous report (Shofner and Young, 1985). The reason for this difference is not clear.Fig. 2 shows the responses of a low-SR PL/Type I/III unit to single clicks and click pairs. Responses of this unit to single clicks at 65 and 85 dB SPL peak are shown in the top PSTHs of the two left-most columns. The response was characterized by a large peak followed by a gradual decay of the response over 5–6 ms. Responses to click pairs at short ICIs (1–3 ms), shown in the remaining rows of the two columns, tended to be slightly more prolonged than the response to single clicks. At larger ICIs the response to the second click was evident. As the ICI increased, the second-click response gradually increased in strength such that by 16-ms ICI, the second-click response was similar to the first-click response. The lower right panel provides the magnitude of the second-click response as a function of ICI at 45, 65 and 85 dB SPL. The second-click response gradually recovered from near 25% at 1-ms ICI to above 50% by 4-ms ICI and to near a complete recovery (80–100%) by 16-ms ICI. This PL/Type I/III unit exhibited a slower recovery of the second-click response as click level was increased from 45 to 85 dB SPL.The behavior of the sample PL/Type I/III unit shown in Fig. 2 was typical of a majority of the units recorded from the AVCN in this study (to be shown further below) and resembled the behavior of AN fibers recorded under similar conditions (Parham et al., 1996). However, a minority of the units exhibited responses to click pairs that were distinct from the typical behavior resembling AN fibers. The next five figures (Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7) illustrate units characterized by sustained and transient responses to pure tones that exhibited click pair responses that were distinct from that of the AN fibers.The three examples shown in Figs. 3–5 exhibited faster recovery of the second-click responses than AN fibers. The responses of a Chop-S/Type III AVCN unit to single clicks and click pairs is shown in the two left-most panels of Fig. 3. This unit's responses preserved the representation of both clicks of a pair at low and moderate stimulus levels (45 and 65 dB SPL), even at ICIs as short as 1 ms. At 45 dB SPL, the PSTH of the response of this unit to single clicks consisted of a single narrow peak. At 1-ms ICI the response to the second click is distinct and nearly equal to that of the first click. At 85 dB SPL, the single click response consisted of two peaks, separated by an interval (1.8 ms) which is approximately equal to the interpeak interval of the PSTH of the response to the CF tone (upper right panel). The response to click pairs at 1-ms ICI was similar to that of the single-click response except that the second peak of the PSTH was narrower and taller (i.e., distributed across fewer bins). A distinct response to the second click was present at 2-ms ICI which gradually recovered as ICI was increased to 32 ms. The lower right panel shows the second-click response recovery functions of this unit. At 45 and 65 dB SPL the second-click response remained near 100% for all ICIs, but at 85 dB SPL the second-click response recovered from 0% at 1-ms ICI to 100% at 32 ms.The responses of another Chop-S/Type III AVCN unit are shown in Fig. 4. This unit exhibited a poor recovery of the second-click response at a low stimulus level for ICIs even up to 32-ms ICI, but strong second-click response at high stimulus level even at 1-ms ICI. At 45 dB SPL a weak response to the second click was present by 2-ms ICI. Unlike previous examples, however, second-click response was completely absent by 8-ms ICI and remained below 50% for ICIs up to 32 ms. In contrast, at 105 dB SPL the second-click response was present at 1-ms ICI in the form of a broad peak. By 2-ms ICI the second-click response was nearly the same as that of the first. The second-click recovery functions of this unit are shown in the lower right panel. At 45 dB SPL the second-click response remained below 50%, but at 105 dB SPL the second-click response was recovered to 85–100% across all ICIs examined. At intermediate levels of 65 and 85 dB SPL the second-click response exhibited more rapid recovery than that expected from AN-like recoveries.Fig. 5 shows the responses of an On-C/Type I/III unit. At 65 dB SPL the second-click response was strong (>75%) across all ICIs. At other click levels the recovery of the second-click response was more gradual, although somewhat faster than those exhibited by AN fibers. For example, at 85 dB SPL the response to the second click was greater than 75% at ICIs beyond 3 ms.The next two examples shown in Figs. 6 and 7 exhibited slower recovery of the second-click response recovery function than AN fibers. The responses of a Chop-T AVCN unit is shown in Fig. 6. The second-click response of this unit at 45 dB SPL exhibited a strong response at 1-ms ICI, but weak responses at 3–8-ms ICIs. The second-click response at 65 dB SPL was similar to that of 45 dB SPL (lower right panel). At 85 dB SPL this unit exhibited a more gradual recovery (middle column and lower right panel).The responses of an On-L/Type I/III AVCN unit are shown in Fig. 7. This unit was characterized by a weak second-click response up to 32-ms ICI for all stimulus levels tested. Interestingly, the second-click response increased between 2- and 8-ms ICIs, but decreased for longer ICIs.Fig. 8 shows recovery functions for populations of primary-like (left column), chopper (middle column) and onset (right column) units at 45–105 dB SPL. Individual curves in Fig. 8 represent separate units, while the thick line in each panel represents the mean second-click response recovery function. In general, the majority of the units exhibited recovery functions qualitatively similar to those observed at the level of the AN. However, a small number of non-AN-like recovery functions were observed in all three populations. Examples of fast recovery to the second click can be seen in primary-like (panel d), chopper (panels b, e and k) and onset (panels c and f) units. Examples of weak response to the second click can also be seen in primary-like (panel g), chopper (panels b, e, h and k) and onset (panels f, i and l) units.The mean recovery functions of the three main AVCN PSTH types are shown in Fig. 9a–c. Primary-like units showed little change in the mean second-click response with the click level (Fig. 9a). Only at 16- and 32-ms ICIs were the second-click responses to 45 and 65 dB SPL significantly greater than those at 105 dB SPL stimuli (Tukey HSD, P<0.05). Chopper units exhibited an orderly weakening of the mean second-click response as click level was increased from 45 to 85 dB SPL for 1–3-ms ICIs (Fig. 9b). However, at 105 dB SPL the chopper units showed the strongest mean second-click responses for 1–3-ms ICIs. The mean second-click response of the onset units also decreased with increasing stimulus level (Fig. 9c). However, the level-dependent differences among chopper and onset units were not statistically significant (P>0.05).The mean second-click recovery functions of the three unit types were similar at 65 and 85 dB SPL. At 45 dB SPL the onset units exhibited more rapid recovery of the second-click to above 75% than the primary-like and chopper units (i.e., 3 vs. 4 ms, respectively). However, at 105 dB SPL the onset units exhibited slower recovery of the second-click response than the primary-like and chopper units (75% SCR: 7 vs. 6 ms, respectively). At 45 and 105 dB SPL the mean second-click recovery functions of the primary-like units were intermediate to those of the chopper and onset units. The differences between the three PSTH types were not statistically significant (P>0.05).The mean second-click recovery functions for the three EI-area types are shown in Fig. 9d–f. The mean second-click response of the Type I group increased as the stimulus level increased from 65 to 85 dB SPL (Fig. 9d). Because of the small sample size for Type I units mean second-click response recovery functions are not available at 45 and 105 dB SPL. The mean second-click response decreased from 45 to 85 dB SPL for Type I/III and III units (Fig. 9e,f). However, at 105 dB SPL Type I/III and III units showed an increase in the mean second-click responses relative to those at 85 dB SPL (Fig. 9e,f). The mean second-click response recovery functions of the three EIA types differed little when compared at each click level.Fig. 10 shows the SR distributions for AVCN units recorded in this study and a sample of AN fibers recorded in a previous study (Kim et al., 1991). The AVCN units were divided into low- (<20 spikes/s, N=76, 63%) and high-SR (>20 spikes/s, N=45, 37%) groups, as was previously done for AN fibers (Parham et al., 1996). The mean second-click response recovery functions of these two groups are shown in Fig. 11. Low-SR AVCN units showed little level-dependent changes in the second-click response recovery functions between 45 and 85 dB SPL. Interestingly, at 105 dB SPL the low-SR AVCN units exhibited stronger second-click responses than at lower stimulus levels. One-way ANOVAs at each ICI indicated a significant main effect of level only at the 2-ms ICI (F(3,124)=3.42, P=0.02). In contrast, the high-SR AVCN units exhibited a more noticeable level-dependent decrease in the second-click response between 45 and 105 dB SPL. This decrease was statistically significant at 3-, 4- and 6-ms ICIs (P<0.02). The mean recovery functions of the two AVCN SR groups are compared to one another and their AN counterparts in Fig. 12. At 45 and 65 dB SPL, the four populations exhibited similar second-click response recovery functions. One-way ANOVAs at 45 and 65 dB SPL showed no significant differences in mean second-click responses of the two SR groups. At 85 dB SPL, the high-SR AVCN and AN fibers exhibited weaker second-click response recoveries than their low-SR counterparts. Two-way ANOVAs at each ICI at 85 dB SPL revealed main effects of SR at all ICIs less than 32 ms (P<0.05) without any significant interaction with location (i.e., AN vs. CN).At 105 dB SPL differences in second-click response between the low- and high-SR AVCN units are much more evident, with the high-SR AVCN units having weaker second-click response for ICIs less than 32 ms. The AN data for 105 dB SPL are not available. ANOVAs indicated that the second-click responses of the high-SR AVCN units were significantly weaker than their low-SR counterparts at all ICIs less than 8 ms (P<0.05).Since the second-click response recovery functions presented above are a relative measure, they do not convey information about the absolute neural activities. The latter information can be inferred by combining the relative recovery functions with an absolute measure of neural activities for various stimulus conditions. For this purpose, we examined the number of spikes elicited by single clicks at various levels, as shown in Fig. 13. For all groups, the mean number of spikes elicited by single clicks increased as the stimulus level increased (Fig. 13a). However, this increase was substantially larger for the high-SR AVCN and AN units. Consequently, the low-SR AVCN units and AN fibers consistently exhibited fewer spikes per click than their high-SR counterparts across all levels. The duration of the single-click response is shown in Fig. 13c. Although the response duration increased as stimulus level increased for all four groups, the response durations of high-SR AVCN units and AN fibers were consistently longer than those of their low-SR counterparts.4DiscussionA majority of AVCN units recorded in this study exhibited second-click response recovery time courses that were similar to those of AN fibers. These units had primary-like, chopper and onset PSTHs and Types I, I/III and III EI-areas. A minority of AVCN units, however, exhibited second-click responses whose recovery time courses were markedly reduced or prolonged relative to those of AN fibers. The latter group of units had primary-like, chopper and onset PSTHs and Type I/III and III EI-areas. The mean second-click response recovery functions showed little difference among the three main PSTH and EI-area types. When AVCN units were subdivided into low- and high-SR groups, however, a clear difference in the second-click response recovery functions was found between the two SR groups.In this study, the responses of many AVCN neurons to single click stimuli were often prolonged. For example, multipeaked PSTHs of click responses were relatively common in low-CF (i.e., below 2 kHz) primary-like units. Similar to previous observations in the AN, the PSTH of the responses of these low-CF primary-like units to clicks had a multi-peaked appearance, with the interpeak interval being equal to the 1/CF interval. Some high-CF CN units also had PSTHs of the response to click stimuli that were multi-peaked (e.g., Figs. 3, 5 and 6). However, as previously noted by Kiang (1965), the inter-peak intervals in these high-CF units did not correspond to 1/CF. Kiang's report did not classify CN units according to the PSTH type. In the current study, multi-peaked PSTHs among high-CF units were associated with chopper and onset-chopper type units. The inter-peak intervals in the PSTHs of the response to the clicks in these units approximated those in the PSTHs of the responses to pure-tone bursts at their CFs.The level-dependent weakening of the second-click responses of high-SR AVCN units and their slower recovery relative to low-SR AVCN units are analogous to our earlier observations in the AN (Parham et al., 1996). Spontaneous activities of AVCN neurons appear to be mainly dependent on their AN inputs. Koerber et al. (1966)observed that, after cochlear destruction, spontaneous activity of AVCN neurons was abolished suggesting that SR of AVCN neurons mainly arises from their AN afferent inputs. Therefore, the differences between the two SR groups in the AVCN for the most part appear to be a reflection of their AN afferent inputs.The results presented in this report offer a possible reason for the difference in the dependence of the second-click response on the level between the two SR populations of the AN and AVCN, i.e., neuronal refractoriness. Gaumond et al. (1982)characterized refractoriness of AN fibers in terms of a conditional discharge probability conditioned on the time since the last spike, called tau, for a steady stimulus. The conditional discharge probability of an AN fiber typically was zero (i.e., completely refractory) for tau<0.75 ms, rapidly increased from zero to about 60% of a fully recovered value at tau=4 ms, and more gradually increased to the fully recovered value (i.e., zero refractoriness) between 4 and about 40 ms. Gaumond et al., 1982(Figs. 12 and 13) also observed that, when there were successive short (e.g., <10 ms) interspike intervals, the refractoriness of AN fibers was accumulated, i.e., the conditional discharge probability further decreased. The fact that the time courses (0 to about 40 ms) and general shapes of the two measures, i.e., second-click response vs. inter-click interval and refractoriness vs. time since the last spike, are quite comparable to each other, supports the possibility that neuronal refractoriness is the main mechanism underlying the reduction and a gradual recovery of the second-click response being described in the present report and in Parham et al. (1996). In Fig. 13a, it is seen that, as the stimulus level was increased from 45 to 105 dB SPL, the mean absolute number of spikes increased from about 1 to 3 spikes per click for high-SR AN and AVCN units whereas they changed little for low-SR AN and AVCN units. It is also seen in Fig. 13c that the multiple spikes at high stimulus levels occurred over a period of about 4–10 ms. This corresponds to interspike intervals of about 2–4 ms.In view of the Gaumond et al. study discussed above, the refractoriness of a high-SR unit should be substantially greater during a period following the clicks presented at high levels than that at low levels because additional spikes were discharged with short interspike intervals at high stimulus levels. This expected change in the amount of refractoriness of high-SR units with stimulus level is consistent with the decrease of second-click response observed in high-SR AVCN units (Fig. 11) and AN fibers (Parham et al., 1996, Fig. 7). Likewise, this reasoning as applied to low-SR units regarding their absolute number of spikes per click being nearly independent of stimulus level (Fig. 13a) predicts that their second-click response recovery functions should be nearly independent of stimulus level. Low-SR AVCN units (Fig. 11) and AN fibers (Parham et al., 1996, Fig. 7) indeed exhibited much less dependence of their second-click recovery functions on stimulus level than their high-SR counterparts, largely consistent with the prediction. There is one discrepancy between the refractoriness-based prediction and the observation in this regard. That is, the second-click response of low-SR AVCN units increased somewhat at 105 dB SPL compared with those at lower levels (Fig. 11, left panel) whereas, based on Fig. 13, one predicts that it should remain nearly constant with a slight decrease. The reason for this discrepancy is not clear.When one considers that the discharge behavior of an AVCN neuron is affected by neuronal refractoriness, two components of refractoriness can be considered: (i) refractoriness of AN fibers acting as an input to the AVCN neuron, and (ii) refractoriness of the AVCN neuron. The present observations (Fig. 12) indicate that the second-click response recovery functions are rather similar between the AN fibers and AVCN neurons. In interpreting the second-click response recovery functions in terms of neuronal refractoriness, this similarity implies that the combined total refractoriness observed in AVCN units is similar to that in AN fibers. To our knowledge, direct examinations of neuronal refractoriness (analogous to the Gaumond et al. study) for AVCN neurons have not been reported. We believe that such a study should be useful.The faster recovery of the second-click response of low-SR AN fibers and AVCN neurons than their high-SR counterparts contrasts with findings of forward masking studies using tone bursts at the level of the AN (Relkin and Pelli, 1987; Relkin and Doucet, 1991) and CN (Boettcher et al., 1990; Shore, 1995). Forward masking studies noted that low-SR units required longer times for recovery of responses to tone bursts than high-SR units, opposite of the present observation of the second-click response. Previously, we considered that possible reasons for the discrepancy might include longer signal duration (tone burst) and a large level difference between the masker and probe in the forward masking studies compared with click pairs of the present studies (Parham et al., 1996). Even when the masker and probe levels were nearly equal in the CN forward masking studies, low-SR neurons showed slower recovery than high-SR neurons (Boettcher et al., 1990; Shore, 1995). Therefore, the difference between masker and probe levels does not explain the discrepancy between click-pair and forward masking studies. Different mechanisms may underlie the two phenomena. Adaptation at the inner hair cell and its afferent synapse and suppression of cochlear responses mediated by the olivocochlear efferent system (Bonfils and Puel, 1987) may mainly underlie the forward masking phenomenon whereas neuronal refractoriness may mainly underlie the second-click response reduction phenomenon. We do not expect that the second-click response recovery function is mainly mediated by the efferent system because the click stimulus had a brief duration (Fig. 1) presented with a long (500 ms) repetition period, whereas efferent-mediated effect required a sufficiently long (greater than 35 ms) stimulus (Bonfils and Puel, 1987).The similarity of the recovery rates of low- and high-SR AVCN neurons to their AN counterparts supports the notion that SR may be an important axis in signal representation at the level of the CN. This conclusion is consistent with that of Shore (1995)in her study of forward masking in the VCN. The suggestion of an SR-based segregation of AN fiber projections to the CN, first made by anatomical studies (Leake and Snyder, 1989; Kawase and Liberman, 1992; Liberman, 1991), has received additional neurophysiological support. Recent recordings from the AVCN showed that neurons in the marginal shell were predominantly low SR, whereas the central core included high- and low-SR neurons (Ghoshal and Kim, 1996Ghoshal and Kim, 1997).Since a majority of the AVCN units display AN-like behavior with respect to second-click responses, these neurons do not modify the AN afferent inputs but act to relay AN signals to higher auditory centers. In a forward masking study of CN neurons, Boettcher et al. (1990)arrived at a similar conclusion. A minority of AVCN units, however, showed echo processing behaviors that were distinct from those of the AN fibers. These units had primary-like, chopper and onset PSTH types. Forward masking studies in the CN have also identified neurons whose recovery from short-term adaptation is distinct from those of AN fibers (Boettcher et al., 1990; Shore, 1995). In vivo intracellular recording and labeling experiments have shown that PSTH types tend to be associated with morphological cell types in the AVCN (Rhode et al., 1983; Rouiller and Ryugo, 1984; Smith and Rhode 1987, 1989; Ostapoff et al., 1994). For example, the primary-like responses are generally associated with bushy cells, whereas chopper responses are generally associated with multipolar cells. Since the unusual second-click recovery behaviors of some AVCN neurons were not associated with a distinct response type in the AVCN, the present observation suggests that these novel echo processing strategies may not be associated with a unique cell type in the AVCN.The unusual echo processing patterns of AVCN neurons consisted of neurons with either enhanced or suppressed representation of the echo relative to that of the AN fibers. The enhancement of the second-click response was observed at short ICIs (i.e., less than 4 ms) where individual AN fibers show typically weak responses (less than 50%; Parham et al., 1996). The present study is the first to demonstrate the existence of neurons with very fast second-click response recovery times in the AVCN. One possible way to extract adequate information regarding the temporal position of an echo, is for some AVCN neurons to sample and integrate the responses of many AN fibers. In other words, convergence of several AN fiber inputs onto an AVCN neuron could lead to summation of temporally coincident excitatory post-synaptic potentials thus eliciting an action potential. This is analogous to previously proposed mechanisms for certain VCN neurons (e.g., Smith and Rhode, 1986; Winter and Palmer, 1995; Palmer et al., 1996). Responses of spherical bushy cells in the AVCN are dominated by their AN fiber inputs which are believed to be form one or two calyx-type endings on one bushy cell (e.g., Sento and Ryugo, 1989). Therefore, the spherical bushy cells are a poor candidate for this type of integration. More likely candidates are multipolar cells receiving bouton-type endings from the AN which are distributed over their soma and proximal dendrites (Cant, 1981).The suppression of the second-click responses beyond that exhibited by the AN fibers is likely mediated by circuits releasing inhibitory neurotransmitters such as glycine and/or GABA in some CN neurons (Backoff et al., 1997). Suppression of the second-click response of AVCN neurons could arise partly from intranuclear and/or descending inhibitory projections to AVCN neurons. The intranuclear mechanisms that may underlie such suppression have been discussed in detail elsewhere (Wickesberg and Oertel, 1990; Wickesberg, 1996). Briefly, one hypothesized source is the tuberculoventral neurons of the DCN which provide glycinergic input to the AVCN. In vitro excitation of these neurons through stimulation of AN fibers which project to tuberculoventral neurons, as well as AVCN neurons, showed inhibitory postsynaptic potentials in AVCN neurons about 2 ms after AN induced excitatory postsynaptic potentials. Wickesberg (1996)provided further support for this circuit in a pharmacological perturbation study. Wickesberg showed that, following application of lidocaine in the DCN, responses of 19% of the VCN units (primary-like and chopper type) to the trailing click increased. Such inputs could account for the trough-like recovery functions observed in this study (e.g., Fig. 6). For 62% of the VCN units, however, Wickesberg found that the response to the first click decreased after lidocaine administration. The reason for the latter unexpected finding is not clear.In the AVCN, ICI at 50% recovery of the second-click response ranged from 1.8 ms at 45 dB SPL to 3 ms at 85 dB SPL without any substantial differences between the two AVCN SR groups (Fig. 12). This compares with 1.3–3 ms over the same stimulus level range in the AN (Parham et al., 1996). Thus, there was only a very slight increase in the average 50% recovery time in AVCN relative to the AN. The recovery functions of AVCN units are similar to those reported for units in the SOC by Fitzpatrick et al. (1995). Monaural units in the SOC exhibited second-click recovery functions that were analogous to those of AVCN neurons. The average 50% recovery time of monaural units in the SOC was ∼2 ms (Fitzpatrick et al., 1995). The fact that recovery functions in the SOC are comparable to those in the AVCN with the average 50% recovery times being similar in the two nuclei suggests that no significant modification of the echo processing occurs between these two structures. Substantial alterations apparently occur higher up at the level of the IC as the average 50% recovery time increases to ∼6–20 ms (Yin, 1994; Fitzpatrick et al., 1995). Additional suppression mechanisms are interpreted to be present in the cortex, since in the auditory cortex the average 50% recovery times ranged up to 300–500 ms (Fitzpatrick et al. 1997, 1998).Some of the auditory cortical neurons were observed to exhibit rapid recovery times of <2 ms (Fitzpatrick et al., 1997). Considering that the AVCN also contains neurons exhibiting comparably rapid recovery times (e.g., Figs. 3–5), the projections of such AVCN neurons could form the beginning of a channel that may convey information about the presence and nature of echoes. Such information may underlie the quality of the sound heard, i.e., its timbre (e.g., Blauert, 1997; Harris et al., 1963; Sayers and Toole, 1964). The projections from the majority of AVCN neurons, whose second-click responses recovery times are 2–10 ms, may represent the beginning of a separate channel that may underlie the perception of the sound location, thus mediating the precedence effect which has a time window of about 2–10 ms (Wallach et al., 1949). In addition, certain neurons in the AVCN (present study) and higher auditory centers (e.g., Fitzpatrick et al. 1995, 1997), whose second-click recovery times are in the range of about ten to a few hundred milliseconds, may mediate a time window (`echo window') where a lagging sound (i.e., echo) is heard but its perception is still affected by a leading sound (Fitzpatrick et al., 1998).Based on the findings of our studies in the AN and AVCN, it is important in future studies to keep in mind two important variables: SR and stimulus level. In both AN and AVCN, the 50% recovery time of the second-click response increased from 1.3 and 1.8 ms, respectively, to 3 ms between 45 and 85 dB SPL. At 105 dB SPL, however, the 50% recovery time for low-SR AVCN neurons was less than 1.5 ms, while that of high-SR AVCN neurons was ∼5 ms. Results for the AN at 105 dB SPL are not available for comparison. Given that both SR and stimulus level exert a substantial influence on echo processing, greater attention needs to be paid to these variables when making comparisons across structures.Regarding psychophysical studies of precedence effect which examined the influence of the overall stimulus level, we are aware of only one, Shinn-Cunningham et al. (1993). Using two stimulus levels (80 and 110 dB SPL 1-ms noise bursts), they found: (i) that the stimulus level had relatively small influence on the strength of the precedence effect; (ii) that the strength of the precedence effect was little affected by the stimulus level for 1-ms ICI (or `lag' of Shinn-Cunningham et al.); and (iii) that the strength of the precedence effect tended to decrease somewhat at the higher stimulus level for 10-ms ICI. This psychophysical observation of a decrease of the precedence effect strength with increasing stimulus level for 10-ms ICI appears to be in opposite direction to what one may anticipate from the neurophysiological observations of an increase of the recovery time with increasing stimulus level (Fig. 11 of the present paper; Figs. 7 and 11 of Parham et al., 1996). The reason for this apparent discrepancy is not clear.To summarize, a survey of the available results throughout the auditory pathway suggests that the response to a trailing sound or echo can be either weakened (a characteristic of the AN fibers), enhanced or suppressed. As we ascend the auditory pathway, suppression appears to become more robust or prolonged, but an enhanced representation of a simulated echo continues to persist. These results suggest that, with respect to echo processing, the CN may be the origin of several parallel channels within which the AN signal is either relayed or altered on its way to higher centers.AcknowledgementsThis study was supported in part by the National Institute on Deafness and Other Communication Disorders, NIH, Grant P01-DC-01366. We thank Drs. S. Kuwada, D. Fitzpatrick, and A. Palmer for their helpful comments on this manuscript.ReferencesBackoff et al., 1997P.M.BackoffP.S.PalombiD.M.CasparyGlycinergic and GABAergic inputs affect short-term suppression in the cochlear nucleusHear. Res.1101997155163Von Bekesy, 1960Von Bekesy, G., 1960. Experiments in Hearing. McGraw-Hill, New York, NY.Blauert, 1997Blauert, J., 1997. Spatial Hearing: The Psychophysics of Human Sound Localization. MIT Press, Cambridge, MA.Boettcher et al., 1990F.A.BoettcherR.J.SalviS.S.SaundersRecovery from short-term adaptation in single neurons in the cochlear nucleusHear. 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