Article history:Accepted 18 July 2006Available online 17 August 2006
Duration selectivity appears to be a fundamental neural encoding mechanism foundthroughout the animal kingdom. Previous studies reported that band-pass duration-tunedneurons typically show offset responses and occupy a small portion of auditory neurons innon-echolocation mammals relative to echolocation bats. Therefore, duration tuning isgenerally weaker in non-echolocation mammals. In the present study, duration tuning wasanalyzed for 207 neurons recorded in the inferior colliculus (IC) of guinea pigs. Durationtuning was found to be stronger in the onset component of the responses from sustained,on–off and pause neurons than had been reported previously, when a short analysiswindowwas applied. The need for an appropriate time window for duration tuning analysiswas also supported by the fact that the on and off responses from an on–off neuron mayshow different duration tuning features. Therefore, duration tuning appears to be atransient neural coding process in the IC of guinea pigs. Duration tuning for these types ofneurons may have been blurred by the use of a relatively unselective, long window.
Duration is a salient temporal feature of acoustic stimuliincluding human speech. It provides critical communicationcues for terrestrial and flying vertebrates. Neurons tuned toduration were first reported in frogs (Narins and Capranica,1980; Potter, 1965). The best durations of these neuronscorrespond to the durations of conspecific vocalizations,especially calls for mating (Gooler and Feng, 1992). Durationcoding was more intensively investigated in several speciesof bats in which this coding mechanism has beenconsidered as a specific adaptation for echolocation (e.g.Casseday et al., 1994; Ehrlich et al., 1997; Fuzessery, 1994;
).o this paper.
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Mora and Kossl, 2004; Pinheiro et al., 1991). Recently,duration tuning has also been reported in different stationsof the auditory pathway in several non-echolocationmammals including the auditory cortex (AC) of cats (He etal., 1997), the inferior colliculus (IC) of chinchillas (Chen,1998), mice (Brand et al., 2000) and rats (Perez-Gonzalez etal., 2006), as well as the medial geniculate body (MGB) ofguinea pigs (He, 2002).
Although these previous studies have demonstrated thatduration tuning is a common feature in vertebrate auditorysystems, the neurons that show clear duration tuning occupya much smaller portion of the total population in these non-echolocation species than in the echolocation mammals that
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have been reported. In echolocation bats, band-pass andshort-pass units comprised approximately 30–60% of all ICneurons recorded and most of these band-pass neuronsshowed responses after the offset of signal, and thereforewere offset neurons (Casseday et al., 1994; Ehrlich et al., 1997;Faure et al., 2003; Fuzessery, 1994; Fuzessery and Hall, 1999;Galazyuk and Feng, 1997; Mora and Kossl, 2004; Pinheiro et al.,1991). However, in non-echolocationmammals, the band-passand short-pass tuned neurons were less than 20%. Consistentwith what has been reported in echolocation bats, band-passneurons in the IC of non-echolocation mammals were mostlikely offset units (Brand et al., 2000; Chen, 1998; Perez-Gonzalez et al., 2006). To date, duration tuning of theseneurons is much better documented and the potentialmechanisms for their duration tuning are much betterexplored than neurons in other categories of peristimulustime histogram (PSTH). Since offset neurons represent arelatively small population in non-echolocation mammals,duration tuning appears to be a less important signal processmechanism in these animals, unless duration tuning can beidentified in other neurons.
Therefore a question is raised as to whether the signalduration can be coded by other types of neurons. Thispossibility is supported by a study in the dorsal zone of theauditory cortex of cats (He et al., 1997). In this study, 13 out of132 long latency neurons were found to be band-pass tuned.Furthermore, duration tuning analysis methods utilized foroffset neurons may not be reasonable for other types ofneurons. For example, the total spikes of a sustained neuronusually increase with an increase of stimulus duration. Themeasures of the spikes/trial as a function of signal duration(the method for duration tuning used in the most previousstudies) will likely show a long-pass pattern. Whether thislong-pass behavior really represents duration tuning is ques-tionable. Rather, it may simply represent the accumulation ofthe neural spikes with elongated stimulation, the same as thebehavior of the primary auditory neurons, which are generallyconsidered not duration selective. On the other hand, it ispossible that the neural responses at different time segmentswith respect to the signal may be different in term of durationtuning. However, analyzing in a long time window may fail toreveal the duration tuning if it is only associated with a shortperiod of response time, or is a transient process. In our
Table 1 – Categorization of IC units according to PSTH and dura
a W stands for window.b For on–off neurons, the subtotal was counted in each duration-tuningresponses.
preliminary experiments, we did see that onset response peakof some sustained neurons changes with signal duration. Wethought that duration tuning might be a transient neuralcoding mechanism that could be demonstrated better in ashort time window, close to the temporal response peak inPSTH.
In the present study, we re-evaluated duration tuning inthe IC of guinea pigs, a species in which duration tuning hasnot been investigated in IC, in an attempt to verify if durationtuning is associated with the transient response. To do this,we analyzed and compared the duration tuning in differenttime windows, including short time windows related to theonset response and other temporal peaks. We found thatduration selectivity of IC neurons in guinea pigs is indeedstronger within short analysis windows that surrounded thetemporal response peaks.
2. Results
A total of 207 neurons were examined for duration tuningfrom the IC of guinea pigs. The best frequencies of theseneurons ranged from 2 to 40 kHz. Table 1 shows theclassifications of the neurons recorded in both duration tuningand PSTH. The classification of duration tuning was based onthe results obtainedwith broadband noise burst at 50 dB abovethreshold (a.T., the noise burst was the standard signal in thisreport) and evaluated through the short 30-ms time windowaround the temporal response peaks, which in most cases,was around the onset peaks, with the exception of the offsetneurons to which a sliding window of 30-ms that followed theoffset response peaks was used.
2.1. Sustained neurons
Of the total of 207 neurons, 64 (31.0%) had a sustainedresponse to the broadband noise burst at 50 dB a.T. Theseneurons appeared to have an onset peak followed by quicklydeclining, but sustained responses. The relative spike rate ofthe sustained response and that of the onset peak showedlarge variations among neurons. For the purpose of simplicity,we do not differentiate neurons that have a marked onsetpeak from those that do not.
category according to the analyses through the window at the onset
Fig. 1 – Duration tunings of a sustained unit. (A) Durationtuning curve for onset responses in a short window from 0 to30ms after onset of the stimulation, (B) duration tuning curvefor all responses in a long window from 0 to 300 ms. (C to F)PSTHs of the same neuron obtained at four signal durations.The stimuli arewhite noise at 50 dB a.T. The “0ms” in panelsC to F indicates the onset of stimuli.
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Similar to previous reports (Brand et al., 2000; Chen, 1998;Xia et al., 2000), most of the neurons in this group were notconsidered to be duration-tuned when the spikes werecalculated in a long window that covered the longeststimulation duration. However, we found that the onset
Fig. 2 – Duration tuning curves of 10 sustained neurons that showin a 300 ms long window (right).
responses appeared to be duration-tuned in some neuronsof this group. Fig. 1 shows the duration tunings of a sustainedneuron analyzed through two time windows: a short windowof 30 ms around the onset peak (A) and a long window of300 ms that covered the longest stimulus duration (B, thelongest signal duration was 256 ms). When the short timewindow was used, the neuron was band-pass tuned to the16ms (more than 50% drop in spike rate was seen on both sideof this best duration). However, when the long window wasused, the spike rate was roughly proportional to the signalduration and did not reach a plateau even at the longestduration tested. Of course, such a neuron would have beenjudged as long-pass or untuned if examined by using the longwindow. In our sample of total 64 sustained neurons, 10 werejudged as band-pass in duration tuning when the spike rateswere calculated in a 30-ms short time window (Fig. 2, left).These neurons would have been judged as long-pass or all-pass, if the spike rates were calculated in the long timewindow (Fig. 2, right). It was noted that the long-pass tuningcurves from more than half of neurons in this subgroup neverreached a plateau within the durations tested.
In addition to the band-pass response pattern, we foundtwo neurons in the sustained group whose duration tuningswere classified as a band-reject pattern, if their responseswere examined in the 30-ms window around the onset peak.Again, these two neurons would have been categorized asbeing long-pass or untuned if examined with the longwindow. Fig. 3 compares the duration tuning curves obtainedwithin the two timewindows for these two neurons. Using thelong window, the spike rate for unit 17-03 increased linearlywith the increased stimulus duration, while for unit 11-19, thespike rate reached a plateau when the duration was beyond16 ms.
Analyzed in the short time window around their onsetresponses, 14 out of 64 (21.9%) sustained neurons showedtuning to short durations. Fig. 4 compares the duration tuningcurves obtained with the two time windows for two neuronsin this subgroup. Analyzing through the short time windowdemonstrates that the onset responses of these two sustainedneurons were tuned to very short signal durations: 4 and 8ms,respectively. However, their onset responses dropped whenthe signal duration was elongated beyond the best durations.These neurons would otherwise be categorized as all-pass or
band-pass in a 30-ms shortwindow (left) but long- or all-pass
Fig. 3 – Duration tunings from two sustained neurons. Band-rejecting tuning was found when analyzed in a short timewindow, but it turned to long-pass pattern in a long time window. Left: the duration tunings; Right: PSTHs.
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long-pass, if the spike count was collected in the long timewindow.
In order to verify if the duration tuning was related withresponse latency, we compared the minimal latency betweenthe sustained neurons that showed no duration tuning (all-pass and long-pass, n=38) and these that were duration-tuned(band-pass, short-pass, and notch neurons, n=26). It wasfound that the duration-tuned sustained neurons had arelatively longer latency (mean±SD: 15.31±5.79 ms versus10.92±4.89 ms for duration-tuned and not duration-tuned
Fig. 4 – Duration tuning curves of two sustained neurons that apwindow. Long-pass or all-pass duration tuning was seen whenneurons in response to stimuli of four different durations.
A total of 24 (11.6%) neurons in this sample showed an on–offpattern in their PSTHs. As shown in the Table 1, 11 (45.8%), 3(12.5%) and 1 (4.2%) of the neurons in this subgroup presentedshort-pass, band-pass and long-pass duration tuning patternsrespectively in their onset responses analyzed in the 30-ms
peared to be short-pass when examined in a shortexamined in a long window. Right: the PSTHs of these two
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window around the onset peaks, therefore showing clearduration tuning. It is of interest to note that the offsetresponses of these neurons generally showed stronger dura-tion tuning than the onset responses. In this report, theduration tuningof theoffset responsewasanalyzed in a slidingwindow of 30-ms around the peaks of the offset responses.Among the total of 24 neurons in this PSTH category, 3 (12.5%),9 (37.5%) and 11 (45.8%) of the neurons appeared to be short-pass, band-pass, and long-pass respectively in their offsetresponses and only one neuron's offset response showed anall-pass pattern. Among the band-pass neurons, only oneneuron showedband-pass response in both its onset andoffsetresponses. To further appreciate the duration tuning differ-ence between the onset and the offset responses, we calcu-lated the ratio between the spike rates obtained at the “optimalduration” and the averaged rate for the rest of the durations.We called this ratio the “duration tuning index”. The indexesfor both onset and offset responses were 1.76±0.51 and 2.98±1.63, respectively. A paired t test showed that the differencewas statistically significant (p=0.003), suggesting a strongerduration tuning in offset responses. Fig. 5 presents theduration tuning curves of the onset and offset responsesfrom two neurons of this group. In these two cases, theduration tunings of the onset responses were all-pass patternand those of the offset responses were band-pass. It was alsonoted that the duration tuning of neurons in this groupappeared to be much weaker if the spike count was calculatedin a long-time window that covers the longest stimulusduration (Table 1). In this long window, 16 out of 24 neurons(62.5%) appeared to be all-pass; with the remaining neuronsbeing short-pass (n=5) and band-pass (n=3).
Since the offset responses inmost cases were clustered in ashort time window, it is predictable that the use of a slidingwindow or a long window for duration tuning analysis doesnot impact the apparent duration tuning. In our sample of 10offset neurons, 5 (50%) showed band-pass tuning, and 1 (10%)was short-pass, 2 (20%) were long-pass (Table 1). Thepercentage of band-pass offset neurons was comparable to
Fig. 5 – Duration tuning curves (upper panel) of two neurons thatto both on and off responses separately.
the percentage of the band-pass duration tunings seen in theoffset responses of on–off neurons, which was 37.5%.
2.3. Pause neurons
The selection of the analysis window also exerted greatimpact on the duration tuning for pause units. With the useof the short 30-ms window around the onset peaks, out of atotal of 19 pause units in this sample, 7 (36.8%), 6 (31.6%) and 2(10.6%) neurons appeared to have a short-pass, band-pass orlong-pass duration tuning pattern, respectively. However,only 2 out of the 6 band-pass neurons were also classified asband-pass using the long window, while the others becameall-pass or long-pass; similarly, only 2 out of 7 short-passneurons remained short-pass with the long window. Fig. 6compares the duration tuning curves of two pause units in thissample. Unit 05-11 showed a short-pass duration tuning whenanalyzed in the 30-ms short window. However, the durationtuning of this neuron became long-pass when the spikes werecalculated in the long window. Similarly, the band-passduration tuning of unit 06-15 seen with the short windowchanged to long-pass pattern when analyzed with the longwindow.
2.4. Onset neurons
Similar to previous reports, neurons with an onset responsePSTH pattern represent the largest population in IC: 90 out of207, or 43.5%. In our study, we did not differentiate the onsetunits that showed restrictive time locking (spikes distributedin a very narrow time bin) from those that showed burstresponses, because we found no difference in duration tuningbetween these two subgroups. Due to the specific PSTHpattern of these neurons, the length of analysis window wasnot an influencing factor in determining the duration tuningas long as the window covered all of the responses. However,unlike what was reported previously, we did find that almost50% of neurons in this group showed some kind of duration
showed on–off PSTHs (lower panel). Duration tuning is shown
Fig. 7 – Normalized duration tuning curves from 15 onsetunits that showed band-pass duration tuning. Thenormalized response at each point in the curve is calculatedas the ratio to the peak response of each unit, which istaken as 100%.
Fig. 6 – Duration tuning curves measured in the short onset window (on) and the long window (long) from two pause neurons.One of them (05-11) showed a short-pass pattern and the other (06-15) showed a band-pass pattern with the short onsetwindow. They both showed long-pass pattern when analyzed with the long window. Right: the PSTHs of these two neuronsresponding to the signals of four different durations.
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tuning according to the 50% drop-off criterion, among which15 (16.7%) showed band-pass duration tuning and 20 (22.2%)short-pass. Fig. 7 shows the normalized duration tuningcurves of the 15 band-pass neurons in this group. Since thebest durations are biased to short durations, the x axis ispresented in log scale to demonstrate the tuned peaks better.
In the attempt to determine if the duration tuning for onsetneurons was associated with the response latency, wecompared the latency between all-pass onset neurons (n=48)and band-pass onset neurons (n=15). It was found that theband-pass onset neurons had a significantly longer latency(mean±SD: 11.43±6.1 ms versus 5.3±1.21 ms for band-passand all-pass respectively; p<0.001, Mann–Whitney Rank SumTest).
2.5. Effect of signal type and level
In a small portion of our sample, we were able to test theneurons' duration tunings to both noise bursts and CF tones,or to the same type of signals but at multiple intensity levels.For the total 42 neurons that were tested with differentsignals, 22 were classified as band-pass in response to noisebursts. Eighteen out of these 22 neurons remained band-passin response to CF tones, while the other four became all-pass.Twelve neurons out of 42 were short-pass when measuredwith noise bursts, 10 of which remained short-pass inresponse to CF tones. The remaining 8 neurons showed all-pass or long-pass duration tuningswhenmeasuredwith noise
bursts; they all retained the same pattern in response to CFtones. The tuning indexes of these 42 neurons were 1.82±0.52(mean±SD) and 1.77±0.68 for the noise burst and the CF tone
Fig. 8 – The distribution of the best durations for neuronsthat showed a band-pass duration tuning pattern.
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respectively. The differences resulted from the use of differentsignals were not significant (paired t test, p>0.05).
Thirty-eight neurons were tested for duration tuning tonoise bursts at two levels (50 dB a.T and 10 dB a.T.). Most ofthese neurons (29 out of 38) showed the same duration tuningpatterns at the two different levels. The duration tuningindexes obtained for these neurons were 1.66±0.42 and 2.32±1.09 for 50 dB a.T and 10 dB a.T., respectively, slightly better forthe lower level. However, this difference was not statisticallysignificant either (paired t test, p>0.05).
2.6. Distribution of the best duration
A great portion of the neurons appeared to be best tuned toshort durations. This is first evident by the proportion ofneurons that showed short-pass duration tuning. In total, 53neurons (25.6%) were short tuned to durations below 8 ms(Table 1). In addition, 40 (19.3%) neurons that showed band-pass duration tuning were remarkably biased to short dura-tions. Fig. 8 shows the distribution of the best durations ofneurons in this subgroup, most of which (29 out of 40) weretuned to durations at or below 32 ms.
3. Discussion
In the auditory periphery, the duration of acoustic signals isrepresented by the duration of the neural responses thatgenerally correspond to the signal duration. Therefore, noauditory-nerve fiber is tuned to signal duration. Along theascending pathway of the auditory system, duration tuningneurons appear first in the midbrain and have not been foundin the lower levels (Condon et al., 1991; Feng et al., 1990; Halland Feng, 1991). For these duration-tuned neurons, theinformation about the duration of the signal is not necessarilyrepresented by the duration of the neurons' response, but bythe changes in the number of spikes or firing rate as a functionof signal duration. Duration tuning was first reported in themidbrain of the frog (Feng et al., 1990; Potter, 1965), and wasmore intensively investigated in echolocation bats (e.g.,Casseday et al., 2000; Ehrlich et al., 1997; Mora and Kossl,2004). In several recent studies, duration tuning neurons werealso found in the central auditory system of non-echolocationmammals (Brand et al., 2000; Chen, 1998; He, 2002; He et al.,1997; Perez-Gonzalez et al., 2006). However, the duration
tuning in these species is generally weaker than what hadbeen reported from echolocation bats, in the sense that asmaller proportion of neurons appeared to be duration-tuned.This is especially true for the number of neurons that showband-pass tuning. The results reported in the present studysuggest that this superficial differencemight have been due tothe method of duration tuning analysis. A new approach maybe needed to look at duration coding in non-echolocationmammals.
3.1. Analyzing duration tuning in a short time window
In all previous reports, duration tuning was examined as theduration-related changes in the total spikes collected in a longtime window that covered the longest signals used forduration tuning measurement. The data analyses in thepresent study challenge the use of such a long window. Ouranalyses suggest that duration tuning may be a transientprocess that is favorably associated with a certain timesegment in the response and therefore should be demon-strated better in a correctly selected time window.
The importance of selecting an appropriate time window isclearly demonstrated in neurons that show non-phasicresponse PSTHs, especially those with sustained responses.In the midbrain of mammals, many neurons have a sustainedresponse pattern that is similar to the pattern of the primaryauditory neurons. Sustained neurons fire throughout theduration of the stimulus signal. As a consequence, the spikecount from such a neuron keeps increasing with the elonga-tion of the signal duration. Therefore, most of these neuronsare considered to be long-pass or all-pass in duration tuningclassification (Brand et al., 2000; Chen, 1998; He et al., 1997; Xiaet al., 2000). Whether this “long-pass/all-pass” behaviorshould be considered as duration tuning has been questionedin previous reports. For example, long-pass neurons were notconsidered as duration-tuned by some authors studying bats(e.g., Casseday et al., 2000; Ehrlich et al., 1997). This isreasonable because for many sustained neurons, the largerspike count obtained with longer signal duration simplyrepresents the accumulated output with time, which is notexactly a feature of duration selectivity.
He et al. (1997) further classified the long-pass neurons intotwo groups: those with so-called duration threshold, beyondwhich neurons started firing, and those without (He et al.,1997). In a more recent paper, Mora and Kossl (2004) definedlong-pass neurons as those that required more than 5 ms ofstimulus duration to reach 25% of maximum spike count(Mora and Kossl, 2004). Taking these considerations in mind,none of the sustained neurons in our sample would beconsidered as duration-tuned if the spikes were counted inthe long window. However, a great portion of neurons in thisgroup appeared to be short-pass or band-pass tuned whenonly the responses within the short time window around theonset were taken into account for duration tuning. Therefore,the responses in this short window appeared to be bettertuned to the signal duration than the overall responses.Similarly, a greater portion of pause units, which otherwisewould not show clear duration tuning, appeared to be short-pass or band-pass tuned when analyzed with such a shortwindow around the onset response.
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The idea that neurons may use their transient responsesfor signal coding is not new. Geisler et al. (1969) were probablythe first to suggest a separate analysis between onset and latecomponents (Geisler et al., 1969). However, this notion wasgenerally ignored until the 1980s when several studies werepublished on the dynamic behavior of onset responses ofauditory nerve fibers and neurons in the cochlear nuclei. Theonset responses of the single auditory-nerve fibers appear tohave a larger dynamic operating range than the late steady-state components (Brachman, 1980; Smith, 1983, 1984; Smithand Brachman, 1980; Smith et al., 1985;Westerman and Smith,1984). The advantage in dynamic range at the onset isenhanced in central auditory neurons such as in the cochlearnuclei (Frisina, 2001; Frisina et al., 1985, 1990a,b; Smith, 1984).This dynamic response feature at the onset obviously providesa potential mechanism for the auditory neurons to code thelarge intensity range of acoustic signals. Moreover, the onsetdynamic feature in auditory responses has also been con-sidered as an important player in other aspects of auditorycoding. For example, Henry (1998) reported that the onsetresponse tuning of the auditory-nerve fibers to differencetones showed higher sensitivity to the forward masking thanthe later responses (Henry, 1998). Heil (1998) has reported thatit is the onset response component of IC neurons thatrepresents the interaural transient envelope disparities,whereas the sustained response component does not, but iswell associated with the binaural combination of steady-stateSPLs (Heil, 1998). These reports also stress the necessity forseparating the onset responses from the later responses inanalyzing the auditory coding of transient signals. In terms ofduration tuning, the fact that a higher portion of IC offsetneurons show strong duration tuning than neurons of othertemporal categories also suggests that duration coding isactually a transient function of the auditory system: theduration information of acoustic signals can be embedded inthe clusters of responses that occurred in a short period oftime. In a recent study of IC neurons of bats M. molossus, Moraand Kossl (2004) reported that band-pass duration tuningoccurred at either the onset or the offset responses. Moreimportantly, in neurons that showed both onset and offsetresponses, the onset component might show different dura-tion selectivity from the offset component. The authorspresented one example of such neurons in which the offsetcomponent was band-pass, while the onset was all-pass. Itwas not clear though, how many neurons in this subgroupshowed such preference to the offset component. Never-theless, the result suggests that a neuron's responses indifferent time segmentmay behave differently with respect toduration coding. This idea is supported by more data obtainedin the present study. We indeed found that the onset and theoffset responses of an on–off neuron can have independentduration tuning, but using a long analysis window, we werenot be able to reveal the duration tuning in the twocomponents.
3.2. Comparison with data from echolocation bats
Previous studies showed that duration tuning in non-echolo-cation mammals is generally weaker than what has beenreported in bats. This especially appeared as the fact that a
smaller proportion of neurons show clear duration tuning inthe non-echolocation mammals. One of the reasons for thisdifference is related to the fact that, in the non-echolocationmammals, neurons that show an offset response pattern arefewer. However, the results reported in the present studysuggest that the duration tuning in the non-echo locatingmammals may be much stronger than previously reported. Inour sample of 24 on–off neurons, band-pass duration tuningwas evident in 9 neurons in their offset responses, in 3neurons in their onset responses and in only one neuron inboth its onset and offset responses. A band-pass pattern wasalso evident in 10 out of 64 sustained neurons, and 6 out of 19pause neurons when analyzed in a short window around thetransient response peaks. As shown in Table 1, the proportionof neurons that show both short-pass and band-pass durationtunings comprise ∼45% of the sample, which is comparable tothe values from bats.
3.3. Possible mechanisms for the duration tuning
In previous studies, the mechanisms for duration tuning havebeen comprehensively explored for offset responses. A coin-cidencemodel was proposed and has beenwidely accepted forthis purpose (Casseday and Covey, 1995; Ehrlich et al., 1997).This model requires that a rebound from a sustained inhibi-tion (or an offset excitation) overlaps with a long latency sub-threshold onset excitation. Such a coincidence has beenverified in IC neurons with patch-clamp recording (Covey etal., 1996). The role of inhibitory circuitry in this model has alsogained support from the fact that the band-pass durationtuning of some IC neurons could be eliminated by blockingGABAergic or glycinergic inhibition (Casseday et al., 1994;Fuzessery and Hall, 1999; Jen and Feng, 1999).
3.3.1. Sustained and pause neuronsThe results in the present study suggest that the onsetresponses of sustained neurons can be duration-tuned whenanalyzed through a short window. The mechanisms under-lying this duration tuning are not clear. However, the role ofinhibition has been indicated by some previous studies. In anevoked potential (EP) study, the amplitude of the peakresponse was found to decrease with the elongated signalduration. This decrease was much smaller for animals whohad received a traumatizing tone and this desensitivity tostimulus duration was found to be related to the loss of GABA-mediated inhibition (Szczepaniak and Moller, 1995). Thedecrease in EP responses to longer durations is similar to theshort-pass duration tuning in our single unit results. Althoughthe EP response peak is not equivalent to the onset responsesof single neurons, the latter must make the main contributionto the former, because it is the onset responses that are bettersynchronized than the responses in any other time segment.Therefore, it is reasonable to assume that the “short-passduration tuning” in EP somehow reflects the similar changesin the onset responses in the single units.
3.3.2. Onset neuronsIn the present study, we found that a great portion of onsetneurons were actually duration-tuned. This might, partially atleast, be due to the difference in the signals used for
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evaluating duration tuning. In the present study, the standardsignal was broadband noise, while in most of the previousstudies, pure tones were the signals of choice for durationtuning evaluation. In any case, the possibility that onsetneuronsmay also be duration selective has not generally beensupported. He et al. (1997) reported a great proportion ofcortical neurons in cats that showed clear duration selectivity(He et al., 1997). However, their study was in the dorsal zone ofcat auditory cortex, and all neurons reported had longresponse latency (>30 ms). The study raised two questions:(1) How do neurons in the primary auditory cortex achieveduration tuning? (2) Is the duration selectivity of onsetneurons associated with their response latency? In a recentstudy on the IC neurons of bats (Mora and Kossl, 2004),duration selectivity was also reported from neurons withonset response patterns or from the onset components of on–off neurons. However, no latency information was provided inthat report. In the present study, we found that the onset andsustained neurons that showed clear duration selectivitytended to have longer response latencies. The longer latencyof the onset responses in these neurons may be partially dueto a stronger inhibitory innervation to them. This hypothesisis supported by the fact that blocking inhibitory inputsdecreases the response latency to sound for many brainstemauditory neurons (Manis, 1990). In addition, whole-cell patch-clamp recordings from IC neurons (Covey et al., 1996) indicatethat synaptic inhibition dominates the early part of theresponse, resulting in an early unresponsive period correlatedwith the onset of the stimulus. Therefore, the longer latencyfor the onset band-pass neurons suggests a role of inhibitionfor their duration tuning. The role of inhibition in the durationtuning of neurons' onset responses was also supported by astudy of big brown bats (Casseday et al., 2000), in which short-pass duration tuning was found in some neurons that showedan onset pattern. Also, the short-pass tuning appeared to beregulated by GABA-mediated inhibition: many such neuronsbecame all/long-pass after the GABA-A antagonist, bicucul-line, was applied (Casseday et al., 2000).
3.3.3. Mechanisms for short-pass duration tuningOf all neurons in this sample, 53 neurons were classified asshort-pass in response to the noise bursts at 50 dB a.T. Theseneurons show robust responses for stimuli shorter than 8 msbut the firing rates are reduced at longer signal durations. Inour samples, most of the short-pass neurons do not totallystop firing at longer duration. Tested within the short timewindow, we found more than 20% of neurons in each PSTHgroup showing short-pass duration patterns, except the offsetgroup, in which we found that only 1 out of 10 neurons wasshort-pass (Table 1). In addition, many neurons sampleshowed a sharp peak for best duration at a short duration,even though they would be judged as all-pass neuronsaccording to the 50% drop off criterion (example in Fig. 4).
As discussed above, GABAergic inhibition appears to play arole in the formation of short-pass duration tuning (Cassedayet al., 2000). It is reasonable to assume that the quick dropdown in firing rate to longer duration signals is due toincreased inhibitory input evoked by the longer durationsignals. A so-called anti-coincidence model was proposed toexplain the role of inhibition in the short-pass selectivity
(Fuzessery and Hall, 1999). It was so named as it is opposite tothe coincidence model for the band-pass duration selectivityof offset neurons. In the anti-coincidence model, excitatoryinput and inhibitory input are not coincident, with thetransient excitatory input arrives first and the inhibitoryinput builds up late.
3.3.4. Mechanisms for long-pass duration tuningBrand et al. (2000) proposed two possible mechanisms toexplain the long-pass duration tuning (Brand et al., 2000). Thefirst mechanism is linked to the requirement of neurons tohave a high degree of temporal summation. Because of this,neurons show no responses at all when the signal duration istoo short. The authors suggested that, under this mechanism,the low cut-off for these neurons should shift when the signalchanges in spectrum and amplitude. In our experiments thestandard signal was 50 dB a.T. At this relatively high soundlevel, most of the long-pass responses did not show durationthresholds, as compared with the responses to lower signallevels reported by others, which generate spikes in response tothe shortest signal duration tested. Unfortunately, we do nothave adequate data to verify shifts to lower cut-offs associatedwith reductions in sound level. Since the overall signal level ispartly a function of signal duration, we cannot rule out thepossibility that the duration sensitivity threshold is a functionof sound levels. The secondmechanismproposed for the long-pass duration tuning of sustained neurons is a transient onsetinhibitory input that prevents neurons from responding toshort duration signals. This mechanism is also applicable tothe onset responses or onset components. The existence ofsuch onset inhibition has been reported in bats (Bauer et al.,2000; Klug et al., 1999; Park et al., 1998).
3.4. Behavior consideration
Duration tunings provide another type of auditory filtering.Presumably, this selectivity operates during the processing ofbiologically important signals. The behavioral relevance ofduration tuning has been well documented in bats and frogs.In several species of bats, for example, the best durations ofneurons that are duration-tuned are in the same range ofduration values of their echolocation calls (Ehrlich et al., 1997;Fuzessery and Hall, 1999; Galazyuk and Feng, 1997; Kossl et al.,1999; Mora and Kossl, 2004). Although duration tuning hasbeen confirmed in several non-echolocation mammals andtherefore appears to be a general mechanism of signalprocessing throughout the animal kingdom, the significanceof duration selectivity in these species that do not rely uponecholocation is still not clear. In our study, we found manyneurons that were short-pass tuned to signal duration <10 msand the best durations for most of the band-pass neurons inthis sample were between 8 and 100 ms (Fig. 8). This timerange of duration selectivity is consistent with what has beenreported in mice (Brand et al., 2000), and rats (Perez-Gonzalezet al., 2006). It is suggested that the cut-off durations of short-pass neurons and the best durations of band-pass neurons inrats are consistent with the durations of rats' vocalizationduring many different behaviors (Perez-Gonzalez et al., 2006).Detailed analysis is needed to confirm the correlation.According to Happer (1976), guinea pigs can produce 11
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distinct calls that are important for their communication(Happer, 1976). Among them, 3main stereotypes (purr, chutterand chirp) contain acoustic impulses of short durations below100ms (Happer, 1976; Suta et al., 2003; Syka et al., 1997). This isconsistent in general with the bias of duration tunings wereported in Fig. 8. However, we do not know the behavioralrelevance for the duration tuning to the extremely shortdurations (cf. <10 ms) that are represented by a great portionof neurons that are short-pass tuned. It is possible that theseshort-pass tuned responses are responsible for processing thesharp peaks in these calls, or calls from other species, such aspredators, that contain sound segments of shorter durations.
3.5. Summary
Duration tuning appears to be a transient process in theresponses of IC neurons of guinea pigs. This filtering functionis demonstrated in both onset (or onset component) andoffset responses. The results of this study show the impor-tance of selecting an appropriate time window for durationtuning analysis. The duration tuning at the onset or onsetcomponent of the responses can be explained by an anti-coincidence model. Inhibitory inputs and their timely inter-action with excitatory circuits appear to be critical for thegeneration of duration tuning in both onset and offsetresponses. Further studies are needed to confirm the role ofinhibition in duration tuning of different response types. Thecontributions from lower stations in the ascending auditorypathway are obviously important but go beyond the scope ofthe present investigation. The range of best durations of the ICneurons in guinea pigs generally corresponds to durations ofsome rodent species-specific calls that are important forsurvival and communications. However, the behavioralrelevance for the responses tuned to very short durations isstill unclear.
4. Experimental procedures
4.1. Subjects and surgical procedures
A total of 23 healthy adult albino guinea pigs of either sexwereemployed in this study. All procedures involving the use ofanimals conformed to NIH guidelines and were approved bythe University Committee of Laboratory Animal of DalhousieUniversity.Anesthesiawas inducedwithsodiumpentobarbital(40mg/kg, i.m.) andmaintained by ketamine (30mg/kg/h, i.m.)plus xylazine (5 mg/kg/h). The maintenance dose of ketamineand xylazine was first given 3 h after the administration ofpentobarbital. Core temperaturewasmaintainedat 38.5±0.5 °Cthroughout the experiment.
The animal's head was held still in a stereotaxic frame.Initially, both ear canals were closed with ear bars thatprovided support to the head. A 2-cm incision was madealong the midline of the scalp; the skin and the muscles wereretreated laterally to expose the area over the posterior part ofthe brain. To provide support to the head, a metal barequipped with a screw tip was fixed into the skull contral-aterally to the IC that would be recorded and the contralateralear bar was removed. The contralateral ear canal was then
remained open throughout the experiment to allow sounddelivery. The ipsilateral ear canal remained closed by the earbar, which provide 30–40 dB acoustic attenuation as judged bythe threshold evaluation in the test of auditory brainstemresponses. A hole of 3-mm diameter was drilled through theskull lateral to the midline and centered at the parietal-occipital suture. The dura-mater was excised with a scalpel topermit smooth electrode penetration into IC through thecerebrum. The surgery preparation took approximately30 min. The recording started immediately after the surgeryin a sound-proof booth. A typical recording session lastedaround 8–10 h. At the end of each experiment, the animal wassacrificed with an overdose of pentobarbital and the skull wasopened to visualize the penetrationmark on the surface of thecerebrum and the IC.
4.2. Acoustic stimuli
Acoustic stimuli were generated digitally with Tucker-DavisTechnologies hardware and software (TDT System III). Thesound level was calibrated with a Larson-Davis sound levelmeter (model 824) and TDT hardware and software. Thecalibration was performed using a quarter inch free-fieldmicrophone (B&K, 4939), whichwas placed at the position thatwould be occupied by the ear of the animals. Stimuli weredelivered in open field through a broadband electrostaticloudspeaker (ES1 from TDT) positioned 10 cm away from thecontralateral ear of the animal. This particular speakerprovides a flat frequency response from 1 kHz to 110 kHz. Allstimuli used in this study were bursts of pure tones or whitenoise with duration varied from 2 ms to 256 ms (with a 0.5 msrise/fall time) in exponent steps.
4.3. Single unit recording
The single unit activity was recorded with microelectrodesmade of glass-pipettes. The electrode impedance rangedbetween 5 and 12 MΩ. The electrode was positioned perpen-dicular to the surface of the cerebrum that covered the IC, andadvanced by a remote micro-drive. The output of therecording electrode was amplified, filtered and routed to anoscilloscope, audio monitor and the A/D converter of the TDTsystem. Spikes were discriminated using Brainware software,and their time of occurrence was stored with a resolution of2.5 μs re:stimulus onset. A noise burst with a duration of 40mswas presented at a moderate-to-high sound level (40–70 dBSPL) to search for responsive neurons while the electrode wasadvanced through the IC. When a unit was isolated, thecharacteristic frequency (CF) was determined either byrecording iso-intensity response curves at moderate intensitylevel (40–70 dB SPL), or by manually adjusting the intensityand the frequency of tone bursts andmonitoring the responseaudio-visually. Depending on the stability of the neurons,duration tuning was recorded in response to the noise bursts,first at 50 dB and then 10 dB above the neuron's threshold (dBa.T.) and then at 10 dB a.T., followed by the responses to CFtone, and then to tone(s) away from CF. When measuringduration tuning, the signals of different durations werepresented randomly. At each duration, the spike rate wascalculated as the average of 10 stimulus responses. The neural
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activities were recorded in a 600 ms time window, and thestimulation interval was 700 ms. The recording site wasverified in some animals by injecting fast-green through therecording electrode at the end of recording and dissecting thebrain after the experiment. The central nucleus of IC wasfocused in this experiment.
4.4. Data analysis
IC neurons reported were first categorized according to theirperistimulus time histogram (PSTH) into five categories: (1)onset (responses concentrated within a short durationfollowing the onset of the stimulus), (2) onset–offset(responses time-locked to both onset and offset of thestimulus), (3) offset, (4) sustained (responses throughout thestimulus period) or (5) pause (shows a silent gap betweenonset and sustained portions of the response). The durationtuning of neurons was classified into four categories: (1) all-pass (no duration tuning), (2) short-pass, (3) long-pass, and (4)band-pass. The criteria for classification of duration selectiv-ity are similar to what were proposed previously (Fuzesseryand Hall, 1999; Narins and Capranica, 1980; Potter, 1965). Aneuron was classified as duration-tuned if the spike ratereached a peak at a sound duration (the best duration), andthe rate decreased by more than 50% of the response to thebest duration at longer and/or shorter durations. Band-passduration tuning was defined as a response in which the spikerate dropped more than 50% at both shorter and longerdurations. Short-pass (or long-pass) duration tuning wasdefined as a response in which the spike rate dropped morethan 50% of peak value at longer (or shorter) durations. Inlong-pass responses, the spike rate either reached a plateau ata particular duration, or continued to increase over the rangeof the durations tested.
The duration tuning was determined differently fromprevious studies in two aspects. Firstly, driven spike ratewas obtained by subtracting the spontaneous spike rateobtained in a “quiet” window from the total spike rate,instead of using the total spikes per trial. Secondly, theresponse spike rate was calculated in a short time window(30 ms) around the peak responses in PSTH, instead ofmeasuring the total spikes per trial in a long time windowthat covered the entire range of stimulus durations. The use ofa 30 ms window is somehow arbitrary, but this windowincluded the durations of the onset bursts of most onset units.The window was set up around the onset responses (theresponse transient). However, for the neurons that showed anoffset response pattern, a sliding window was used to followthe temporal response peak. The results of duration tuningobtained using this short window were compared with thoseobtained in a long window for sustained, on–off neurons andpause units to further verify the need of using this shortwindow.
Acknowledgments
We gratefully thank Dr. Douglas Rasmusson's comments andsuggestions on the writing of this paper. This study was
supported by the grant of The Natural Sciences and Engineer-ing Research Council of Canada 250088-02 and the grant of TheNational Natural Science Foundation of China 30271410/C030310.
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