Brain Research 973 (2003) 131–141www.elsevier.com/ locate/brainres

Research report

T he effect of bicuculline application on azimuth-dependent recoverycycle of inferior collicular neurons of the big brown bat,

Eptesicus fuscus*Xiaoming Zhou, Philip H.-S. Jen

Division of Biological Sciences, University of Missouri at Columbia, Columbia, MO 65211,USA

Accepted 5 March 2003

Abstract

The recovery cycle of auditory neurons is an important neuronal property, which determines a neuron’s ability to respond to pairs ofsounds presented at short inter-sound intervals. This property is particularly important for bats, which rely upon analysis of returningechoes to extract the information about targets after emission of intense orientation sounds. Because target direction often changesthroughout the course of hunting, the changing echo direction may affect the recovery cycle and thus temporal processing of auditoryneurons. In this study, we examined the effect of sound azimuth on the recovery cycle of inferior collicular (IC) neurons in the big brownbat,Eptesicus fuscus, under free-field stimulation conditions. Our study showed that the recovery cycle of most IC neurons (42/49, 86%)was longer when determined with sounds delivered at 408 ipsilateral (i408) than at 408 contralateral (c408) to the recording site. To studythe contribution of GABAergic inhibition to sound azimuth-dependent recovery cycle, we compared the recovery cycle of IC neuronsdetermined at two sound azimuths before and during iontophoretic application of bicuculline, an antagonist for GABA receptors.A

Bicuculline application produced a greater decrease of the recovery cycle of these neurons at i408 than at c408. As a result, theazimuth-dependent recovery cycle of these neurons was abolished or greatly reduced. Possible mechanisms underlying these observationsand biological relevance to bat echolocation are discussed. 2003 Elsevier Science B.V. All rights reserved.

Theme: Sensory systems

Topic: Auditory systems: central physiology

Keywords: Bat; Inferior colliculus; Recovery cycle; GABAergic inhibition; Bicuculline

1 . Introduction ing echo direction and in processing increasing number ofechoes, past studies have examined the directional sen-

During hunting, insectivorous bats systematically vary sitivity and recovery cycle of bat auditory neurons.the frequency, intensity, duration and repetition rate of To determine the directional sensitivity of auditoryemitted sounds and analyze the returning echoes to extract neurons, studies have been conducted to measure variationinformation about the direction, location, and velocity of in the minimum threshold or in the number of impulses ofprey during different phases of hunting [12,48]. To under- neurons with sound direction [9,14–16,19,21,23,25,stand the neural basis underlying a bat’s ability in localiz- 31,41,42,45,52,56]. Most of these studies have been per-

formed at the inferior colliculus (IC) because the IC is anobligatory relay station that receives excitatory and inhib-

Abbreviations: IC, Inferior colliculus; BF, Best frequency; SPL, Sound itory inputs from all lower auditory nuclei and is thuspressure level; GABA, Gamma-aminobutyric acid; c408 or i408, 408 critical for auditory information processing [4,36]. Thesecontralateral or ipsilateral to the recording site; PST, Peri-stimulus time; studies showed that the minimum threshold of IC neuronsISI, Inter-sound interval

increases by 25–30 dB when a sound azimuth changes*Corresponding author. Tel.:11-573-882-7479; fax:11-573-884-from 408 contralateral (c408) to 408 ipsilateral (i408) to the5020.

E-mail address: jenp@missouri.edu(P.H.-S. Jen). recording site within the frontal auditory space. These

0006-8993/03/$ – see front matter 2003 Elsevier Science B.V. All rights reserved.doi:10.1016/S0006-8993(03)02575-7

132 X. Zhou, P.H.-S. Jen / Brain Research 973 (2003) 131–141

studies also showed that the slope of directional sensitivity tanyl 0.08 mg/kg, b.w. /Droperidol 4 mg/kg, b.w.), andcurves of IC neurons increases with sound frequency. placed inside a bat holder (made of wire mesh) that wasBecause sound pressure transformation at the pinna varies suspended in an elastic sling inside a double-wallwith sound direction [17] and pinna position affects the soundproof room (Industrial Acoustics Company, Inc.,maximal spatial sensitivity of auditory neurons [19,21,52], temperature 28–308C). The bat’s head was immobilizedit has been suggested that directional characteristics of the and oriented with the eye–snout line pointed to 08 inpinna and binaural interaction contribute to the directional azimuth and 08 in elevation with respect to the frontalsensitivity of IC neurons [9,42,53]. auditory space [23]. Small holes were bored in the skull

Previous studies have shown that the bat IC neurons above the IC for insertion of piggyback multibarrel elec-have different periods of recovery cycles when determined trodes to record auditory responses of IC neurons and forwith identical or pulse-echo pair stimuli iontophoretic application of bicuculline. Additional doses[4,7,13,30,37,50,51,57]. They have shorter recovery cycles of Innovar-Vet were administered during later phases ofwhen stimulated with 3.4–4.0 ms than 1–2 ms sounds [37] recording if the bat showed signs of discomfort. Each bator when stimulated with two identical sounds than with was used for one to five recording sessions on separatepulse-echo pairs [7,13,50,51]. Previous studies have also days and each recording session typically lasted for 4–6 h.shown that increasing sound level either increases or These procedures were conducted in compliance with NIHdecreases the recovery cycle of peripheral and central publication No. 85-23, ‘Principles of Laboratory Animalauditory neurons [6,33,34]. Furthermore, GABAergic inhi- Care’ and with the approval of the Institutional Animalbition shapes the recovery cycle of IC neurons because Care and Use Committee ([1438) of the University ofblockage of GABA receptors by bicuculline application Missouri–Columbia.A

decreases the recovery cycle and increases the ability of IC Sound stimuli (4 ms with 0.5 ms rise–decay times) wereneurons in following the pulse repetition rate [29,30]. formed with an oscillator (KH model 1200) and a

Because the minimum threshold of IC neurons increases homemade electronic switch driven by a stimulator (Grass25–30 dB when sound azimuth changes from c408 to i408 S88). These stimuli were then amplified after passing[9,14,21,45,56], this change in minimum threshold with through a decade attenuator (HP 350D) before being fed tosound azimuth in essence changes the level of a given a small condenser loudspeaker (AKG model CK 50, 1.5sound stimulus. Since the recovery cycle of auditory cm diameter, 1.2 g) that was placed 23 cm away from theneurons varies with sound level [6,33,34], it follows that bat. The loudspeaker could be positioned at 08 elevationsound azimuth should affect the recovery cycle of auditory and in any specific azimuth within6908 in the frontalneurons. However, all past studies on the recovery cycle of auditory space through a remote control system driven bybat IC neurons were conducted at single sound azimuth so two small electric motors. Calibration of the loudspeakerthat they can not predict if sound azimuth affects the was performed with a 1/4 inch microphone (B & K 4135)recovery cycle of bat IC neurons. placed at the position where the bat’s head would be

Under free-field stimulation conditions, the main objec- during recording. The output of the loudspeaker wastive of this study is to examine how the recovery cycle of expressed in dB SPL in reference to 20mPa root meanIC neurons may vary with sound azimuth and whether square.GABAergic inhibition contributes to azimuth-dependent Upon isolation of an IC neuron with 4-ms soundsvariation of recovery cycle. To achieve this objective, we delivered at a rate of two per second from c408, whichmeasured the recovery cycle of IC neurons at two sound typically elicited maximal responses from IC neurons ofazimuths (c408 and i408) before and during bicuculline this bat species [25,39], its best frequency (BF) wasapplication using piggybacked multibarrel electrodes. We determined by changing the frequency and intensity ofthen examined how the recovery cycle of these neurons sound stimuli. The minimum threshold at the BF waschanged with sound azimuth before and during bicuculline defined as the sound level that elicited 50% responseapplication. probability from the neuron. A neuron’s recovery cycle

was then studied with a pair of identical BF sounds at 20dB above the minimum threshold at seven selected inter-

2 . Materials and methods sound intervals (ISIs at 5, 10, 15, 20, 40, 60, and 100 ms).The neuron’s number of impulses in responding to each

Six Eptesicus fuscus (17–21 g, body weight, b.w.) were sound at these ISIs was systematically recorded. Thisused for this study. As in previous studies [25], 1 or 2 days procedure was then repeated after the loudspeaker wasbefore the recording session, a 1.8 cm nail was glued onto changed to i408.the exposed skull of each Nembutal-anesthetized (45–50 As in our previous study [30], a recovery cycle wasmg/kg, b.w.) bat with acrylic glue and dental cement. plotted with the percent recovery against the ISIs. TheExposed tissue was treated with an antibiotic (Neosporin) percent recovery was calculated by dividing the number ofto prevent infection. During the recording session, the bat impulses discharged to the second sound by the number ofwas administered the neuroleptanalgesic Innovar-Vet (Fen- impulses discharged to the first sound. In this study, we

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used the ISI corresponding to 50% recovery (i.e., the 50% was adjusted to include only the part of the PST histogramrecovery time, see Fig. 3) to characterize the recovery that was generated by the first or the second sound tocycle of each neuron. The 50% recovery time of all IC obtain a neuron’s number of impulses discharged to eachneurons obtained at each azimuth both before and during sound at each ISI. This adjustment of sampling windowbicuculline application was then compared statistically allowed us to easily exclude the spontaneous impulses.using the paired Student’st-test (two-tailed) or the Kol- The number of impulses discharged to each sound wasmogorov–Smirnov test (non-parametric) atP,0.01. then used to plot a neuron’s recovery cycle.

Iontophoretic application of bicuculline has been de-scribed in our previous studies [29,30]. Briefly, a three-barrel electrode (tip: 10–15mm) was piggybacked to a 3 3 . ResultsM KCl single-barrel electrode (tip: less than 1mm;impedance: 5–10 MV) whose tip was extended about 10 We isolated 57 IC neurons at depths between 124 andmm from the tip of the three-barrel electrode. The 3 M KCl 1672mm. Their BFs and minimum thresholds weresingle-barrel electrode was used to record neural re- between 16.5 and 71.8 kHz and 19 and 68 dB SPL. Theysponses. One of the barrels of a three-barrel electrode was were tonotopically organized along the dorsoventral axis offilled with bicuculline methiodide (10 mM in 0.16 M the IC such that BFs increased with recording depths (Fig.NaCl, pH 3.0). The bicuculline was prepared just prior to 1A). Their minimum thresholds also increased significantlyeach experiment and the electrode filled immediately with BFs (Fig. 1B). These findings are similar to thosebefore use. This bicuculline channel was connected via obtained from previous studies on the same species of batsilver–silver chloride wire to a microiontophoresis con- [3,18,25,30,35,39,55].stant current generator (Medical Systems Neurophore BH- We plotted recovery cycles of 49 neurons with pairs of2) that was used to generate and monitor iontophoretic BF sounds at both c408 and i408 before bicucullinecurrents. During bicuculline application, a 1-s pulse of application. However, we only plotted recovery cycles ofpositive 40 nA at 0.5 pps was applied until the effect of 36 neurons at both azimuths during bicuculline applicationbicuculline reached a steady state when three consecutive because of loss of neurons through the course of study.responses did not vary by more than 15%. The applicationcurrent was then changed to 10 nA during data acquisition. 3 .1. Recovery cycle of IC neurons at c408 and i408The other two barrels were filled with 1 M NaCl (pH 7.4),one of which was used as the ground and the other as the Fig. 2 shows the PST histograms of a representative ICbalanced barrel. The balance electrode was connected to a neuron in responding to single BF sounds and to pairedbalance module. The retaining current was negative 8–10 identical BF sounds at seven ISIs at both c408 and i408.nA. When stimulated by paired BF sounds at both sound

Recorded action potentials were amplified, band-pass azimuths, this neuron always discharged impulses to thefiltered (Krohn-Hite 3500), and fed through a window first sound at each ISI but the number of impulses wasdiscriminator (WPI 121) before being sent to an oscillos- always larger at c408 than at i408. However, this neuroncope (Tektronix 5111) and an audio monitor (Grass AM6). did not always discharge impulses to the second sound atThey were then sent to a computer (Gateway 2000, 486) each ISI. For example, the neuron discharged only 2 andfor acquisition of peri-stimulus time (PST) histograms (bin 13 impulses to the second sound at ISIs of 5 and 10 mswidth: 500 ms, sampling period: 300 ms) to 32 stimulus when sound was delivered at c408 (Fig. 2 left panel). Thepresentations. During data analysis, the sampling window number of impulses greatly increased to 42 at the ISI of 15

Fig. 1. Scatter plots showing the relationship between best frequency (BF) and recording depth (A), best frequency and minimum threshold (B) of ICneurons. The linear regression line and correlation coefficient of each plot are shown by a straight line andr. N, number of neurons;P, significant level.

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than 5 ms. The average 50% recovery time of these 42neurons significantly increased from 26.6618.2 ms at c408to 42.0623.7 ms at i408 (pairedt-test,P,0.0001). For theremaining seven neurons, the 50% recovery time either didnot change (4/49, 8%) (Fig. 3C) or decreased (3/49, 6%)(Fig. 3D) when sound azimuth changed from c408 to i408.

A comparison of 50% recovery time of all 49 ICneurons at two sound azimuths is shown in Fig. 4. It isclear that most (42, 86%) data points are above theequal-value diagonal line indicating a longer 50% recoverytime at i408 than at c408. A Kolmogorov–Smirnov test(non-parametric) showed that the 50% recovery time ofthese neurons determined at two sound azimuths wassignificantly different (P,0.005).

To determine if 50% recovery time of IC neurons iscorrelated with the BF or minimum threshold, we plotted50% recovery time of IC neurons obtained at c408 and i408in relation to BF (Fig. 5A) and minimum threshold (Fig.5B). The range of 50% recovery time was larger for lowBF neurons than for high BF neurons. Linear regressionanalysis indicated that 50% recovery time obtained at bothazimuths significantly decreased with increasing BF (Fig.5A, P,0.0001). The 50% recovery time of most ICneurons obtained at both azimuths scattered over a widerrange in relation to minimum threshold than to BF (Fig.5B vs. A). Linear regression analysis indicated that 50%recovery time obtained at both azimuths also significantlydecreased with increasing minimum threshold (P,0.005).Fig. 5C and D show the difference in 50% recovery timeof IC neurons between two azimuths in relation to BF (Fig.5C) and minimum threshold (Fig. 5D). It is clear that thedifference in 50% recovery time significantly decreased

Fig. 2. Peri-stimulus time (PST) histograms of an inferior collicular with increasing BF (P,0.0001) and minimum thresholdneuron (IC) obtained with single sound and with a pair of identical (P,0.005).sounds (BF sounds at 20 dB above the neuron’s minimum threshold)delivered at 408 contralateral (c408, left panel) and 408 ipsilateral (i408,

3 .2. The effect of bicuculline application on recoveryright panel) to the recording site. The number of impulses in respondingcycle of IC neurons at c408 and i408to each sound and the inter-sound interval (ISI, ms) are shown within

each histogram. To save space, the sampling period beyond 150 ms is notshown (see text for details). Consistent with a previous study [30], bicuculline

application produced an increase in the number of im-ms and then ranged between 46 and 52 with further pulses and a decrease in the recovery cycle of IC neurons.increase of the ISI from 20 up to 100 ms. The neuron did The PST histograms at four ISIs and recovery cycles of anot respond to the second sound at ISIs shorter than 10 ms representative IC neuron obtained at both sound azimuthswhen the sound was delivered from i408. The number of before and during bicuculline application are shown in Fig.impulses then progressively increased from 4 to 37 as the 6. This neuron discharged more impulses to each sound atISI increased from 15 to 100 ms (Fig. 2, right panel). As c408 than at i408 regardless of ISI. Bicuculline applicationdescribed in the Materials and methods, these numbers of produced an increase in the number of impulses to eachimpulses discharged to each sound at different ISIs were sound at all ISIs at both sound azimuths. Based upon theused to plot the neuron’s recovery cycles (Fig. 3A). It is recovery cycles plotted under each stimulation condition,clear that this neuron’s 50% recovery time was longer at the neuron’s 50% recovery time decreased from 18.5 toi408 than at c408 (Fig. 3Ab vs. a, i.e., 28.5 vs. 12.5 ms). 12.5 ms (a change of 32.4%) at c408 and from 30 to 13 ms

Among 49 IC neurons studied, 50% recovery time of 42 (a change of 56.7%) at i408 during bicuculline application(86%) neurons increased when sound azimuth changed (Fig. 6 bottom).from c408 to i408 (Fig. 3A,B). The increase in 50% Fig. 7 shows the recovery cycles of this and two otherrecovery time ranged from 0.5 to 46.5 ms (mean6S.D.: IC neurons determined at both sound azimuths before and15.5611.2 ms) with most (32/42, 72%) increased more during bicuculline application. The 50% recovery time of

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Fig. 3. The recovery cycles of four IC neurons obtained with a pair of identical BF sounds delivered at c408 (filled circles) and i408 (unfilled circles).Ordinates and abscissae represent percent recovery and inter-sound interval in ms. Each horizontal dashed line indicates the 50% recovery. The arrowslabeled a and b indicate the 50% recovery time (shown at the lower right corner) for each curve. Note that the 50% recovery time of these neurons eitherincreased (A, B), remained unchanged (C), or decreased (D) when the sound azimuth changed from c408 to i408.

all three neurons increased as sound azimuth changed from line application was minimal (Fig. 7Aa vs. b, unfilledc408 to i408 before bicuculline application (Fig. 7A, B, Ca circles at lower right) or small (Fig. 7Ba vs. b, Ca vs. b,vs. b, filled circles, values indicated by filled circles at unfilled circles at lower right).lower right) but decreased at both sound directions during A comparison of the 50% recovery time of 36 ICbicuculline application (Fig. 7A, B, Ca vs. b, unfilled neurons obtained before and during bicuculline applicationcircles, values indicated by unfilled circles at lower right). at each sound azimuth is shown in Fig. 8. Most data pointsHowever, the decrease in the 50% recovery time during are distributed below the equal-value diagonal line indicat-bicuculline application was greater at i408 than at c408 ing a decrease of 50% recovery time during bicuculline(Fig. 7, %D at lower right). As a result, the difference in application. However, data points of IC neurons with short50% recovery time of these three neurons during bicucul- 50% recovery time are distributed more closely to the

equal-value diagonal line than those neurons with long50% recovery time. This asymmetrical distribution of datapoints indicates that bicuculline application produced agreater decrease of 50% recovery time for neurons withlong than with short recovery cycles. The pre-drug average50% recovery time of these neurons at c408 was29.3618.3 ms (range: 8–79 ms) which significantly de-creased to 15.667.7 ms (range: 6–41 ms) during bicucul-line application (pairedt-test, P,0.0001). Similarly, thepre-drug average 50% recovery time of these neurons ati408 was 46.3622.8 ms (range: 17.5–95 ms) which alsosignificantly decreased to 19.869.0 ms (range: 8–46 ms)during bicuculline application (pairedt-test, P,0.0001).

Fig. 9 shows a comparison of bicuculline producedchanges in 50% recovery time of these 36 IC neurons attwo azimuths. A few data points are at the equal-valuediagonal line indicating an equal amount of change in 50%Fig. 4. A comparison of 50% recovery time (50% RT) of 49 IC neuronsrecovery time produced by bicuculline application at twodetermined at c408 and i408. Note that most data points are above the

equal-value diagonal line indicating a longer 50% RT at i408 than at c408. azimuths. However, most data points are distributed above

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Fig. 5. (A, B) Scatter plots showing 50% recovery time of 49 IC neurons determined at c408 (filled circles) and i408 (unfilled circles) in relation to bestfrequency (A) and minimum threshold (B). (C, D) Scatter plots showing the changes (D) in 50% recovery time when a sound azimuth changed from c408

to i408 in relation to best frequency (C) and minimum threshold (D) (see Fig. 1 for legends).

the equal-value diagonal line indicating a greater change in tion significantly increased when sound azimuth changed50% recovery time produced by bicuculline application at from c408 to i408 (paired t-test, P,0.0001). Bicucullinei408 than at c408. The average change in 50% recovery application produced a significant decrease in the 50%time was 26.5618.6 ms (range: 0.5–66 ms) at i408 which recovery time of these two groups of neurons at bothis significantly larger than 13.7612.6 ms (range: 0.5–47 sound azimuths (pairedt-test,P,0.001), and the decreasems) at c408 (paired t-test, P,0.0001). was greater at i408 than at c408 (45.2–55.8% vs. 38.6–

Since bicuculline application affected the 50% recovery 38.8%, pairedt-test, P,0.005). The decrease in the 50%time of these 36 IC neurons in different degrees at two recovery time of group I neurons during bicucullineazimuths, the average difference in the 50% recovery time application was so much greater at i408 than at c408 thatof these neurons between two azimuths decreased sig- significantly azimuth-dependent variation in 50% recoverynificantly from 17.0611.3 ms (ranged: 0.5–46.5 ms) to time was abolished (pairedt-test, P.0.01). However,4.864.7 ms (ranged: 0–22 ms) during bicuculline applica- bicuculline application only produced a moderate decreasetion (paired t-test, P,0.0001). The number of neurons in the 50% recovery time of group II neurons at i408 suchwith azimuth-dependent difference in the 50% recovery that the significantly azimuth-dependent variation in 50%time larger than 5 ms also greatly decreased from 30 recovery time was only reduced but not abolished (paired(83%) to 15 (42%) while that with azimuth-dependent t-test, P,0.0001).difference smaller than 5 ms increased from 6 (17%) to 21(58%) during bicuculline application. To determine theeffect of bicuculline application on azimuth-dependent 4 . Discussion50% recovery time of IC neurons, we arbitrarily dividedthese 36 IC neurons into two groups with 50% recovery 4 .1. Azimuth-dependent recovery cycle of IC neuronstime difference between two azimuths during bicucullineapplication either smaller (Group I,n521) or larger Sound localization and temporal structure recognition(Group II, n515) than 5 ms. These two groups of IC are two important tasks during acoustic communicationneurons did not differ in BF, minimum threshold, and and echolocation. Because a neuron’s recovery cyclerecording depth (data not shown). determines its ability to follow repetitive sound pulses, the

As shown in Table 1, the 50% recovery time of both recovery cycle is thus important for discriminating tempo-groups of neurons determined before bicuculline applica- ral structure of a sound. To the best of our knowledge, this

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Fig. 6. PST histograms (top) and recovery cycles (bottom) of an IC neuron obtained with a pair of BF sounds delivered at c408 (A) and i408 (B) before(pre-drug) and during bicuculline application. Note that bicuculline application greatly decreased the neuron’s 50% recovery time at both sound azimuthsand the decrease was greater at i408 (17 ms) than at c408 (6 ms) (see Fig. 2 for legends).

is the first study to show that the recovery cycle of IC reported that increasing sound level either increases orneurons varies with sound azimuth (Figs. 3,4,6,7; Table 1). decreases the recovery cycle of peripheral and centralThis observation suggests interdependence between spatial auditory neurons of cats [6,33,34].and temporal processing in the IC. This interdependence In this study, azimuth-dependent recovery cycle of ICbetween signal parameters has been shown in many neurons was only tested at two sound azimuths. This effectprevious studies. For example, many studies showed that of sound azimuth on the recovery cycle of IC neurons mayselectivity of bat IC neurons in frequency, intensity, vary when tested at other azimuths because the transferduration, and direction domains improves with increasing function at the pinna changes in a complex way at differentpulse repetition rate [10,22,24,26,42,49,56,61]. Other azimuths [54]. Further studies need to be conducted tostudies showed that sound azimuth affects frequency determine the effect of different sound azimuths on thetuning of most central auditory neurons in bats [20], mice recovery cycle of IC neurons.[2], and frogs [11,58,60].

We observed that minimum threshold of IC neuronsincreased with BF (Fig. 1B) and the 50% recovery time of 4 .2. GABAergic inhibition shapes the recovery cycle ofIC neurons decreased with BF (Fig. 5A). It follows that the IC neurons50% recovery time of most IC neurons would alsodecrease with minimum threshold (Fig. 5B). Because we We observed that bicuculline application significantlyalways used BF sounds at 20 dB above a neuron’s decreased the 50% recovery time of IC neurons (Figs. 6–8;minimum threshold to measure the recovery cycle, abso- Table 1); similar to a previous study [30]. This observationlute sound level for measuring recovery cycle varied with indicates that GABAergic inhibition, which is long-lastingIC neurons that had different minimum thresholds. The (.5 ms) in the IC [59], shapes the recovery cycle of ICfact that 50% recovery time varied with minimum thres- neurons. It is conceivable that a decrease in the recoveryhold (e.g. Fig. 5B) suggests that sound level may affect the cycle of IC neurons during bicuculline application may berecovery cycle of IC neurons. Previous studies have due to removal of this long-lasting GABAergic inhibition

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Fig. 7. The recovery cycles of three IC neurons obtained before (filled circles) and during (unfilled circles) bicuculline application at c408 (Aa, Ba, Ca) andi408 (Ab, Bb, Cb). The 50% recovery time determined before (filled circles) and during (unfilled circles) bicuculline application and the percent change(%D) are shown within each plot (see Fig. 3 for legends).

thus allowing IC neurons to respond to the second sound at observation indicates that GABAergic inhibition shapes theshorter ISIs (Figs. 6–8). recovery cycle of IC neurons more effectively at i408 than

Although bicuculline application typically decreased the at c408. What might be the possible mechanisms underly-50% recovery time of IC neurons, the application was ing this different strength of GABAergic inhibition withmore effective on IC neurons with long than with short sound azimuth?recovery cycles at both sound azimuths (Fig. 8). A similar Under ear-phone stimulation conditions, a previousobservation has been reported in a previous study [30]. We study has shown that most IC neurons of the same batshowed that IC neurons with short recovery cycles typical- species are EI (excitation–inhibition) neurons that arely had high BFs while those with long recovery cycles had excited contralaterally and inhibited ipsilaterally [28]. Itlow BFs (Fig. 5A). Because these neurons were tonotopi- has been suggested that inhibition of EI neurons includescally organized along the dorsoventral axis of the IC (Fig. GABAergic inputs from the contralateral dorsal nucleus of1A), the former neurons were located at deeper IC than the lateral lemniscus and glycinergic inputs from the ipsilaterallatter neurons. A previous study has shown that neurons lateral superior olivary nucleus [1,27,38,40,46]. Previouswith GABA receptors are mostly distributed in the studies have also suggested that the directional sensitivityA

dorsomedial region of the IC but are sparsely distributed in of IC neurons is mainly due to changes in binauralthe ventrolateral region, which contains mostly glycinergic interaction and head shadowing with sound azimuthneurons [8]. For this reason, high BF neurons would [9,19,21,42,52,53]. Thus when a sound azimuth changesreceive less GABAergic inhibitory inputs than low BF from c408 to i408, the strength of contralateral excitation toneurons. It is conceivable that this different strength of EI neurons decreases but the strength of ipsilateral inhibi-GABAergic inhibition might contribute to the different tion increases. For this reason, IC neurons typically haveeffect of bicuculline application on the recovery cycle higher threshold and discharge fewer number of impulsesbetween high and low BF neurons (Fig. 8). at i408 than at c408 (Figs. 2,6). A previous study suggested

that a long-lasting and complex temporal pattern of4 .3. Possible mechanisms underlying different strength excitatory and inhibitory inputs is the main factor thatof GABAergic inhibition with sound azimuth shapes a neuron’s recovery cycle [4]. It is therefore

conceivable that variation in the strength of excitation andBicuculline application produced a greater decrease of inhibition with sound azimuth might contribute to the

the recovery cycle of IC neurons at i408 than at c408 such different strength of GABAergic inhibition and hence thethat azimuth-dependent recovery cycle of IC neurons was azimuth-dependent recovery cycle in the IC.abolished or greatly reduced (Fig. 9; Table 1). This Azimuth-dependent variation of the 50% recovery time

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of one group of IC neurons was abolished while that of theother group of IC neurons was only substantially reducedduring bicuculline application (Table 1). This observationindicates that these two groups of neurons might receivedifferent strengths of GABAergic inhibition in shaping therecovery cycle. Because these two groups of neurons didnot differ in BF, minimum threshold, and recording depth,their different strengths of GABAergic inhibition may bedue to different aural types or they may simply haveinherited directional response properties from lower audit-ory nuclei. For example, they may be EE (excitation–excitation) neurons that are excited bilaterally or EO(monaural excitation) neurons that are excited contralater-ally only [5,9,28,43,44]. Conversely, they may inheritdirectional response properties created in the lateral su-perior olive or the dorsal nucleus of the lateral lemniscusthrough the crossed excitatory projections [38]. Furtherwork with combined the ear-phone system and free-fieldstudy would be necessary to confirm this speculation.

4 .4. Biological significance of azimuth-dependentrecovery cycle

While a bat relies upon directional responses of auditoryneurons to determine target direction, it depends uponrecovery cycles of auditory neurons to process rapidlyreturning echoes. As shown in Fig. 5A, IC neurons withBF of 20–30 kHz had wide range of recovery cyclesincluding some long ones. These neurons would besuitable for processing returning echoes during early phase

Fig. 8. Comparisons of 50% recovery time of 36 IC neurons determined of hunting when pulse-echo delay is long and the pre-before and during bicuculline application at two sound azimuths. Note

dominant frequency of echoes is at 20–30 kHz [47,48].that all data points are below the equal-value diagonal line indicating aConversely, IC neurons with BF higher than 30 kHz anddecrease in 50% recovery time during bicuculline application.some low BF neurons had very short recovery cycles.These neurons would be suitable for processing rapidlyreturning echoes at the final phase of hunting when thefundamental of brief multiple harmonic FM sounds sweepfrom |60 to 23 kHz [47].

We have also shown that sound azimuth had less effecton the recovery cycle of IC neurons with high BF than lowBF (Fig. 5C). Previous studies showed that the bestazimuth of high BF neurons is located more medially inthe frontal auditory space than that of low BF neurons[25,39]. All these observations further suggest that high BFneurons can effectively process echoes during the finalphase of hunting.

Variation of recovery cycle of IC neurons with soundazimuth (Fig. 3A,B,D and Figs. 4, 6 and 7) suggests thattemporal analysis of returning echoes might be affectedwhen the bat alters its head orientation during targetlocalization. However, the bat has two ICs so that degra-dation of recovery cycles of neurons in one IC may becompensated by an improvement of recovery cycles of

Fig. 9. A comparison of changes in 50% recovery time of 36 IC neuronsneurons in the other IC. Furthermore, the recovery cycle ofproduced by bicuculline application at two azimuths. Note that most dataIC neurons would be affected very little during the finalpoints are scattered above the equal-value diagonal line indicating a

greater change in 50% recovery time at i408 than at c408. phase of hunting when echoes return within6108 lateral

140 X. Zhou, P.H.-S. Jen / Brain Research 973 (2003) 131–141

Table 1The average 50% recovery time of two groups of IC neurons determined at c408 and i408 both before and during bicuculline application

Difference in 50% recovery time c408 i408 t-test, Pduring drug application

Group I (,5 ms) Pre-drug 25.7615.3 38.7617.8 ,0.0001*(n521, 58%) Bicuculline 13.665.5 14.565.0 0.0343

t-test, P 0.0001* ,0.0001*% change 38.8625.0 55.8622.6 0.0002*

Group II (.5 ms) Pre-drug 34.4621.3 56.9625.3 ,0.0001*(n515, 42%) Bicuculline 18.469.5 27.368.0 ,0.0001*

t-test, P 0.0005* 0.0001*% change 38.6620.0 45.2622.4 0.0040*

*significant difference. Note that bicuculline application significantly decreased the 50% recovery time of two groups of IC neurons at both sound azimuths(P,0.001). Bicuculline application abolished azimuth-dependent 50% recovery time of group I neurons (P.0.01) but only substantially decreasedazimuth-dependent 50% recovery time of group II neurons (P,0.0001).

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