Abstract This study examined the effect of GABAergicand glycinergic inhibition on excitatory frequency tuningcurves (FTCs) of inferior collicular (IC) neurons of thebig brown bat, Eptesicus fuscus. The excitatory FTCs of70 IC neurons were either V-shaped (57, 81%), closed(11, 16%), or double-peaked (2, 3%). By means of atwo-tone stimulation paradigm, inhibitory FTCs wereobtained at one frequency flank only (low-frequencyflank: 11, 16%; high-frequency flank: 7, 10%), at bothfrequency flanks (36, 51%) of excitatory FTCs, or be-tween two excitatory FTCs (2, 3%). IC neurons that hadinhibitory FTCs typically had larger Q10 and Q30 values(i.e., sharper excitatory FTCs) than neurons that did nothave inhibitory FTCs. Neurons with inhibitory FTCs atboth frequency flanks had larger Q10 and Q30 values thanneurons with inhibitory FTCs at one frequency flank on-ly. IC neurons with a small difference between excitatoryand inhibitory best frequencies typically had sharper ex-citatory frequency tuning. Bicuculline (an antagonist forGABAA) application produced a greater degree of abol-ishing inhibitory FTCs than strychnine (an antagonist forglycine) application. Application of both drugs was mosteffective in abolishing the inhibitory FTCs of IC neu-rons. The implications of these findings for bat echoloca-tion are discussed.

Keywords Bicuculline · Frequency tuning curves · Inferior colliculus · Neural inhibition · Strychnine · Bat

Introduction

In sensory signal processing, neural inhibition plays animportant role in shaping response properties. For exam-ple, neural inhibition contributed to color contrast andedge contrast in the retina, the lateral geniculate nucleus,and the visual cortex (Singer and Creutzfeldt 1970; Kelly1973; Eysel et al. 1987). In the somatosensory system, ex-citatory areas surrounded by inhibitory areas were mappedout over the entire somatosensory pathway, including theskin, the dorsal column nuclei, the thalamus, and the so-matosensory cortex (Mountcastle and Powell 1959). In thecentral auditory system, Katsuki and his associates (1958)have found that the sharpness of excitatory frequency tun-ing curves (FTCs) increases along the auditory pathway,and they suggest that this sharpening of excitatory fre-quency tuning is due to synaptic inhibition from neighbor-ing auditory neurons. Since then, many studies have useda two-tone stimulation paradigm to examine neural inhibi-tion in excitatory FTCs of central auditory neurons. Thesestudies showed that many central auditory neurons haveinhibitory FTCs on one or both frequency flanks of theirexcitatory FTCs (Suga and Tsuzuki 1985; Ehret and Mer-zenich 1988; Vater et al. 1992; Suga et al. 1997). This typeof neural inhibition, which has been called afferent, or lat-eral, or side-band inhibition, sharpens excitatory FTCsand provides a means to reduce ambiguity in encodingfrequency at high intensities (see review, Suga 1995). Be-cause auditory neurons are organized tonotopically in eachauditory center, inhibition from neurons with small differ-ences in best-tuned frequencies arrives earlier than neu-rons with large best-frequency (BF) differences. For thisreason, we hypothesize that IC neurons with small differ-ences between excitatory and inhibitory BFs (abbreviatedas “BF difference”) should have sharper excitatory FTCs.

In the ascending auditory pathways, the central nucle-us of the inferior colliculus (IC) receives and integratesexcitatory and inhibitory inputs from a large number oflower auditory nuclei (Adams and Mugnaini 1984; Pollakand Casseday 1989; Covey and Casseday 1995; Cassedayand Covey 1996). The two main forms of inhibition are

Y. Lu · P.H.-S. Jen ( )Division of Biological Sciences, University of Missouri-Columbia, MO 65211, USAe-mail: jenp@missouri.eduTel.: +1-573-8827479, Fax: +1-573-8845020

Present address:Y. Lu, Department of Otolaryngology-HNS, University of Washington, Seattle, WA 98195, USA

Exp Brain Res (2001) 141:331–339DOI 10.1007/s002210100885

R E S E A R C H A RT I C L E

Yong Lu · Philip H.-S. Jen

GABAergic and glycinergic neural inhibition in excitatory frequency tuning of bat inferior collicular neurons

Received: 5 March 2001 / Accepted: 10 August 2001 / Published online: 18 October 2001© Springer-Verlag 2001

GABAergic and glycinergic (Roberts and Ribak 1987a,1987b; Saint Marie et al. 1989; Fubara et al. 1996), whichcontribute to shaping many response properties of ICneurons, including the discharge rate, discharge pattern,response latency, duration tuning, and binaural signalprocessing (Pollak and Park 1993; Casseday et al. 1994;Klug et al. 1995; Le Beau et al. 1996; Jen and Feng1999). GABA- and glycine-mediated inhibition alsoshapes selectivity for frequency modulation (FM) of ICneurons in FM bats (Fuzessery and Hall 1996; Koch andGrothe 1998) and delays tuning for IC neurons in themustached bats (Wenstrup and Leroy 2001).

In frequency tuning, GABAergic inhibition contrib-utes importantly to sharpening excitatory FTCs (Vater etal. 1992; Yang et al. 1992; Palombi and Caspary 1996).However, the role of glycinergic inhibition in frequencytuning has been only briefly mentioned for the bat ICneurons (Vater et al. 1992). Nevertheless, judging fromthe fact that glycinergic inhibition shapes many auditoryresponse characteristics as cited above, it is reasonable toexpect that glycinergic inhibition may also contribute toexcitatory frequency tuning in bat IC neurons.

In this study, we examined the importance of neuralinhibition in frequency tuning of IC neurons by compar-ing the sharpness of excitatory FTCs of IC neurons withand without inhibitory FTCs. We then determined thecontribution of GABAergic and glycinergic inhibition toexcitatory frequency tuning by comparing the excitatoryFTCs of IC neurons plotted before and during iontopho-retic application of the antagonists bicuculline andstrychnine (Cooper et al. 1982; Bormann 1988).

Materials and methods

Six bats (three males, three females; 15–20 g) were used for thisstudy. As in previous studies (Jen et al. 1989), the flat head of a1.8-cm nail was glued onto the exposed skull of each Nembutal-anesthetized bat (45–50 mg/kg) with acrylic glue and dental ce-ment 1 day or 2 days before the recording session. Exposed tissuewas treated with an antibiotic (Neosporin) to prevent inflamma-tion. During recording, the bat was administered the neuroleptan-algesic Innovar-Vet (fentanyl 0.08 mg/kg, droperidol 4 mg/kg) andwas placed inside a bat holder (made of wire mesh), which wassuspended in an elastic sling inside a sound-proof room (tempera-ture 28–30°C). The ceiling and inside walls of the room were cov-ered with 3-inch convoluted polyurethane foam to reduce echoes.A local anesthetic (lidocaine) was applied to the open wound area.After orienting the bat with its eye-snout line pointed to 0° in azi-muth and 0° in elevation of the frontal auditory space, its head wasimmobilized by fixing the shank of the nail into a metal rod with aset screw (Suga and Schlegel 1972). Small holes were bored in theskull above the IC for insertion of piggy-back multibarrel elec-trodes, to record auditory responses of IC neurons and to ejectbicuculline or strychnine iontophoretically to the recording site.The recording depth was read from the scale of a microdrive (Da-vid-Kopf). A reference electrode (silver wire) was placed at thenearby temporal muscles. Each bat was used in one to six record-ing sessions on separate days, and each recording session typicallylasted for 6–8 h. The experiments were conducted in compliancewith NIH publication No. 85–23, Principles of Laboratory AnimalCare and with the approval of the Institutional Animal Care andUse Committee (1438) of the University of Missouri-Columbia.

The construction of piggy-back multibarrel electrodes for re-cording and iontophoretic application of drugs has been described

in previous studies (Lu et al. 1997). Briefly, a five-barrel electrode(tip 10–15 µm) was “piggybacked” to a 3 M KCl single-barrelelectrode (tip less than 1 µm; impedance 5–10 MΩ), whose tipwas extended about 10 µm beyond the tip of the five-barrel elec-trode. The 3 M KCl single-barrel recording electrode was connect-ed by a silver wire to an amplifier (HP 465A) followed by an elec-tronic filter (KH 3500). One of the barrels of the five-barrel elec-trode was filled with bicuculline methiodide (10 mM in 0.16 MNaCl, pH 3.0; Sigma). A second barrel was filled with strychnine(10 mM in 0.16 M NaCl, pH 3.5; Sigma).

Drug solutions were prepared just prior to each experiment andthe electrode filled immediately before use. The two drug chan-nels were connected via silver-silver chloride wire to a microion-tophoresis constant-current generator (Medical Systems Neuro-phore BH-2), which was used to generate and monitor iontopho-retic currents. The iontophoretic current for bicuculline andstrychnine application ranged from 10 to 50 nA, and from 20 to100 nA, respectively. During single-drug application, 1-s pulses at0.5 pulses/s (pps) were applied and data were collected after a sta-ble effect was observed. When both drugs were applied, 1-s pulsesat 0.5 pps were applied to the two channels alternatively. The oth-er two barrels were filled with 1 M NaCl (pH 7.4), one of whichwas used as the ground and the other as the balanced barrel. Thebalance electrode was connected to a balance module. The retain-ing current was negative 8–10 nA.

Generation of sound stimuli has been described in previousstudies (Jen et al. 1989). Briefly, continuous sine waves from anoscillator (KH model 1200) were formed into tone pulses by ahomemade tone burst generator (electronic switch) driven by astimulator (Grass S88). The tone pulses were then amplified (HP465A) after passing through a decade attenuator (HP 350D), be-fore they were fed to a small condenser loudspeaker (AKG modelCK 50; 1.5-cm diameter, 1.2 g). The loudspeaker was placed23 cm away from the bat and 30° contralateral to the recordingsite. The loudspeaker was calibrated with a quarter-inch micro-phone (B&K 4135) placed at the bat’s ear using a measuring am-plifier (B&K 2607). The output of the loudspeaker was expressedin decibels of sound pressure level (SPL), referred to 20 µPa root-mean-square. A frequency characteristics curve was plotted for theloudspeaker to determine the maximal available stimulus intensityat each frequency.

Upon isolation of an acoustically evoked IC neuron with 4-mssounds (0.5-ms rise-decay times, at 2 pps), the BF and minimumthreshold (MT) of the neuron were determined by changing the fre-quency and intensity of sound pulses. The MT was defined as theintensity at which the neuron responded to each of two consecutiveBF sounds. The neuron’s excitatory FTC was then measured audio-visually by determining the threshold of each responsive frequency.

Using a two-tone stimulation paradigm, an inhibitory FTC wasaudiovisually measured by first delivering a 4-ms excitatory BFtone at 10 dB above excitatory MT (the probe tone). Then the fre-quency and intensity of a 4-ms tone (the masker) delivered with a2-ms gap to the excitatory tone were systematically changed to de-termine the limit at which the neuron’s response to the excitatoryBF tone was reduced by at least 20%.

To determine the contribution of GABAergic and glycinergicinhibition to excitatory frequency tuning, the neuron’s excitatoryFTC was measured before and during iontophoretic application ofthe antagonists bicuculline and/or strychnine. The sharpness of ex-citatory FTCs obtained under different stimulation conditionswere then determined and compared. The sharpness of an excitato-ry FTC was expressed with Q10 and Q30 values, which were ob-tained by dividing the excitatory BF by bandwidths at 10 dB and30 dB above the excitatory MT.

Recorded action potentials were amplified, band-pass filtered(Krohn-Hite 3500), and fed through a window discriminator (WPI121) before being sent to an oscilloscope (Tektronix 5111) and anaudio monitor (Grass AM6). They were then sent to a computer(Gateway 2000, 486) for acquisition of peristimulus-time (PST)histograms (bin width, 500 µs, sampling period, 100 ms) to 16sound presentations. The total number of impulses in each PSThistogram was used to quantify a neuron’s response under eachstimulus condition.

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Results

Excitatory FTCs

Seventy IC neurons responding to presented acousticstimuli were isolated at depths of 266–1863 µm, with ex-citatory BFs of 10.1–87.6 kHz (59/70; 84% between 20and 70 kHz) and excitatory MTs of 5–74 dB SPL (54/70;77% less than 50 dB SPL). The excitatory FTCs of theseneurons can be classified into three groups (Fig. 1). Thefirst group (57/70; 81%) had V-shaped FTCs that were ei-ther broad (29/70; 41%) or narrow (28/70; 40%). At high

intensities, broad FTCs were tuned to a frequency band-width of 30–45 kHz (Fig. 1a), while narrow FTCs weretuned to a bandwidth of less than 30 kHz (Fig. 1b, c).Two neurons had extremely narrow FTCs (BF, 22.6 and23.6 kHz), with sharp slopes on both high-frequency(1,516 dB/octave and 402 dB/octave) and low-frequencyflanks (419 dB/octave and 625 dB/octave; Figs. 1c, 6d).The second group (11/70; 16%) had closed (upper thresh-old) FTCs which had thresholds at both low and highstimulus intensities. The ascending flanks from the lowestMT widened to a certain degree before closing at higheststimulus intensity (Fig. 1d). The third group (2/70; 3%)had double-peaked FTCs (Fig. 1e). Each of these 2 neu-rons were tuned to two different frequency regions.

Q10 values of these 70 IC neurons ranged from 1.0 to39.4 (mean, 7.3±6.2), with only 3 (4%) above 20 and 55(79%) below 10. Q30 values were obtained for 54 ICneurons that ranged between 0.8 and 13.1 (mean,4.0±2.7), with only 3 (6%) between 10 and 20, and 51(94%) below 10.

Inhibitory FTCs plotted with a two-tone stimulation paradigm

The excitatory and inhibitory FTCs as well as PST histo-grams of a representative IC neuron obtained under dif-ferent experimental conditions are shown in Fig. 2. Thisneuron had inhibitory FTCs (hatched areas) at both high-and low-frequency flanks of its excitatory FTC (opencircles). Both inhibitory MTs are higher than the excita-tory MT. The neuron discharged a total of 24 impulses toan excitatory BF tone at 10 dB above the excitatory MT(1st PST histogram in Fig. 2). The number of impulses inresponse to the excitatory tone decreased to 10 when amasker was delivered within the high-frequency flankinhibitory FTC (2nd PST histogram in Fig. 2). The num-ber of impulses was affected very little when the maskerwas delivered outside the inhibitory FTCs (3rd and 4th

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Fig. 1a–e Representative excitatory frequency tuning curves(FTCs) of inferior collicular (IC) neurons of the big brown bat,Eptesicus fuscus. These FTCs were broad (a), narrow (b, c),closed (upper threshold, d), or double-peaked (e). Ordinates andabscissae represent stimulus intensity (dB SPL) and stimulus fre-quency (kilohertz; see text for details)

Fig. 2 An IC neuron with inhib-itory FTCs (hatched areas) atboth frequency flanks of its ex-citatory FTC (unfilled circles).These inhibitory FTCs wereplotted with a two-tone stimula-tion paradigm. At the bottom areshown peristimulus-time (PST)histograms obtained during fivedifferent stimulation conditions.The frequency and intensity of aprobe tone (an excitatory bestfrequency tone at 10 dB abovethe excitatory minimum thresh-old) was inside the excitatoryFTC (1) while that of a maskertone was either inside (2) or outside (3, 4) the inhibitory FTCof high-frequency flank or inside (5) the excitatory FTC (N the number of impulses per16 stimuli)

PST histograms in Fig. 2). When the masker was deliv-ered within the neuron’s excitatory FTC prior to the ex-citatory tone, the masker evoked response from the neu-ron (the first prominent response in the 5th PST histo-gram in Fig. 2), which decreased the neuron’s responseto the excitatory tone (the second small response in the5th PST histogram).

Using a two-tone stimulation paradigm, inhibitoryFTCs were obtained either at both frequency flanks(36/70; 51%; Fig. 3, neurons 1–36) or at low-frequencyflank only (11/70; 16%; Fig. 3, neurons 37–47) or high-frequency flank (7/70; 10%; Fig. 3, neurons 48–54) ofexcitatory FTCs. There were 2 neurons (3%) whose in-hibitory FTCs were sandwiched between two excitatoryFTCs (Fig. 3, neurons 55 and 56). The remaining (14/70;20%) neurons did not have inhibitory FTCs (Fig. 3, neu-rons 57–70).

Contribution of neural inhibition to excitatory frequency tuning

A cursory comparison of all excitatory and inhibitoryFTCs of these 70 neurons shown in Fig. 3 suggests thatneurons with inhibitory FTCs appear to have sharper ex-citatory FTCs than those without. Neurons with inhibito-ry FTCs at both frequency flanks also appear to havesharper excitatory FTCs than those with inhibitory FTCsat one frequency flank only. To quantitatively determinethe contribution of neural inhibition to excitatory fre-quency tuning, we compared the Q10 and Q30 values ofexcitatory FTCs with and without inhibitory FTCs. Wealso compared the Q10 and Q30 values of excitatory FTCswith inhibitory FTCs at both frequency flanks and at onefrequency flank only.

As shown in Fig. 4, the distributions of Q10 and Q30 ofIC neurons in relation to excitatory BF show a peak ataround 20 kHz, which declines to low values at high andlow excitatory BFs. Nine neurons which were tuned be-tween 20 and 25 kHz had a broad range of Q10 (1.6–39.4)and Q30 (0.9–13.1) values. These distributions show thatneurons with larger Q10 and Q30 values typically had BFsof 20–35 kHz. Larger Q10 and Q30 values (sharper fre-quency tuning) were also more often obtained from ICneurons with inhibitory FTCs than IC neurons without in-hibitory FTCs (Fig. 4a, b, unfilled circles vs filled trian-gles) and from IC neurons with two inhibitory FTCs thanfor IC neurons with one inhibitory FTC (Fig. 4c, d un-filled circles vs filled triangles). However, because of thesmall sample size of IC neurons that either did not haveinhibitory FTCs or had inhibitory FTCs at one frequencyflank only, such a comparison was not always statisticallysignificant (see insets in Fig. 4).

In order to determine whether closer inhibitory FTCssharpen the excitatory FTCs more than farther inhibitoryFTCs, we plotted the distributions of Q10 and Q30 valuesagainst the BF differences (Fig. 5). These distributionsshow that IC neurons with small BF differences had larg-er Q10 and Q30 values than IC neurons with large BF dif-ferences. The Q10 and Q30 values of these neurons de-creased drastically with increasing BF differences. Lin-ear regression analyses showed a significant decrease inQ30 values with increasing BF differences (P<0.05).

Effects of application of bicuculline, strychnine, or bothon inhibitory FTCs

Representative excitatory and inhibitory FTCs of five neu-rons obtained under different experimental conditions are

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Fig. 3 Excitatory (solid lines)FTCs of all 70 IC neurons with(neurons 1–56) or without(neurons 57–70) inhibitoryFTCs (hatched). InhibitoryFTCs were obtained either atboth frequency flanks (neurons1–36), at one frequency flankonly (neurons 37–54) of excita-tory FTCs or sandwiched be-tween two excitatory FTCs(neurons 55, 56). The frequen-cy and intensity scales areshown for one neuron (70)

shown in Fig. 6. Application of bicuculline broadly ex-panded the excitatory FTCs (unfilled circles vs filled cir-cles) and literally abolished the inhibitory FTCs (hatchedareas) of two neurons (Fig. 6a, b). Further application ofstrychnine to one neuron produced little effect on the al-ready expanded excitatory FTC (Fig. 6b, filled circles vsfilled triangles). In another neuron, application of strych-nine alone broadly expanded the excitatory FTC (unfilledcircles vs filled squares) and almost completely abolishedthe inhibitory FTC (hatched area; Fig. 6c). Figure 6d–fshows the effect of application of both drugs on frequencytuning of two IC neurons that had either a sharp (Fig. 6d)or a closed excitatory FTC (Fig. 6e, f). Application of bothdrugs expanded the sharply tuned excitatory FTC (Fig. 6d,unfilled circles vs filled triangles) but did not completelyabolish the inhibitory FTCs at both high- and low-frequen-cy flanks (Fig. 6d, hatched areas). For the neuron with aclosed excitatory FTC, bicuculline application unevenlyexpanded the upper portion of the excitatory FTC such thatthe inhibitory FTC at the low-frequency flank was com-pletely abolished but the inhibitory FTC at the high-fre-quency flank was only partially abolished (Fig. 6e, unfilled

circles vs filled circles, hatched areas). Further applicationof strychnine greatly expanded the high-frequency flank ofexcitatory FTC and completely encompassed the predruginhibitory FTC at the high-frequency flank (Fig. 6f, un-filled circle vs filled triangles, dashed areas). Thus, appli-cation of both drugs converted the closed excitatory FTCinto a V-shaped FTC, similar to previous findings (Vater etal. 1992; Fuzessery and Hall 1996).

Among the 56 neurons that had inhibitory FTCs: 30neurons were tested by bicuculline application alone; 7neurons were tested by bicuculline application alone andthen with additional strychnine application; 16 neuronswere tested by strychnine application alone and thenwith additional bicuculline application; and 9 neuronswere tested by bicuculline and strychnine application.The excitatory FTCs of these IC neurons plotted beforeand during application of bicuculline (neurons 1–31),strychnine (32–42), or both drugs (neurons 43–74) areshown in Fig. 7. It is clear that application of these drugseither did not affect excitatory FTCs (e.g., Fig. 7, neu-rons 29, 33, 34, 71) or expanded the excitatory FTCs tovarying degrees (e.g., Fig. 7, neurons 2, 37, 49). Table 1

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Fig. 4a–d Scatter plots show-ing the distributions of Q10 andQ30 values of excitatory FTCsof IC neurons with (unfilledcircles) or without (filled trian-gles) inhibitory FTCs (a, b)and with one (filled triangles)or two (unfilled circles) inhibi-tory FTCs (c, d) in relation tothe excitatory best frequency(EBF). The number of neurons,range, mean Q10 and Q30 valuesand the t-test significance levelfor each distribution are shownwithin the inset (see text for details)

Fig. 5a, b Scatter plots showingthe distributions of Q10 and Q30values in relation to the differ-ence between inhibitory and ex-citatory best frequencies (IBFand EBF difference). Data ob-tained between low-frequencyflank IBF and EBF are shown atthe left and those between high-frequency flank IBF and EBF areshown at the right. The linear re-gression line and correlation co-efficient for each distribution areshown by a solid line and r

336

Fig. 7 Excitatory FTCs of ICneurons plotted before (dashedlines) and during (solid lines)application of bicuculline (neu-rons 1–31), strychnine (32–42)or during application of bothdrugs (neurons 43–74). Thefrequency and intensity scalesare shown for one neuron (74)

Fig. 6a–f Excitatory and inhibitory FTCs of five IC neurons de-termined under different drug application conditions (shown withdifferent symbols). Unfilled circles, excitatory FTCs obtained be-fore drug application; solid line with hatched area, inhibitoryFTCs plotted with a two-tone stimulation paradigm before drug

application; solid circles, excitatory FTCs obtained during bicu-culline (bic) application; solid squares, excitatory FTCs obtainedduring strychnine (stry) application; solid triangles, excitatoryFTCs obtained during application of both bicuculline and strych-nine; dashed lines, predrug inhibitory FTCs (See text for details)

Discussion

Excitatory FTCs of IC neurons

The three different types of excitatory FTCs of IC neuronsobtained from the present study (Fig. 1) have been reportedin earlier studies (Jen and Schlegel 1982; Casseday andCovey 1992). They are probably the result of excitatoryand/or inhibitory integration of as many as ten separatepathways arising from lower auditory nuclei (Pollak andCasseday 1989). We found that neurons tuned at around20–25 kHz had a wide range of Q10 and Q30 values(Fig. 4a–d). Some of these neurons had extremely sharpfrequency tuning and low MTs (Figs. 1c, 6d). These neu-rons are similar to “filter neurons” found in the constantfrequency-frequency modulated (CF-FM) bats (Neuweilerand Vater 1977; Suga and Jen 1977; Suga and Tsuzuki1985). The 20- to 25-kHz tuning is the first harmonic of15–20-ms echolocation sounds emitted by the big brownbat during the search phase of hunting (Griffin 1958; Simmons et al. 1979). Casseday and Covey (1992) havesuggested that these sharply tuned neurons are specializedfor detecting echoes during the search phase of hunting.

Neural inhibition sharpens excitatory frequency tuning

In this study, we showed that neurons with inhibitoryFTCs often have larger Q10 and Q30 values than neuronswithout inhibitory FTCs (Fig. 4a, b), and neurons withtwo inhibitory FTCs have larger Q10 and Q30 values thanneurons with one inhibitory FTC only (Fig. 4c, d). Thesefindings support the contention that neural inhibitionsharpens excitatory FTCs (see review, Suga 1995).

We also found that the Q10 and Q30 values increasedwith decreasing BF differences (Fig. 5). This is expectedbecause auditory neurons are organized tonotopically,and this tonotopic organization dictates that the inhibi-tion from neurons with small BF differences arrives ear-lier with less attenuation than neurons with large BF dif-ferences. The fact that only Q30 values increase signifi-cantly with decreasing BF differences suggests that neu-ral inhibition contributes more to sharpening of upperthan lower portions of excitatory FTCs (Fig. 5, right).This is corroborated by the fact that most inhibitory MTswere higher than corresponding excitatory MTs (Figs. 2,3, 6). Drug application also expanded the upper portionmore than the lower portion of excitatory FTCs (Figs. 6,7). Previous studies have suggested that sharpening ofthe upper portion of excitatory FTCs by inhibition reduc-es the ambiguity in encoding frequency at high stimulusintensities (Suga and Tsuzuki 1985; Suga 1995).

GABAergic and glycinergic inhibition contributes to excitatory frequency tuning

Previous studies show that the main inhibitory neuro-transmitters for the IC are GABA and glycine (Roberts

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Table 1 Effect of application of bicuculline (Bic), strychnine (Stry),or both drugs (Bic/Stry) on inhibitory frequency tuning curves(FTCs) of inferior collicular neurons (N number of neurons studied)

Drug Effect on inhibitory FTCsapplication

Completely Partially Not abolished abolished affected

N % N % N %

Bic (N=37) 11 30.0 23 62.0 3 8.0Stry (N=16) 2 12.5 10 62.5 4 25.0Bic/Stry (N=32) 22 69.0 6 19.0 4 12.0

Fig. 8 Distributions of the recording depth of IC neurons whose ex-citatory FTCs were affected by bicuculline (unfilled bars) andstrychnine (filled bars) application. Most (24/34; 71%) IC neuronswhose excitatory FTCs were affected by bicuculline applicationwere recorded at depths of less than 1,200 µm. However, most (8/12;67%) IC neurons whose excitatory FTCs were expanded by strych-nine application were recorded at depths of more than 1,200 µm

summarizes the effect of different drug applications onthe inhibitory FTCs of IC neurons. Bicuculline applica-tion affected the excitatory FTCs of a larger percentageof IC neurons than strychnine application. Bicucullineapplication was also more effective in completely abol-ishing the inhibitory FTCs than strychnine application.However, application of both drugs was most effective incompletely abolishing the inhibitory FTCs.

Because IC neurons were recorded at different depths,we were able to examine the effect of bicuculline andstrychnine application on excitatory FTCs in relation totheir recording depths. The 34 neurons whose excitatoryFTCs were expanded by bicuculline application were re-corded over a wide range of depths (Fig. 8, unfilledbars). Most of them (24/34; 71%) were recorded atdepths of less than 1,200 µm. For those 12 neuronswhose excitatory FTCs were expanded by strychnine ap-plication, most (8/12; 67%) were recorded at depths ofmore than 1200 µm (Fig. 8, filled bars).

and Ribak 1987a, 1987b; Saint Marie et al. 1989; Fubaraet al. 1996). We found that bicuculline and/or strychnineapplication expanded the excitatory FTCs into frequencyregions that were covered by predrug inhibitory FTCs(Figs. 6, 7). This finding indicates that both GABAergicand glycinergic inhibition sharpens excitatory FTCs ofIC neurons. However, GABAergic inhibition contributedmore significantly to excitatory frequency tuning thanglycinergic inhibition, because bicuculline applicationwas more effective than strychnine application in affect-ing the inhibitory FTCs (Table 1). A similar finding hasbeen reported for frequency tuning of IC neurons inhorseshoe bats, Rhinolophus rouxi (Vater et al. 1992).

It has been reported that the density of GABAA recep-tors is high dorsomedially and low ventrolaterally, whilethe opposite is true for the density of glycine receptors(Fubara et al. 1996). Because we isolated IC neurons atdepths of 266–1863 µm along the dorsoventral axis ofthe IC, these IC neurons probably received both GABA-ergic and glycinergic inhibition to varying degrees. Thisis supported by our findings that bicuculline applicationaffected the excitatory FTCs of more neurons in the up-per than in the deeper IC, but strychnine application af-fected the excitatory FTCs of more neurons in the deeperthan in the upper IC (Fig. 8).

Application of both bicuculline and strychnine did notabolish inhibitory FTCs of a small (12%) number of ICneurons (Table 1). GABAergic and glycinergic inhibitioneither did not contribute to excitatory frequency tuningof these neurons or they may simply inherit the frequen-cy tuning properties from lower-order auditory neurons.

Implications in echolocation

Eptesicus fuscus emits ultrasonic signals and analyses thereturning echoes to extract information on the insects(Griffin 1958). During this hunting process, fine-frequen-cy analysis is essential for prey feature discrimination. Wehave demonstrated that GABAergic and glycinergic inhi-bition contributes to sharpening of frequency tuning of ICneurons (Figs. 6, 7, Table 1). For example, neural inhibi-tion is responsible for extremely fine frequency tuningproperties of 20-kHz neurons found in the big brown bats(Figs. 1c, 6d) and the “level tolerant” neurons in CF/FMbats (Suga and Tsuzuki 1985; Suga 1995; Suga et al.1997). These sharply tuned neurons are particularly effec-tive for target detection and fine-frequency analysis (Sugaand Tsuzuki 1985; Casseday and Covey 1992; Suga 1995;Suga et al. 1997). Neural inhibition is also responsible forcreating FM sweep selectivity (Suga 1965; Fuzessery andHall 1996) and closed FTCs (Figs. 1d, 6e, f). Neuronswith closed FTCs are both frequency and intensity specif-ic such that they are specialized in responding to either theemitted pulse or the returning echo only (Grinnell 1963).

In summary, we found that IC neurons with inhibitoryFTCs had sharper excitatory FTCs than neurons withoutinhibitory FTCs. Neurons with inhibitory FTCs at bothfrequency flanks of excitatory FTCs had sharper fre-

quency tuning than neurons with inhibitory FTCs at onlyone frequency flank. IC neurons with small BF differ-ences typically had sharper excitatory frequency tuning.Bicuculline application produced a greater degree ofabolishing inhibitory FTCs than strychnine application.However, application of both drugs was most effective incompletely abolishing the inhibitory FTCs.

Acknowledgements This work was supported by a research grantfrom the National Science Foundation (NSF IBN 9907610) and agrant from the Research Board of the University of Missouri (RB98–014 to Jen) and the Research Council (URC 01–012 to Jen).We thank Dr. J. Maruniak and two anonymous reviewers for theircritical comments on an earlier version of this manuscript.

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