Leading inhibition to neural oscillation is important for ... · PDF filecutaneous respiration. The board was contoured such that the frog’s head was raised slightly

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Leading inhibition to neural oscillation is important for time domain processing in the

auditory midbrain

Alexander V. Galazyuk*, Wenyu Lin, Daniel Llano, and Albert S. Feng

Department of Molecular and Integrative Physiology and Beckman Institute, University of Illinois,

Urbana, IL 61801

Corresponding author (*present address):

Dr. Alexander V. Galazyuk

Northeastern Ohio University College of Medicine

4209 State Route 44

Rootstown, OH 44272

Tel: 330-325-6640; Fax: 330-325-5916

Email: [email protected]

Keywords: Bat, frog, inferior colliculus, paradoxical latency shift, neural oscillation

Articles in PresS. J Neurophysiol (March 16, 2005). doi:10.1152/jn.00056.2005

Copyright © 2005 by the American Physiological Society.

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Abstract:

A number of central auditory neurons exhibit paradoxical latency shift (PLS), a response

characterized by longer response latencies at higher sound levels. PLS neurons are known to

play a role in target ranging for echolocating bats that emit frequency-modulated sounds. We

recently reported that early inhibition of unit’s oscillatory discharges is critical for PLS in the

inferior colliculus (IC) of little brown bats. The goal of this study was to determine in

echolocating bats and in non-echolocating animals (frogs): 1) the detailed characteristics of PLS

and whether PLS was dependent on sound level, frequency and duration, 2) the time course of

inhibition underlying PLS using paired pulse paradigm. We found that 22% of IC neurons in bats

and 15% in frogs exhibited periodic discharge patterns in response to tone pulses at high sound

levels. The firing periodicity was unit specific and independent of sound level and duration.

Other IC neurons (28% in bats; 14% in frogs) exhibited PLS. These PLS neurons shared

several response characteristics: 1) PLS was largely independent of sound frequency, 2) the

magnitude of shift in first-spike latency was either duration-dependent or duration-tolerant. For

PLS neurons, application of bicuculline abolished PLS and unmasked the unit’s periodical firing

pattern that served as the building block for PLS. In response to paired sound pulses, PLS

neurons exhibited delay-dependent response suppression confirming that high threshold

leading inhibition was responsible for PLS. Results also revealed the timing of excitatory and

inhibitory inputs underlying PLS and its role in time domain processing.

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Introduction

Animals that echolocate, such as bats and dolphins, utilize a sonar system to orient in

3D space and to detect and track moving prey. For this, they must assess the static and

dynamic characteristics of each target in their surroundings, such as the target range, size,

shape, azimuth, elevation, direction and speed of target movement. Target ranging is especially

important for tracking a moving prey, and has been extensively studied behaviorally and

physiologically. Physiological studies have shown that central auditory neurons of echolocating

bats are tuned to echo delay, showing response facilitation to pulse-echo pairs at particular

echo delays (Feng et al., 1978; O'Neill and Suga, 1979; Sullivan, 1982 a, b; Mittmann and

Wenstrup, 1995; O'Neill, 1995). Although delay-tuned response characteristics have been

described in detail, the underlying mechanisms are not well understood.

In the auditory cortex of little brown bats, a species that uses frequency modulated (FM)

ultrasonic pulses for echolocation, Sullivan (1982a,b) proposed that delay-tuned responses are

attributed to a coincidence detection mechanism. For this, the input neurons must show a

delayed response to a loud sonar signal such that this response can align temporally with the

units' response to a weak and late arriving echo. Neurons exhibiting paradoxical latency shift

(PLS), i.e., longer response latencies for intense sounds than for weaker sounds, are therefore

well suited for construction of delay-tuned responses. There are two lines of evidence

suggesting that PLS plays a role in target ranging. First, behavioral experiments in bats

suggested that neurons exhibiting PLS may be involved in target ranging (Denzinger and

Schnitzler, 1994). Second, Sullivan (1982b), and later Berkowitz and Suga (1989), found that for

the majority of neurons showing PLS in the little brown bat auditory cortex, the magnitude of the

latency shift for each neuron corresponded to the unit’s best echo delay to which this neuron is

tuned.

Sullivan proposed two models which may account for the PLS in the cortex of little brown

bats (the Delay Line model and the Timed Inhibitory model), but neither of these models was

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experimentally validated. Galazyuk and Feng recently (2001) reported that, for the inferior

colliculus in the same species, PLS can be created by high-threshold early inhibition of neural

oscillatory discharges. Their result provides support for the Timed Inhibitory model of Sullivan.

Early inhibition (which shall be referred to as “leading inhibition”) that precedes an excitatory

response therefore, represents a key building block for the PLS. At this time, our understanding

of the basic characteristics of leading inhibition and its role in PLS is limited. The goal of the

present study is to fill this knowledge gap. Given that the inferior colliculus is the first station

along the central auditory pathway where PLS neurons are found (Berkowitz and Suga, 1989;

Klug et al., 2000), our study was focused there. Additionally, because PLS appears to be a

common phenomenon among animals (cats, Rose et al., 1963; gerbils, Klug et al., 2000; and

insects, Krahe et al., 2002), the present study will investigate PLS phenomena in the IC of the

little brown bats (an echolocating species for which PLS is known to have a role in delay-tuned

response and target ranging) and the northern leopard frog (a non-echolocating vertebrate).

METHODS

Surgical and Recording Procedures

Bats - Experimental subjects comprised 23 little brown bats, Myotis lucifugus. Details of

experimental methods are given in Galazyuk and Feng (2001). For surgery, the animal was

anesthetized via halothane inhalation (4% halothane administered by a precision vaporizer).

After incision of the skin and clearing of the tissues above the skull, a small metal rod was glued

to the skull using glass ionomer cement. Following the surgery, animals were allowed to recover

for 2-4 days in individual holding cages.

Recordings were made from awake bats. During the recording session the animal was

placed inside a sound attenuating chamber. The metal rod on the bat’s head was secured to a

small holder for restraining the animal's head atraumatically, leaving the ears unobstructed for

free-field acoustic stimulation. A small hole (~ 50 µm) was then made in the skull overlying the

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IC through which a recording electrode was inserted to reach the IC. Throughout the recording

session, the animal was offered drinking water periodically and monitored for signs of

discomfort. After a recording session of 6-8 hours, the exposed skull was covered with sterile

bonewax, and the animal was returned to its holding cage. Such experiments proceeded every

2-3 days for a maximum of 3 weeks.

Extracellular single-unit recordings were made with glass micropipettes (10-20 MΩ, 2-3

µm tip) filled with horseradish peroxidase (5% in 0.2 M tris buffer). Such electrodes produce

stable single-unit recordings over long durations, and thus are preferred over other types of

recording electrodes. The electrode was positioned above the IC by means of a precision (1

µm) digital micromanipulator and lowered to the dorsal brain surface. The relative position of

each electrode was monitored from the readouts of digital micrometers using a common

reference on the skull. Vertical advancement of the electrode was made by a precision

piezoelectric microdrive from outside the sound attenuating chamber. Recorded action

potentials were amplified (Dagan 2400 preamplifier), monitored audiovisually, and stored on the

computer hard-drive, and processed off-line (100 µs bin width) using data acquisition

software/hardware from RC Electronics.

Frogs - Experimental methods generally adhered to those described in Lin and Feng (2003).

Briefly, 32 Northern leopard frogs (Rana pipiens), weighing 12-32 g, were used in this study. For

surgery, the animals were anesthetized by immersion in tricaine methanesulfonate (MS-222),

and immobilized with an intra-muscular injection of d-tubocurarine chloride (10 µg/g body

weight). The animal was placed on a Styrofoam board and wrapped in moist gauze to facilitate

cutaneous respiration. The board was contoured such that the frog’s head was raised slightly

above the body in order to approximate its natural sitting position. Under anesthesia, a skin flap

was incised on the dorsal surface of the head, and a small opening was made in the skull above

the left optic tectum. Lidocaine (4%) was applied topically to the wound area. The dural,

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arachnoid, and pial membranes were removed to expose the surface of the tectum. After the

surgery, the animal was placed in a sound attenuated chamber and allowed to recover from

anesthesia. Immobilization was maintained during the recording session by periodic injections of

d-tubocurarine chloride.

Extracellular recordings were made from single neurons in the left torus semicircularis (a

homolog of mammalian inferior colliculus, IC) using the same type of electrode described for

bats. The electrode was attached to a precision piezoelectric microdrive and positioned

stereotactically onto the dorsal surface of the left optic tectum. The action potentials were

amplified and monitored using similar audiovisual devices, and stored on a computer hard drive

but processed using Tucker Davis data acquisition software (Brain-Ware).

Our experimental protocols for bats and frogs are in compliance with the "Guide for the

Care and Use of Laboratory Animals" publication No. 86-23 of the National Institutes of Health

and with the Animal Welfare Act of 1966 and its amendments of 1970 and 1976. These were

reviewed and approved by the University of Illinois Lab Animal Use and Care Committee.

Acoustic Stimulation

Bats - Acoustic stimuli, comprising tone pulses at the unit’s characteristic frequency (CF), were

delivered to the bat via a free field ultrasonic loudspeaker located 60 cm in front of the bat. Tone

pulses had a constant rise-fall time of 0.5 ms. The duration of tone pulses ranged from 2 to 8

ms. Sound pulses of a wide range of sound levels, from 10 to 90 dB SPL, were presented at a

rate of 1 per second in increments of 2-4 dB. The range of sound level was extended to lower

values if unit’s response threshold was below 10 dB SPL. The absolute sound pressure level

(dB re: 20 µPa) was measured with a 1/4-inch microphone (Brüel and Kjaer 4135) situated near

the concha of the ear opposite to the recording side. Typically we presented 10-20 tone pulses

at each sound level.

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Frogs - Acoustic stimuli comprising tone bursts at the unit’s CF having a rise-fall time of 5 ms

and duration of 20 - 80 ms, were presented once every 2.5 sec. The stimulus parameters were

controlled by a PC-based Tucker Davis System-2 hardware and software. Frog was stimulated

via a free field loudspeaker (ADS 200 LC) located 55 cm on the frog’s right side (90˚ from the

frog’s longitudinal axis which corresponds to the best azimuth of most IC units). The absolute

sound pressure level (dB re: 20 µPa) was calibrated at the location of the frog’s left tympanum

using a free-field condenser microphone (Brüel and Kjaer 4165). Ten tone bursts were

presented at each sound level; the sound level ranged from 20 to 80 dB SPL.

Data analysis

Dot raster histograms were used to depict a unit’s temporal discharge pattern for each epoch at

each level. Each dot in a raster histogram indicated a spike at the relative time instant with

respect to the stimulus onset. Dot raster histograms from different sound levels were combined

to create a unit’s composite dot histogram for visualization of how the response latency and the

firing pattern changed globally with sound level. To quantify the response latency, we

determined the average and the standard deviation of latency of the first spike of a unit’s

responses to 10-20 epochs within the “response window” (defined as the window wherein the

spike count and/or firing rate is > 25% above the background spontaneous firing). The

spontaneous firing rate was the average response over a 50 ms window prior to each stimulus

epoch.

Iontophoretic drug injection

For a number of neurons in the bat and frog IC showing PLS, we applied bicuculline (i.e.,

a GABAa receptor blocker) iontophoretically in order to assess the role of GABAergic inhibition

in unit’s temporal firing pattern and/or PLS. For this, the glass micropipette that was used for

extracellular single-unit recording was piggy-backed to a 5-barrel glass pipette with a tip

diameter of ~10 µm (Havey and Caspary, 1980) - the tip of the former protruded about 20 µm

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beyond the tip of the latter. Two of the 5 barrels were filled with bicuculline methiodide (or BIC,

10 mM, pH 3.0; Sigma), a GABAa receptor blocker. The central barrel was filled with 0.64%

sodium chloride (pH 7.4) for maintaining electrical balance. The 5-barrel pipette was connected

to a six-channel iontophoresis current generator (Dagan 6400). A retaining current of -15 nA (or

–20 nA) was used to prevent drug leakage at all times except during drug injection. To apply

bicuculline, we used a current of +5 to +20 nA. A unit's response was evaluated before, during,

and after drug application.

RESULTS

Extracellular responses were obtained from 186 neurons in the IC of M. lucifugus and

202 neurons in the IC of Rana pipiens pipiens. Data from the bat IC will be described first,

followed by data from the frog IC. Results from a smaller sample in the bat IC have been

described briefly in a short report (Galazyuk and Feng, 2001).

In agreement with previous reports (Condon et al. 1994; Galazyuk et al. 2000), IC

neurons in little brown bats had CFs ranging from 7 to 80 kHz. Thresholds at CF ranged from 2

to 68 dB SPL, with a modal value of 49 dB SPL. Units with low CFs were represented in the

dorsal aspect of the IC and higher frequencies progressively more ventrally in the IC.

For the majority of IC neurons (132 units, or 71%), an increase in sound level elicited a

monotonic increase in spike count and a concomitant decrease in the first spike latency. The

increase in spike count and the decrease in latency shift were most pronounced at low sound

levels, within 20 dB of the threshold at the unit’s CF; the spike count and the latency did not

change appreciably with a further increase in sound level. Similar level-dependent latency

changes have been described by others (Park and Pollak, 1993; Heil and Irvine, 1997).

A subset of the total population (40 units, or 22%) exhibited periodic discharge patterns

at high sound levels. An increase in sound levels prolonged the firing duration of these neurons,

due to the addition of later spikes at a fixed period. A notable feature was the constancy of their

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firing periodicity. Responses of two representative neurons exhibiting periodic discharge

patterns are shown in Figure 1. Unit GGF6 in Figure 1A fired on average 1-2 spikes at 10-20 dB

SPL, and it consistently fired 4-5 spikes at regular firing intervals from 24 to 90 dB SPL. The

firing periodicity of this neuron was level independent and remained constant at 1.9 ms at 24-90

dB SPL, as evidenced by the tight distribution of the inter-spike interval histogram (Fig. 1B). Unit

GK12 in Figure 1C fired 1 cycle at 10-30 dB SPL, and this grew to 2 firing cycles at 34-60 dB

SPL. Further increase in sound level led to a progressive increase in the number of firing cycles

ending up with 4 at 80-90 dB SPL. Its firing periodicity remained the same (~4.8 ms) over a wide

range of sound levels (Fig. 1D). The firing periodicity was unit specific, ranging from 1.3 to 8.4

ms (Fig. 1E). Figure 1E shows that the firing periodicity was not evenly distributed within this

range; the majority of 40 units had firing periodicities in the range from 1 to 3 ms.

For 26 neurons exhibiting periodic discharges, we evaluated whether the unit’s firing

periodicity depended on sound duration ranging from 1 to 8 ms. We did not use longer sound

durations for this test because the majority of IC neurons did not respond to tone bursts having

longer sound durations. For 25 of 26 units, a change in sound duration did not change the unit’s

firing periodicity. Although the unit’s firing periodicity was unchanged, the firing duration was in

some cases (12 of 25) was dependent on stimulus duration (Fig. 2C), and in other cases (13 of

25) independent of the stimulus duration (Fig. 2A). Unit GGY4 in Figure 2A discharged one

spike at low sound levels (<34 dB SPL) but gave three spikes at higher sound levels at a

constant inter-spike interval (~1.7 ms) for tone pulses with 1 to 8 ms duration, as shown by

inter-spike interval histograms at different sound durations (Fig. 2B). The above neuron was

therefore duration-independent, both in terms of its firing periodicity and duration. In contrast, for

unit GGT9 (Fig. 2C), its firing periodicity also remained constant but its response duration

increased progressively with an increase in stimulus duration (Fig. 2D).

Paradoxical latency shift (PLS)

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For 52 of 186 IC neurons (28%), an increase in sound level elicited an increase in the

first spike latency, i.e., these neurons exhibited PLS. A large majority of these neurons (48/52)

exhibited latency shifts in quantum steps that were unit-specific (Figs. 3A, B). For

example, unit AGO5 in Fig. 3A gave a single spike to tone pulses at 20-60 dB SPL with average

latency of 13 ms; this response latency was shifted in a quantum step from 13 to ~18 ms when

the sound level was increased to >60 dB SPL (Fig. 3C). For unit AGG3 in Fig. 3B, the average

first spike latency changed from 8-9 ms at low sound levels to 12-13 ms at intermediate sound

levels, to 16-17 ms at high sound levels (Fig. 3D). Sometimes the spike count was sparse in the

zone of latency transition (Fig. 3A). Within each response range, the latency was relatively

stable with variations of <6% from the average latency (see error bars in Figs. 3C,D). The

latency shift appeared to be due to the occurrence of early inhibition at high sound levels.

The distribution of shifts in the first spike latency of PLS neurons, in response to 2 ms

tone pulses, is shown in Figure 3E (range = 0.9 – 10.2 ms). The majority of PLS neurons

showed a shift of 4 to 7 ms, and 12 units exhibited a latency shift of 0.9-3 ms, and five units had

a shift of 7-10.2 ms.

Effect of bicuculline on PLS

The extensive overlap in the distributions of firing periodicities and of PLS indicated that

the two classes of neurons might be functionally related (compare Figs. 1E and 3E). The PLS

response pattern of unit AGG3 (Fig. 3B) additionally suggested that PLS might be attributed to

high-threshold early inhibition of periodic discharges. To test this hypothesis directly, in 21 of the

IC neurons exhibiting PLS, we studied each unit’s responses to tone pulses before, during and

after iontophoretic application of bicuculline, a GABAa blocker. Drug application not only

consistently increased the unit’s spike count as expected, but also abolished PLS. More

importantly, administration of bicuculline transformed the unit’s firing pattern at high sound

levels into periodic discharges (Fig. 4), indicating that the drug unmasked the unit’s periodic

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firing pattern. Prior to drug application, the first spike latency of unit GK10 (Fig. 4A, top panel)

showed a shift from 12.6 to 19.5 ms in quantal steps of ~2.3 ms (see arrow heads in top panel)

when the sound level was raised to >48 dB SPL. Similarly, prior to drug application unit GL26

(Fig. 4B, top panel) also showed a level-dependent shift of 5.3 ms in its first spike latency.

Under bicuculline, these units responded to tone pulses over a broad range of sound levels

with evidence of periodic discharges having multiple firing cycles (middle panels of Figs. 4A and

B). After recovery both neurons once again showed a distinct PLS (bottom panels of Figs. 4A

and B).

For the 21 PLS neurons tested, the average firing periodicity observed during drug

application approximated the average quantal shift in first spike latency during the pre-drug

period (Fig. 4 C). These two values were tightly correlated (correlation coefficient = 0.93).

Effect of sound frequency on PLS

In our pilot study (Galazyuk and Feng, 2001), based on a sample of 4 units, we reported

that the magnitude of the PLS seemed to be independent of sound frequency. The more

extended study on 17 units confirmed this finding. When sound frequency was varied within

10% of a unit’s CF, the amount of the latency shift showed little or no change, as shown by a

representative example in Figure 5. This unit exhibited PLS in response to tone pulses at the

unit’s CF, namely, 48 kHz (middle panel of Fig. 5A). PLS was also evidenced at 49 and 47 kHz,

and the magnitude of latency shift was unchanged. When sound frequency was further shifted

to higher or lower frequencies (to 50 and 46 kHz), PLS was still evidenced in spite of the fact

that the spike count was much weaker. Importantly, the magnitude of latency shift was stable for

these sound frequencies, as shown by the dashed lines across the composite PST histograms

in Figure 5B.

Effect of sound duration on PLS

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For 33 neurons exhibiting PLS, we studied the effects of stimulus duration on the PLS

pattern. Sound duration affected the unit’s response in one of two ways. For 19 of 33 units

(58%), the PLS was duration tolerant. Increasing sound duration did not appreciably altered the

magnitude of the PLS although it often suppressed the unit’s responses to tone pulses at high

sound levels, and to a lesser extent the unit’s responses at low sound levels (Fig. 6A). Unit

GGN5 in Fig. 6A exhibited a distinct PLS, with a shift of 5.1 ms in response to 1 ms tone pulses.

When the sound duration was increased to 2 or 4 ms, the PLS remained distinct, and the

amount of latency shift remained 5.1 ms. For 8 ms tone pulses, this neuron stopped responding

to sound above 66 dB SPL.

Latency plots for 19 neurons exhibiting duration-tolerant PLS are shown in Figure 6 B.

For these neurons, the correlation between sound duration and amount of latency shift was very

low (correlation coefficient r = 0.24) suggesting that stimulus duration had little effect on latency

shift.

For the remaining 14 neurons (42%), the PLS was duration-dependent as exemplified by

unit GGX1 in Fig. 6C. This neuron showed a step-wise 3 ms latency shift in response to 1 ms

tone pulses. Increasing the sound duration to 2, 4, and 8 ms increased the latency shift to 3.9

ms, 6.1 ms, and 12.4 ms, respectively. The latency plots for 14 neurons (Fig. 6D) showed that

the change in PLS was tightly correlated with the change in sound duration (correlation

coefficient r = 0.98); the magnitude of latency shift was a monotonically increasing function of

stimulus duration.

Responses of IC neurons to paired sound pulses with various inter-pulse intervals

The bicuculline experiments (Fig. 3) revealed that high-threshold early inhibition is

critical for creation of the PLS. To determine the latency and duration of this inhibition and to

evaluate the functional significance of PLS, we studied the response patterns of 26 IC neurons

to a pair of tone pulses (“pulse“ and “echo”) at the unit’s CF, at inter-stimulus intervals of + 10

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ms (in 1 ms steps). Of the 26 neurons tested, 19 were PLS neurons and 7 were control

neurons. Faure et al. (2003) recently utilized a similar stimulus paradigm and analytical method

to successfully elucidate the timing of synaptic inhibition underlying duration tuning.

Because a unit’s response to high-level sound (the pulse) was often complex,

characterized by early inhibition followed by excitation, whereas a unit’s response to low-level

sound (the echo) was strictly excitatory, we used a unit’s response to the echo to determine the

timing of pulse-induced inhibition. Unit’s responses to the echo (in terms of spike counts) were

measured for different pulse-echo intervals, and unit’s responses to the pulse were ignored.

Spike count to the echo was measured over a time window during which this response was

>50% above the unit’s spontaneous rate.

We observed that the unit’s response to the echo was consistently suppressed when the

echo coincided (or with slight lead) and immediately followed the pulse. In response to 2 ms

tone pulses, unit in Figure 7A showed a PLS of 4.9 ms; the unit’s response to the echo (tone

pulse at 40 dB SPL) had a latency of 7.5 ms but to the pulse (tone pulse at 80 dB SPL) the

latency was 12.4 ms (Fig. 7A). In response to pulse-echo pairs (i.e., tone pulses at 40 and 70

dB SPL respectively), its response to echo was uninhibited when the echo led the pulse by 0.8

ms or greater (Fig. 7D). When the echo delay was 0 - 2 ms, the unit’s response to echo was

completely inhibited. When the echo delay was >2.5 ms, there was a partial release from

inhibition, and even stronger recovery thereafter (Fig. 7G). The above result revealed that this

unit’s response to pulse at 70 dB SPL was preceded by a leading inhibition. The paired pulse

data thus corroborated the bicuculline experiments in revealing the existence of high threshold

leading inhibition for PLS neurons.

The latency and the duration of the inhibition could be estimated. In response to pulse-

echo pairs we first observed response suppression when the echo led the pulse by 0.8 ms or

shorter. This means that inhibition occurred 6.7 ms after the onset of pulse (response latency to

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echo 7.5 ms minus 0.8 ms, the inter-pulse interval when we first observed suppression). This

suppression lasted 3.4 ms, as indicated by dashed vertical lines in Fig. 7G.

For seven control neurons showing short (Fig. 7B) or long (Fig. 7C) burst of spikes to

sound pulses at the unit’s CF throughout 10-90 dB SPL without evidence of PLS, we also

studied their responses to paired tone pulses. Since their responses to CF at 40 and 70 dB SPL

(or 50 and 80 dB SPL) were similar, both pulse and echo were presented at the same intensity

(70 dB SPL). We found that a subgroup of control neurons (N=4) displayed post-excitatory

inhibition, which was evidenced only from their paired-pulse responses, as exemplified by unit

AGN2 in Fig. 7E. This unit showed scattered responses to single tone pulses below 34 dB SPL,

but for tone pulses at 34 to 90 dB SPL it gave periodic discharges over a duration of ~3 ms that

was level independent (Fig. 7B). In response to paired tone pulses, when the echo delay was 3

ms or longer, its echo response was severely inhibited (Fig. 7E), suggesting the existence of a

post excitatory inhibition. Post-excitatory inhibition occurred 2.3 ms after response on-set as

indicated by vertical dashed line in Fig. 7H.

Three of the control neurons showed progressively longer periodic discharges with an

increase in the sound level (Fig. 7C). In response to paired tone pulses at 70 dB SPL, this

neuron gave a more or less constant spike count, independent of the inter-pulse interval (Fig.

7F). Such IC neurons showed no evidence of inhibition, in their responses to single or paired

tone pulses (Fig. 7I).

Frog Inferior Colliculus

Recordings in the frog IC were made primarily from the magnocellular nucleus and the

lateral region of the principal nucleus (Feng and Lin 1991; Endepols et al. 2000). Unit CF

ranged from 200 to 1700 Hz and distributed around three clusters: 300-700 Hz (low-frequency),

900-1300 Hz (mid-frequency), and 1400-1600 Hz (high-frequency). Unit thresholds at CF

ranged from 21 to 47 dB SPL.

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The majority of frog IC neurons (174 of 202 neurons, or 86%) showed a systematic

decrease in the first spike latency when sound level was increased. Of these, a subset of

neurons (n=30; 15%) showed overt periodical discharge patterns at high sound levels, as

exemplified by the composite dot histogram of the unit shown in Fig. 10C. Similar to the

corresponding units in the bat IC, the unit’s firing periodicity, ranging from 5.8 to 60 ms, was

largely independent of the absolute sound level.

Interestingly, 28 IC neurons (14%) exhibited PLS (Figs. 8A, 9A, 9B, 10A). For all but two

of these PLS neurons, the first spike latency shifted in quantum step(s), as exemplified by the

units shown in Figs. 8A, 9A, 9B, and 10A; two PLS neurons showed a gradual increase in the

response latency with sound intensity (not shown). For IC unit in Figure 8A, in response to 20

ms sound pulses at the unit’s CF, it fired a burst of spikes over the range of sound levels tested.

The first spike latency changed from ~39 ms at 20 dB SPL to ~55 ms at 82 dB SPL in quantal

steps of ~7 ms (shown by open arrows on the top panel).

There was a notable difference in the magnitude of latency shift between the bat and

frog IC. In bats, the latency shift ranged from 1.2 to 10.1 ms (mean = 5.6 ms), whereas the

range was 6 to 76 ms (mean = 48 ms) in the frog IC.

Similar to bats, PLS in the frog IC was attributed to high-threshold leading inhibition. We

evaluated the PLS before, during and after application of bicuculine in 5 PLS neurons. Drug

application increased the unit’s spike count and abolished the PLS that was evidenced during

the pre-drug condition (Fig. 8B). The unit’s PLS fully recovered after the drug application was

withdrawn (Fig. 8C). However, in contrast to the bat IC, the PLS in the frog IC was seldom

completely abolished with local application of bicuculline, even with application of a stronger

iontophoeretic current (20-30 nA).

PLS in the frog IC was also unit-specific and reliably reproducible with repeated

measurements of unit’s response over time (compare Figs. 8A and 8C). A change in sound

frequency also had little effect on the magnitude of shift in the unit’s first spike latency (not

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shown). When tone frequency was moved away from the unit’s CF by up to 10%, PLS neurons

showed the same PLS pattern and amount of latency shift as they did in response to tone bursts

at the unit’s CF.

Similar to the bat IC, a change in stimulus duration had two different consequences. For

eleven of PLS neurons (39%), the magnitude of the latency shift was duration-independent. For

example, unit 7/16-7 in Figure 9A showed a distinct PLS in response to 10 ms tone bursts (top

panel); its phasic response at lower sound levels was delayed by ~46 ms when the sound level

was raised to >80 dB SPL. For 20 and 40 ms tone pulses (second and third panels in Fig. 9A),

the unit’s PLS remained unchanged, and there was a latency shift of ~48 ms when sound level

reached beyond 84 dB SPL. At 80 ms, the unit’s response to tone pulses at >82 dB SPL was

completely abolished and so was the unit’s PLS (bottom panel, Fig. 9A).

Seventeen PLS neurons (61%) in the frog IC showed duration-dependent PLS. As

shown for unit W751 in Figure 9B, increasing the stimulus duration from 10 ms to 20, 40, and 80

ms increased the latency shift progressively from 25 ms to 50, 81, and 98 ms, respectively. The

magnitude of the latency shift was roughly proportional to the change in stimulus duration.

For 33 IC neurons (25 PLS neurons and 8 non-PLS neurons), we studied their

responses to paired tone pulses at the unit’s CF with inter-pulse intervals of from 0 to +100 ms

(in 10 ms steps). We found that PLS neurons and non-PLS neurons behaved similarly to the

corresponding neurons in the bat IC (see Fig. 7 for comparison). The PLS neuron in Fig. 10A

was stimulated with a pair of sound pulses (pulse @ 70 dB SPL; echo @ 40 dB SPL). This

neuron showed inhibitory interactions suggesting the presence of leading inhibition (Fig. 10D); it

exhibited response suppression to echo when the pulse-echo interval was -20 ms to 45 ms (Fig.

10G). Thus, with the unit’s response latency to echo of 32 ms (Fig.10A), using simple

calculations (32-20=12; 20+45=65), we deduced that inhibition must have begun 20 ms after the

pulse onset and lasted 65 ms.

17

Non-PLS neurons in the frog IC similarly showed two different response patterns (Figs.

10B, E, H and Figs. 10C, F, I), as did non-PLS neurons in the bat IC. For 3 of 8 non-PLS

neurons, there was evidence of post-excitatory inhibition, as exemplified by the unit in Fig. 10B.

This unit showed tonic discharges in response to 20 ms tone bursts (Fig. 10B). Unlike the PLS

unit in Figure 10A, this unit’s discharge terminated rather abruptly, with little or no delay

response following the initial burst (Fig. 10B). In response to paired tone pulses at 70 dB SPL,

there was a marked response suppression to echo for pulse-echo interval of 20 - 100 ms (Fig.

10E, H). As before, since unit’s response latency to echo had a latency 26 ms, we estimated

that this unit’s post-excitatory inhibitory period must have begun 13 ms after the response

onset, as indicated by the vertical dashed line on the bottom panel of Fig. 10B.

For 5 of 8 non-PLS neurons, there was no evidence of inhibitory interaction over the

entire range of inter-pulse intervals, as exemplified by the paired-pulse responses of a

representative unit in Figure 10C. This unit gave tonic and periodic discharges in response to

tone pulses presented at all sound levels, followed by a delayed non-periodic firing (Fig. 10C).

In response to paired pulses at 70 dB SPL, there was no sign of inhibition across all inter-pulse

intervals (Fig. 10F, I).

Discussion

Oscillatory discharges

A sizable number of IC neurons in frogs (N=30; 15%) and bats (N=40; 22%) show

evidence of oscillatory discharges in response to tone pulses at the unit’s CF at high sound

levels. The firing periodicity is essentially constant, unit-specific, and independent of stimulus

intensity, frequency or duration (Figs. 1, 2, 10C). Whereas the firing periodicity is invariable with

a change in stimulus duration, a unit’s firing duration can be independent (Fig. 2B) or dependent

(Fig. 2D) of the stimulus duration. In all cases, the duration of periodic firing is prolonged with an

elevation of sound level (Figs. 1, 2). Together, these results suggest that periodical firing is likely

18

due to an endogenous origin, e.g., intrinsic oscillation, and that such oscillation is damped over

time.

Additionally, pharmacological results from 21 PLS neurons in the bat IC and 5 PLS

neurons in the frog IC show that the periodic discharge at high sound levels is often masked,

and evidenced only when GABAergic inhibition is suppressed, i.e., when bicuculline is

administered (Figs. 4, 8). As before, after the oscillatory discharge is unmasked, its periodicity is

independent of sound intensity, and its firing duration is longer at higher sound levels - these

again suggest that its origin is endogenous and the intrinsic oscillation is damped. In patch

clamp recordings from the IC of the big brown bat, Covey et al. (1996) have observed damped

oscillatory potentials in neurons having different CFs, across a wide range of stimulus

amplitudes; membrane oscillations occur after sound offset, as observed in the present study.

The rate of oscillations reported by these authors is lower than what we observed in the little

brown bat but longer than that in the leopard frog, however.

The maximum firing duration of bat IC neurons exhibiting periodic discharge patterns

could be as long as 26 ms, which was an order of magnitude longer than the stimulus duration

of 2 ms. Therefore, these neurons differ from that of chopper cells in the brainstem auditory

nuclei in a fundamental way (Rhode and Greenberg, 1992) - the chopper firing pattern in the

cochlear nucleus is usually observed within the period of stimulation. However, it is noted that

studies of chopper cells typically employed long tone bursts at low supra-threshold levels. It is

not clear whether or not chopper cells would elicit prolonged periodic firings in response to short

tone pulses at high supra-threshold levels.

Level-dependent latency change and occurrence of PLS

The response latency of sensory neurons generally shortens with an increase in

stimulus intensity, a phenomenon commonly known as “level-dependent latency change”.

Because many auditory functions depend critically on precise timing of neural inputs (reviewed

19

by Covey and Casseday, 1999; Oertel, 1999), one would expect that level-dependent latency

change would have an especially significant impact on auditory processing. In the auditory

system, level-dependent latency change has been observed at every level investigated,

including the cochlea (guinea pig, Deatherage et al., 1959), cochlear nucleus (cat, Goldberg and

Brownell, 1973; Kitzes et al., 1978; rat, Miller, 1975), superior olivary complex (gerbil, Sanes

and Rubel, 1988), lateral lemniscus (cat, Aitkin et al., 1970; bat, Haplea et al., 1994), inferior

colliculus (cat, Hind et al., 1963; Irvine and Gago, 1990; bat, Bodenhamer and Pollak, 1981;

Haplea et al., 1994), medial geniculate body (cat, Aitkin and Dunlop, 1968), and auditory cortex

(cat, Brugge et al., 1969).

The mechanisms underlying level-dependent latency change in the auditory system are

not completely understood. The extensive studies by Heil and Irvine (Heil, 1997a,b, 1998; Heil

and Irvine, 1996, 1997, 1998) show that the average level-dependent latency shift is essentially

the same at the auditory nerve and at the auditory cortex, thus suggesting that the effect is

established at the periphery. These studies also show that the first spike latency depends more

on the maximum acceleration of peak pressure during the rise time than on the steady-state

peak pressure per se (Heil, 1997a; Heil and Irvine, 1997).

PLS, as observed in the present and previous studies, is characterized by a level-

dependent increase (instead of a decrease) in response latency. In contrast to the common

level-dependent latency change, PLS appears to have a central origin (see below on

mechanisms of PLS). A series of studies have revealed that PLS is more prevalent and overt at

the level of the midbrain and above, especially in bats (Berkowitz and Suga, 1989; Rose et al.,

1959; Suga, 1970; Sullivan, 1982b; Aitkin et al., 1970; Covey, 1993; Klug et al., 2000).

Among vertebrates other than bats, PLS neurons have been observed in the auditory

system of cats (Rose et al., 1963) and gerbils (Klug et al., 2000). The finding that some frog IC

neurons also exhibited PLS suggests that the occurrence of this response characteristic is not

limited to mammals, or to animals that echolocate. Because frogs and mammals evolve along

20

two separate lines of evolution, this feature must have evolved independently.

Neural mechanisms underlying PLS

On the basis of a preliminary study, we previously hypothesized a possible neural

mechanism for construction of PLS (Galazyuk and Feng, 2001). In this hypothesis, we assume

that IC neurons showing unit-specific oscillatory discharges are the basic building blocks for

PLS. When a unit’s oscillatory discharge is subjected to high-threshold leading inhibition, the

inhibition suppresses the early response components of unit’s oscillatory discharge at high

sound levels, one at a time, thereby delaying the unit’s response in quantum steps; quantum

PLS would have been difficult to create without oscillatory discharge as a building block. The

results from a more extended study herein support this hypothesis as one of the mechanisms

for creation of PLS, i.e., duration-independent PLS (see below). We found that about one-half of

PLS neurons in the bat and frog IC exhibit duration-independent PLS (Figs. 6, 9A) for which a

change in sound duration does not change the unit’s PLS. Because application of bicuculline

abolishes PLS and unmasks the unit’s oscillatory discharge of these neurons (Figs. 4, 8), we

conclude that the underlying mechanism for duration-independent PLS is high-threshold

inhibition of unit’s oscillatory discharge.

The other half of PLS neurons, however, exhibit duration-dependent PLS. These

neurons exhibit a greater shift in the first spike latency with an increase in sound duration (Figs.

6, 9B). We believe duration-dependent PLS must be attributed to a different neuronal

mechanism. One possible mechanism involves simple switching of the unit’s firing pattern, e.g.,

responding to stimulus onset at low sound levels, and to stimulus offset at high levels, and as

such, an increase in stimulus duration would produce a delayed offset response. A response

switch at low versus high sound levels has been observed in intracellular recordings from the

cat auditory cortex (Volkov and Galazyuk, 1991) and extracellular recordings from the guinea

pig auditory thalamus (He, 2002). However, whether or not response switch is responsible for

21

duration-dependent PLS in the IC requires direct empirical validation. An alternative

mechanism is having an inhibition whose duration lengthens with the stimulus.

Interplay of excitation and inhibition in processing of time-varying information

The paired pulse experiments reported here allow us to quantify the time course of

inhibition without involving drug application or intracellular recordings. Our results show that the

majority of IC neurons in both groups of animals receive both excitatory and inhibitory inputs.

The timing of these inputs can be used to generate various paired-pulse response functions. For

example, when an IC neuron receives an excitation that is followed by inhibition a few

milliseconds later, it produces a short-lasting firing activity (Figs. 7B, 10B). Such neurons show

suppressed paired-pulse responses at long echo delays. In contrast, as described previously,

high threshold leading inhibition of periodic excitatory discharge can create PLS. PLS neurons

exhibit suppressed paired-pulse responses at short echo delays (including short echo leads).

For units that do not show evidence of inhibition, their paired-pulse responses are characterized

by simple summation of responses to the individual pulses (Figs. 7C, 10C).

As noted above, temporal combination of excitatory and inhibitory inputs has also been

previously shown to be responsible for sound duration tuning in the IC of big brown bats

(Casseday et al., 1994). Interestingly, during whole-cell patch-clamp recordings (Covey et al.,

1996), other combinations of excitatory and inhibitory effects have been observed but their

functional significances are not clarified. For example, sound-evoked excitatory post-synaptic

currents are often preceded, or followed, by an inhibitory post-synaptic current, or both. Our

present studies suggest that such combinations are useful for construction of PLS as well as

non-PLS with short-burst firing pattern, which in turn produce distinct suppression patterns in

paired-pulse responses.

22

Inferior colliculus versus auditory cortex

In the auditory cortex of the little brown bat, Sullivan (1982b) and later Berkowitz and

Suga (1989) reported that one-half of PLS neurons in the cortex show delay-tuned responses

and that the magnitude of a unit’s PLS is correlated with the unit’s best delay. Surprisingly,

although it was not a major aim of our research, in response to paired tone pulses, PLS neurons

in the bat IC do not exhibit delay-dependent facilitation. Instead, PLS neurons in the IC exhibit

paired-pulse inhibition almost exclusively (Figs. 7A, 10A). There are several plausible

explanations for the disparity in experimental results between the IC and auditory cortex. Fist of

all, there are some differences in the experimental paradigms between the various studies.

Paired pulse stimuli in our experiments involved pure tone pulses presented at unit’s CF,

whereas the earlier studies utilized frequency-modulated (FM) sweeps from 80 to 20 kHz. FM

sounds include both unit’s CF and a wide spectrum of non-CF frequencies. It is possible that

side band inhibition (that has been shown to be evoked by non-CF tones) may be influential

shaping a unit’s pulse-echo response pattern. Future research that compares pulse-echo

response patterns of PLS neurons to CF and FM sounds might clarify this point. Second,

paired-pulse processing in the IC can be basically different from that in the auditory cortex. It is

possible that PLS is first created in the IC to generate delay-dependent inhibition - this can in

theory be used to create delay-dependent facilitation at the levels of the auditory thalamus and

cortex, e.g., by means of disinhibition. In the mustached bat, Portfors and Wenstrup (2001)

showed that the organization of delay-tuned neurons in the IC is different from that in the

auditory cortex. Delay-tuned neurons are topographically organized in the auditory cortex but

not in the IC. These authors suggested that the creation and organization of delay-tuned

neurons occur at different stages in the ascending auditory system.

Behavioral significance of PLS

23

In echolocating bats, the existing evidence suggests that PLS neurons are important for target

ranging. Behaviorally, ranging has been shown to involve analysis of the elapsed time between

bat’s ultrasonic emission and its echo, or echo delay (Simmons, 1973). PLS neurons showing

delay-dependent facilitation (or delay-tuned response), i.e., showing maximal response to a best

delay, are therefore well suited for determining target range (Feng et al., 1978). Indeed, as

described above, one-half of PLS neurons in the auditory cortex give delay-tuned responses

with the unit’s best delay matching the magnitude of a unit’s PLS (Sullivan, 1982b; Berkowitz

and Suga, 1989). To evaluate the role of PLS neurons behaviorally, Denzinger and Schnitzler

(1994) studied the ranging performance of big brown bats at different relative sound levels of

echoes with a fixed difference in target range. They reasoned that if PLS underlies ranging, the

bat’s performance in target ranging would be compromised when the echo level approaches the

sound level of bat’s emitted signal. This is exactly what these authors observed – this result is

consistent with the involvement of PLS in target ranging, although other explanations cannot be

ruled out.

That PLS occurs widely in vertebrates suggests that it must have general relevance for

processing sounds. Indeed, PLS neurons in the IC of bats and frogs display delay-dependent

inhibition in response to pulse-echo pairs. Such inhibition can be used to suppress weak

echoes, allowing animals to extract sound with minimal interference. In bats, delay-dependent

inhibition in the IC may be used to create delay-dependent facilitation in the cortex for the

purpose of target ranging as well as processing of communication sounds (Esser et al., 1997).

In contrast, delay-dependent inhibition may be the final product of signal processing in frogs and

non-echolocating mammals such as gerbils and cats.

In the insect auditory system, Krahe et al. (2002) proposed that PLS neurons might be

involved in transmitting the information on onset steepness, a sound attribute that is important

for sexual recognition. The empirical evidence in support of this hypothesis, however, is lacking.

Nonetheless, the result of this study suggests that the auditory system may utilize PLS for yet a

24

different listening task. Clearly, further studies are necessary to determine the actual functional

significance of PLS for the various non-echolocating animals.

Functional significances of duration-dependent and duration-independent PLS in bats

During echolocation when a bat approaches a target, it systematically adjusts many

different parameters of its ultrasonic cries; it increases the emission rate, and concomitantly

decreases the duration and intensity of its ultrasonic pulses, most notably during the final

approach to an insect prey (Griffin et al., 1960; Schnitzler et al., 1987; Kalko and Schnitzler

1989; Kalko 1995). In the auditory cortex of the little brown bats and mustache bats, there are

two types of delay-sensitive neurons (O’Neill and Suga 1979; 1982; Wong et al., 1992; Tanaka

et al., 1992; Chittajallu et al., 1995;). The first, the more common type (so-called delay-tuned

neurons), is sharply tuned to specific echo delays regardless of the repetition rate and duration

of the stimuli. The second type (so-called tracking neurons) shows changing best delays

according to the repetition rate and duration of sound pulses. With increasing pulse repetition

rate or decreasing sound duration, these neurons shift their best delays to smaller values. It has

been hypothesized that delay-tuned neurons presumably respond only to echoes when the

target is at a particular target distance (corresponding to the best delay), and are thus activated

over a limited period during a target pursuit, with different neurons activated during different

periods. In contrast, “tracking neurons” would be activated during the entire period of target

pursuit, regardless of the target distance (O’Neill and Suga 1979).

The present findings from the bat IC provide additional support for the above hypothesis.

In response to sounds having different durations we found two different populations of PLS

neurons. Over one-half of PLS neurons (58%) show duration-independent PLS and the

remainder of PLS neurons (42%) exhibit a greater shift with an increase in sound duration.

Neurons exhibiting duration-independent PLS are useful for constructing delay-tuned

25

responses, neurons showing duration-dependent PLS can be used to construct tracking

neurons.

Acknowledgments:

We thank Jeffrey Wenstrup and two anonymous referees for their valuable comments on earlier

versions of this manuscript. This work was supported by a grant R01DC04998 from the

National Institute on Deafness and Other Communication Disorders of the NIH.

26

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Median of Inter-Spike Interval (ms)

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dB S

PL

10

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0 2 4 6 8 100

150

Inter-Spike Interval (ms)

0

150

unit# GGGY4

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dB S

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Inter-Spike Interval (ms)

0

700

unit# GGG19

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dB S

PL

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0 10 20 30

Time (ms)

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42

Figure Legends

Figure 1. Composite dot raster and inter-spike interval histograms of 2 representative IC

neurons showing periodic firing patterns in response to tone pulses at the unit’s CF (presented

at time of 0 ms) at high sound levels. Each dot represents a spike at a relative time instant (re:

stimulus onset time). For each sound level, responses to 10 trials are shown in the composite

dot histograms. The composite inter-spike interval histogram was constructed by combining the

unit’s responses across the entire range of sound levels with a bin width of 0.2 ms. (A) Unit

GGF6 shows periodic discharges at 20-90 dB SPL with a dominant firing period of 1.9 ms – the

unit’s firing periodicity can be observed by the composite inter-spike interval over the entire

response range as shown in Panel B. Sound stimuli are shown by black horizontal bars below

each of the dot raster histograms (same time scale as dot raster histograms). (C) Same as (A)

but from unit GK12 which has a dominant firing period of 4.8 ms (shown by the composite inter-

spike interval in Panel D). (E) Distribution of inter-spike intervals for a population of IC neurons

showing periodic firing patterns.

Figure 2. Composite dot raster histograms of 2 representative IC neurons in response to tone

pulses at the unit’s CF, having different sound durations (1 ms [top panel] to 8 ms [bottom

panel]), presented over 10-90 dB SPL. See legend of Figure 1 for explanation of composite dot

raster and inter-spike interval histograms. (A) Unit GGGY4 shows a periodic firing pattern

whose firing periodicity of 1.8 ms doesn’t change with sound duration; the unit’s firing periodicity

for respective stimulus duration can be observed by the composite inter-spike interval in panels

B. (C) Unit GGGT9 shows also duration independent firing periodicity (1.8 ms; see the

composite inter-spike interval in panels D). In contrast to unit GGGY4, unit GGGT9 exhibits an

increase in response duration with an increase in sound duration from 1 ms to 8 ms. Sound

43

stimuli are shown by black horizontal bars below each of the dot raster histograms (same time

scale as dot raster histograms).

Figure 3. Composite dot raster histograms (A, B) and distributions of first spike latencies (C, D)

of 2 representative PLS neurons in response to tone pulses at the unit’s CF. See legend of

Figure 1 for explanation of composite dot histogram. (A) Unit GGO1 shows a level-dependent

shift of 5.2 ms in its first spike latency. The magnitude of latency shift is measured as a time

difference between averaged first spike latency within each response component – shown by

vertical bars in Panel C. The vertical length of each bar represents a range of sound levels that

evokes unit responses. (B, D) Unit AGG3 shows latency shifts in two quantum steps. (E)

Distribution of latency shifts for a population of IC neurons showing PLS.

Figure 4. Effects of iontophoretic application of bicuculline (20 nA) on two representative PLS

neurons: (A) and (B). Shown are composite dot raster histograms before (top row), during

(middle row), and after (bottom row) drug iontophoresis. See legend of Figure 1 for explanation

of composite dot histogram. (C) Summary data for 21 IC neurons showing that the average

firing periodicity during bicuculine application is correlated with the average PLS during the

predrug condition (r = 0.93).

Figure 5. Effects of stimulus frequency on PLS for a representative IC neuron (unit GGO1).

Shown are composite dot raster histograms (A) and PST histograms (B) in response to tone

pulses at the unit’s CF (48 kHz) and neighboring frequencies at sound levels from 10 to 90 dB

SPL. See legend of Figure 1 for explanation of composite dot histograms. Vertical dashed lines

in panels in B shows that the magnitude of latency shift is more or less independent of the tone

frequency. Sound stimuli are shown by black horizontal bars below each of the dot raster

histograms (same time scale as dot raster histograms).

44

Figure 6. Examples of duration-tolerant (A) and duration-independent PLS (C). Shown are

composite dot raster histograms in response to tone pulses at the unit’s CF having different

durations (1 ms [top panel] to 8 ms [bottom panel]). See legend of Figure 1 for explanation of

composite dot histogram. (A) This unit (unit GGN5) shows a latency shift of 5.1 ms (at the sound

level around 72 dB SPL), irrespective of the pulse duration. (B) Latency plots as a function of

stimulus duration for 19 PLS neurons show duration-tolerant PLS. For these neurons, the

correlation between sound duration and amount of latency shift is very low (correlation

coefficient, r = 0.24). (C) This unit (unit GGX1) shows a latency shift of 3 ms in response to 1 ms

tone pulses. When the sound duration is increased to 2, 4, and 8 ms, this unit shifts its latency

to 3.9, 6.1, and 12.4 ms, respectively. (D) Latency plots as a function of stimulus duration for 14

PLS neurons showing that the change in first spike latency is tightly correlated with the change

in sound duration (correlation coefficient, r = 0.98).

Figure 7. Paired-pulse response patterns of a representative PLS unit (A), a non-PLS neuron

showing a short bust of spikes (B), and a non-PLS neuron showing a long burst of spikes (C).

Top traces show composite dot raster histograms of these neurons in response to a tone pulse

at unit’s CF. Middle traces (D, E, F) show responses from these 3 neurons to paired pulses at

inter-pulse intervals of + 10 ms. The onset time of one tone (the pulse - grey rectangular) was

fixed, whereas the presentation time of the other tone (the echo - black rectangular) was varied

relative to the onset of the pulse. Negative times indicate that the onset of the echo preceded

the onset of the pulse, whereas at positive times, onset of the echo followed the onset of the

pulse. As the echo is moved to different temporal positions with respect to the pulse, interaction

between the two would be expected to result in spike suppression when the echo response

overlaps with the inhibition evoked by the pulse. Thus, the unit’s response to the echo serves to

measure the strength and time course of the inhibition evoked by the pulse. Y-axis: onset time

45

of the echo with respect to onset time of the pulse. For each of three neurons (single column),

pulse and echo sound levels are indicated on the y-axis in A, B, and C by grey and black

rectangular, respectively. The bottom traces (G, H, I) show unit’s spike count as a function of

pulse-echo interval, based on results of paired-pulse data in D - F. Each data point represents

the average spike count of echo response during paired-pulse stimulation at a given pulse-echo

interval. Dashed line on each graph indicates the 50% level below the maximum spike count. All

responses below dashed line were arbitrary considered as inhibition.

Figure 8. Effects of bicuculline on a PLS neuron in the frog IC. Shown are unit’s composite dot

raster histograms before (A), during (B), and after (B) iontophoretic application of bicuculline (30

nA). To construct the dot histogram, tone pulses (duration = 20 ms) at unit’s CF were presented

at different sound levels, from 10 to 90 dB SPL. For each sound level, 10 tone pulses were

presented. See legend of Figure 4 for other details. Open arrows in panel A represent the times

of first spike latency at different sound levels. Sound stimuli are shown by black horizontal bars

below each of the dot raster histograms (same time scale as dot raster histograms).

Figure 9. (A) Example from a representative PLS neuron (unit 7/16-7) showing that stimulus

duration has little or no influence over the PLS of 46 ms at around 80 dB SPL. Shown are

composite dot raster histograms in response to tone pulses at the unit’s CF having different

durations (10 ms [top panel] to 80 ms [bottom panel]). Increase in sound duration from 10 ms

(top panel) to 80 ms (bottom panel) reduced the spike count to sounds at high levels (>80 dB

SPL) but didn’t change the amount of PLS. (B) Example from a representative PLS neuron (unit

W751) showing duration-dependent PLS. This unit showed a latency shift of 25 ms in response

to 10 ms tone pulses. When the sound duration was increased to 20, 40, and 80 ms this unit

increased the latency shift to 50 ms, 81 ms, and 98 ms, respectively.

46

Figure 10. Paired-pulse response patterns of a representative PLS unit (A), a non-PLS neuron

sowing a short bust of spikes (B), and a non-PLS neuron showing a long burst of spikes (C).

Top traces show composite dot raster histograms of these neurons in response to a tone pulse

at the unit’s CF. Middle traces (D, E, F) show responses from these 3 neurons to paired pulses

at inter-pulse intervals of + 100 ms. The onset time of one tone (the pulse - grey rectangular)

was fixed, whereas the presentation time of the other tone (the echo - black rectangular) was

varied relative to the onset of the fixed pulse. The unit’s response to the echo serves to

measure the strength and time course of the inhibition evoked by the pulse. Y-axis: onset time

of the echo with regard to onset time of the pulse. The bottom traces (G, H, I) show unit’s

response to the echo as a function of pulse-echo interval, based on results of paired-pulse data

in D - F. See legend of Figure 8 for other details.