Hence, for barn owls it is not obvious how sounds will be integrated when low and high frequency cues conflict. Using an approach similar to that of previous human psychophysical studies [3,5], we tested the relative dominance of sound localization cues by presenting owls with simultaneous sounds from different locations. The human auditory system uses temporal coincidence as a strong cue to signify that sound components arise from a single object [12]. By presenting owls with synchronous sounds of different frequencies from different locations, we were able to observe the dynamic resolution of contradictory spatial cues as the central auditory system created a neural representation of the inferred location of the stimuli. Materials and Methods Animals Adult barn owls were housed in flight aviaries. Birds were cared for in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Stanford University Administrative Panel on Laboratory Animal Care (APLAC). Surgeries Owls were anesthetized with 1% halothane mixed with nitrous oxide and oxygen (45:55). A small metal fastener was attached to the rear of the skull and recording chambers (1 cm diameter) were implanted over the optic tectum on both sides, based on stereotaxic coordinates, with dental acrylic. A local analgesic (bupivicaine HCl) was administered to all wounds following surgery. Behavioral Experiments Three owls were used for behavioral testing. During training and testing sessions, an owl was placed on a perch in the center of a darkened sound attenuating chamber. The chamber was equipped with remotely-controlled movable speakers mounted on a narrow, horizontal semicircular track of radius 92 cm. The track held two speakers (Audax TM025F1) mounted on a bar that were separated in space by 30u, either in azimuth (horizontal bar) or elevation (vertical bar). Sound bursts consisted of either low (3–5 kHz) or high (7–9 kHz) frequency narrowband noise, 250 ms in duration, with 5 ms rise and fall times. Bandpass filtering was performed digitally using the ‘‘ellip’’ function in Matlab; stopband attenuation was 50 dB. Sound pressure levels (dBA scale), measured at the center of the chamber with the owl removed, were equal (within61 dB) across frequencies. Head positions were tracked using a head-mounted monitoring device (miniBIRD 500, Ascension Technologies). During an initial training period, an owl learned to first fixate a zeroing light and then orient its head towards the source of a subsequent sound, which was either the low or high frequency sound. Sound levels were randomly interleaved across a range of 10–60 dB above behavioral threshold measured previously for each owl. The location of the sound source was varied randomly across the frontal 640u in azimuth and elevation. The owl was rewarded with a piece of meat for orienting toward the sound with a short latency head movement (,500 ms). During subsequent test sessions, the owl was presented either with one sound alone (as before), or else with two simultaneous sounds (one low and one high frequency narrowband sound) from different locations. When only one sound was presented, the owl was rewarded only when it turned toward the sound source. When simultaneous sounds were presented, the owl was rewarded for any short latency head movement following the onset of the sounds (,500 ms). When both sounds were presented, they were separated either in azimuth or in elevation by 30u. When the Hierarchy of Auditory Cues sounds were separated in azimuth, the elevation was positioned randomly at either +20u, 0u, or 220u; when the sounds were separated in elevation, the azimuth was positioned randomly at either L20u, 0u, or R20u. Each sound was presented at each relative location with equal probability, and sound levels roved randomly from 10–60 dB above behavioral threshold. Single and paired stimuli were randomly interleaved. The data reported in this paper were collected during these test sessions. We collected 20–30 orientation movements for each stimulus configuration from each owl. HRTF measurements To replicate dichotically the frequency-dependent timing and level content of sounds coming from different spatial positions, HRTFs were recorded from 7 owls, using a method similar to that described by Keller et al. [11]. Briefly, each owl was secured in the center of the sound attenuating chamber using the head fastener, and ketamine (0.1 ml/hr) and vallium (0.025 ml/hr) were admin- istered throughout the session. Probetubes (1.5 cm long) attached to microphones (Knowles FG-23652-P16) were inserted into the ears. The tip of each probetube was placed 1–2 mm from the eardrum and the probetube was attached to the edge of the ear canal with superglue. Broadband sounds (2–11 kHz) from a free-field speaker were presented from positions that spanned the frontal hemisphere in 5u increments. For each speaker position, the signal from each microphone was digitally recorded. The HRTF was calculated for each ear and for each location of the speaker by dividing the Fourier transform of the recorded sound waveform by the Fourier transform of the presented sound waveform. The HRTFs were converted into finite impulse response (FIR) filters, or head-related impulse responses (HRIRs) with a linear-phase FIR filter design using least-squares error minimization [13]. We corrected the HRIRs to account for the filtering properties of the speaker, chamber, probetube, microphone, and earphones (Etymotic ER-1, used for dichotic stimulus presentation) by measuring the appropriate transfer functions (see below), and creating inverse FIRs to cancel out their effects. Corrected HRIRs were then used to filter sound waveforms to simulate free field conditions. The phase angle and amplitude from these HRTFs corresponded to the IPD and ILD as a function of frequency. Electrophysiological Experiments Eleven adult barn owls were used for electrophysiological experiments. During a recording session, an owl was suspended in a prone position with its head stabilized using the mounted fastener. Nitrous oxide and oxygen (45:55) were administered continuously so that owls remained in a passive state. Sounds bursts, consisting of low (3–5 kHz) and/or high (7–9 kHz) frequency narrowband noise, 50 ms in duration, with 5 ms rise and fall times, were filtered with head-related transfer functions (HRTFs) from a typical barn owl and were presented dichotically through earphones (Etymotic ER-1). HRTFs from different owls are highly consistent across the frontal region of space that was tested [11,14]. The HRTFs from this owl were chosen because the owl was of average size and its HRTFs closely followed the relationship between ITD and auditory azimuth of the population average. Differences in ILD across the measured HRTFs were on the order of a few decibels. Multiunit and single-unit responses were isolated from the deep layers (layers 11–13) of the OT with insulated tungsten microelectrodes (6–13 MV). The identification of the tectal layers was based on distinct unit properties that have been linked to these layers based on electrode track reconstructions [15]. Site selection was based on two properties: 1) robust responses to broadband (2–10 kHz) search stimuli, and 2) neural thresholds to PLoS ONE | www.plosone.org 2 April 2010 | Volume 5 | Issue 4 | e10396