the low and high frequency narrowband stimuli that differed by no more than 20 dB. Spike times were stored using Tucker-Davis (TDT) hardware (RA-16) controlled by customized MATLAB (Mathworks) software. Auditory stimuli were filtered to match those used in the behavioral experiments. Sound levels were set relative to the minimum threshold for the recording site. Each stimulus set was presented 15–25 times in a randomly interleaved manner. Data Analysis The receptive field (RF) center for each site was defined as the position of the weighted average (center of mass) of responses in azimuth and elevation. The RF center measured with the low frequency narrowband sound is referred to as the ‘‘low frequency center’’, and the RF center measured with the high frequency narrowband sound is referred to as the ‘‘high frequency center’’. When deriving population responses to the sounds, before averaging across sites, responses were centered based on the azimuth and elevation of the RF center measured with broadband sounds (2–10 kHz). The low frequency sound was presented 30u to the right of the high frequency sound for 22 sites, and 30u to the left of the high frequency sound for 24 sites. For this second group of sites, the responses were reversed around the center position so that responses across the two groups could be directly compared. For sounds separated in elevation, the low frequency sound was presented 30u above the high frequency sound for 12 sites, and 30u below the high frequency sound for 11 sites. For this second group of sites, for plotting purposes the responses were reversed around the center position so that responses across the two groups could be directly compared. Before averaging across sites, the response strength at each site was normalized to the maximum response, averaged across time, to either sound presented alone. Results Behavioral responses to two simultaneous sounds separated in space Owls were trained to respond to free-field low frequency (3– 5 kHz) and high frequency (7–9 kHz) narrowband sounds presented alone. Then, they were tested with trials in which low and high frequency sounds were presented simultaneously from different locations, interleaved with trials in which the low and high frequency sounds were presented alone. When paired sounds were presented, the sources were separated in space by 30u either in azimuth or in elevation. The responses of all three owls were similar. Data from Owl L are shown in Fig. 1A,B. Responses to single sound sources (either low or high frequency) were consistent and stereotyped. The owls responded at short latency (interquartile range = 190–281 ms) with a rapid orientation of the head toward the location of the source. Final orientations consistently undershot the location of the source by errors of 2–5u (mean error for each condition and owl), depending on the source location and the owl. When the owls were presented with both low and high frequency sounds simultaneously and at equal levels, localization was dominated by the location of the low frequency sound in azimuth and by the location of the high frequency sound in elevation (Fig. 1). The direction of the head turn was highly predictable and the latencies were short (interquartile range = 214–256 ms). When the sources were separated in azimuth, the owls oriented in the direction of the location of the low frequency source, regardless of whether it was to the left or to the right (Fig. 1A, open circles; p,1025 for each owl; 2-tailed t- test; hypothesis that the mean response was zero). In contrast, when the same stimuli were separated in elevation, the owls Hierarchy of Auditory Cues oriented in the direction of the high frequency source, regardless of whether it was above or below the horizon (Fig. 1B, open circles; p,1025 for each owl; 2-tailed t-test; hypothesis that the mean response was zero). Significant differences in orientation latencies were not observed for single and multiple sounds. The robustness of frequency-dependent localization dominance to changes in the relative levels of the two sounds was tested for azimuthal separations in two owls (Fig. 2). When the low and high frequency sounds were presented at equal levels, the owls oriented toward the location of the low frequency sound: the endpoints of the orienting movements did not differ statistically from those generated in response to the low frequency sound alone (Fig. 2, top histograms; p.0.05, 2-tailed t-test). On interleaved trials, the relative levels of the high and low frequency sounds were altered randomly in 10 dB intervals. As the relative level of the high frequency sound increased, the probability of the owl orientating toward the high frequency sound increased. This effect was more pronounced for Owl L than for Owl D (Fig. 2). Increasing the relative level of the high frequency sound also increased the variance of the responses for both owls. When the level of the high frequency sound had been increased by 15 dB and the level of the low frequency sound decreased by 15 dB (difference = 30 dB; Fig. 2, bottom histograms), Owl D still maintained an orientation bias towards the location of the low frequency sound (p,0.001, 2- tailed t-test, hypothesis that the mean response was zero), whereas Owl L no longer displayed a statistically significant bias in either direction (p..05, 2-tailed t-test). Neural responses to sounds separated in azimuth We recorded neural activity in the OT space map in response to the same low and high frequency narrowband sounds that were used in the behavioral experiments. Auditory neurons in the OT are sharply tuned for space, broadly tuned for frequency, and their spatial tuning is predicted by their tuning to frequency-specific IPDs and ILDs [16,17]. For these experiments, the owls were sedated and the sounds were presented in virtual space (through earphones; Materials and Methods) to permit rapid interleaving of stimuli from various locations. As in the behavioral experiments, either the low or the high frequency sound was presented alone, or both sounds were presented together with a fixed spatial separation (in virtual space) of 30u in azimuth or elevation. The stimuli were positioned relative to the center of the recording site’s RF, and different positions were randomly interleaved. This method of sampling of stimulus space allowed us to infer the distribution of responses across the OT space map to the two stimuli separated in space by 30u. The responses of the site shown in Fig. 3 were representative of the sites that we sampled (sites that responded well to both the high and the low frequency sounds; Materials and Methods). In all of these experiments, the low and high frequency sounds were presented at sound levels that were equal before being filtered by the transfer functions of the ears, thereby mimicking the stimulus conditions in the behavioral experiment. The site illustrated in Fig. 3 exhibited sharp spatial tuning to either the low or the high frequency narrowband sound when presented alone. The site responded at a latency of 14 ms with a burst of spikes that lasted about 8 ms, followed by a brief decline in firing rate, and then a sustained discharge. Both the phasic and sustained components of the neural response were tuned for the azimuth of the stimulus. The site responded most strongly to either a low frequency sound (Fig. 3A, top raster) or a high frequency sound (Fig. 3A, middle raster) at the RF center (azimuth = 0u). When both sounds were presented together, the site responded at the beginning of the stimulus when either sound was positioned at the RF center PLoS ONE | www.plosone.org 3 April 2010 | Volume 5 | Issue 4 | e10396